Pharmaceutical formulations of anti-ILT4 antibodies or antigen-binding fragments thereof and methods of use thereof

Abstract

The present invention relates to stable formulations of antibodies or antigen-binding fragments thereof that bind to human immunoglobulin-like transcript 4 (ILT4). The formulations provided herein may also include buffers, non-ionic surfactants, non-reducing sugars, antioxidants, and other compositions and adjuvants. Also provided are methods of treating various cancers using the formulations disclosed herein. The formulations can be prepared as a medicament and administered to a patient in need thereof.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 336,670, filed on April 29, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference to an Electronically Submitted Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on March 21, 2023, is named 25401_WO_PCT_SL.XML and is 131,718 bytes in size. Field of the Invention

[0005] The present invention relates to pharmaceutical formulations of antibodies or antigen - binding fragments thereof that bind to human interleukin - like transcript 4 (ILT4) and their use in the treatment of various disorders such as cancer. Background of the Invention

[0007] Common strategies used by tumor cells to evade innate and adaptive immune responses are associated with aberrant expression of human leukocyte antigen (HLA)-G (Curigliano et al. Clin Cancer Res. 2013;19(20):5564 - 5571 and Gonzalez et al. Crit Rev Clin Lab Sci. 2012;49(3):63 - 84). HLA-G can directly inhibit immune cell function through receptor binding and / or through the attenuation of trogocytosis and chemotaxis (Morandi et al. Cytokine Growth Factor Review. 2014, 25:327 - 335 and Lin et al. Mol Med. 2015, 21:782 - 791). High expression of HLA-G in multiple tumor types, including, for example, colorectal cancer, pancreatic cancer, endometrial cancer, lung cancer, breast cancer, ovarian cancer, and gastric cancer, is associated with advanced disease stage, tumor aggressiveness, metastatic potential, and poor prognosis (Lin et al. Mol Med. 2015, 21:782 - 791 and Loumange et al. Int J Cancer. 2014, 135, 222:581 - 597). Antibody-mediated blockade of HLA-G function in transgenic mouse models has been shown to inhibit tumor progression and block the expansion of myeloid-derived suppressor cells (MDSC) (Loumange et al. Int J Cancer. 2014, 135, 222:581 - 597, Lin et al. Hum Immunol. 2013, 74:439 - 446, and Agaugue et al. Blood. 2011, 117:7021 - 7031). The binding of HLA-G to ILT4 can directly inhibit the function of monocytes, dendritic cells, and neutrophils, thus attenuating the innate immune anti-tumor response. The interaction between HLA-G and monocytes through ILT4 inhibits the maturation of human monocyte-derived antigen-presenting cells (APC), resulting in reduced expression of MHC class II antigens and co-stimulatory molecules activated by Stat3 (Colonna et al. J Immunol. 1998, 160:3096 - 3100; Allan et al. J Exp Med. 1999, 189(7):1149 - 1155, and Liang et al. Proc Natl Sci USA. 2008, 105(24):8357). Using human monocyte-derived dendritic cells (DC) and ILT4 transgenic mice, HLA-G has been shown to induce the development of tolerogenic APC, accompanied by a maturation / activation arrest of myeloid DC, and the induction of tolerogenic DC by HLA-G is through disruption of the MHC class II presentation pathway (Ristich et al. Eur J Immunol. 2005, 35:1133 - 1142).

[0008] There is an unmet medical need for patients with cancer that does not respond to T cell therapy but may benefit from the alleviation of tissue-associated macrophage / MDSC-mediated tumor tolerance (e.g., "myeloid-rich" tumors). ILT4 blockade would meet this need and is distinguished from current T cell-targeting antibodies (e.g., anti-PD1, anti-TIGIT) by alleviating the inhibition of tolerogenic myeloid cells in the tumor microenvironment. SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention provides a formulation of an anti-human ILT4 (anti-ILT4) antibody or an antigen-binding fragment thereof, comprising: (i) from about 10 mg / mL to about 200 mg / mL of the anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) from about 5 mM to about 20 mM of a buffer; (iii) from about 6% to about 8% weight / volume (w / v) of a non-reducing sugar; (iv) from about 0.01% to about 0.10% (w / v) of a non-ionic surfactant; and (v) from about 1 mM to about 20 mM of an antioxidant, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, wherein X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21), wherein the buffer is an L-histidine buffer, an acetate buffer or a citrate buffer, wherein the non-reducing sugar is a disaccharide, wherein the non-ionic surfactant is polysorbate 20 or polysorbate 80, and wherein the antioxidant is methionine.

[0010] In certain embodiments, the buffer is selected from an L-histidine buffer, an acetate buffer and a citrate buffer. In one embodiment, the buffer is an L-histidine buffer. In another embodiment, the buffer is an acetate buffer. In yet another embodiment, the buffer is a citrate buffer.

[0011] In some embodiments, the non-reducing sugar is sucrose.

[0012] In certain embodiments, the nonionic surfactant is polysorbate 80 (PS-80) or polysorbate 20 (PS-20). In one embodiment, the nonionic surfactant is PS-80. In another embodiment, the nonionic surfactant is PS-20.

[0013] In some embodiments, the antioxidant is L-methionine.

[0014] Thus, in another aspect, the present disclosure provides a formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 10 mg / mL to about 200 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) from about 5 mM to about 20 mM L-histidine buffer; (iii) from about 6% to about 8% (w / v) sucrose; (iv) from about 0.01% to about 0.10% (w / v) PS-80; and (v) from about 1 mM to about 20 mM L-methionine, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, wherein X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0015] In some embodiments, the formulation comprises from about 8 mM to about 12 mM L-histidine buffer.

[0016] In certain embodiments, the formulation comprises from about 5 mM to about 10 mM L-methionine.

[0017] In other embodiments, the formulation comprises from about 0.01% to about 0.05% (w / v) PS-80.

[0018] In still other embodiments, the formulation comprises from about 10 mg / mL to about 150 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof. In yet other embodiments, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 10 mg / mL, about 12.5 mg / mL, about 15 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL or about 150 mg / mL. In one embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 10 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 12.5 mg / mL. In still another embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 15 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 25 mg / mL. In one embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 50 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 75 mg / mL. In still another embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 100 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 125 mg / mL. In still yet another embodiment, the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 150 mg / mL. In a specific embodiment, the anti-ILT4 antibody or an antigen-binding fragment thereof is a monoclonal antibody.

[0019] Thus, in one specific embodiment, the formulation comprises about 25 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80 and about 10 mM L-methionine.

[0020] In another specific embodiment, the formulation comprises about 50 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80 and about 10 mM L-methionine.

[0021] In still another specific embodiment, the formulation comprises about 75 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80 and about 10 mM L-methionine.

[0022] In another specific embodiment, the formulation comprises about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0023] In yet another specific embodiment, the formulation comprises about 125 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0024] In certain embodiments of the various formulations provided herein, the formulation has a pH range of about 5.0 to about 6.8. In some embodiments, the formulation has a pH range of about 5.5 to about 6.0. In other embodiments, the formulation has a pH of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0. In one embodiment, the formulation has a pH of about 5.5. In another embodiment, the formulation has a pH of about 5.6. In yet another embodiment, the formulation has a pH of about 5.7. In another specific embodiment, the formulation has a pH of about 5.8. In another embodiment, the formulation has a pH of about 5.9. In yet another specific embodiment, the formulation has a pH of about 6.0.

[0025] Thus, in one specific embodiment, a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof comprises: (i) about 50 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer, pH about 5.5; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) polysorbate 80; and (v) about 10 mM L-methionine, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, wherein X1 is N, Q, E, or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0026] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, where X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0027] In some embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0028] In other embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:58.

[0029] In still other embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO:2 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0030] In yet other additional embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO:80 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0031] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:58.

[0032] In some embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:7.

[0033] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody that comprises: two heavy chains comprising the amino acid sequence shown in SEQ ID NO:2 and two light chains comprising the amino acid sequence shown in SEQ ID NO:7.

[0034] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody that comprises: two heavy chains consisting of the amino acid sequence shown in SEQ ID NO:2 and two light chains consisting of the amino acid sequence shown in SEQ ID NO:7.

[0035] In other embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:80 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:7.

[0036] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody that comprises: two heavy chains comprising the amino acid sequence shown in SEQ ID NO:80 and two light chains comprising the amino acid sequence shown in SEQ ID NO:7.

[0037] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody that comprises: two heavy chains consisting of the amino acid sequence shown in SEQ ID NO:80 and two light chains consisting of the amino acid sequence shown in SEQ ID NO:7.

[0038] In one embodiment, the formulation is a liquid formulation. In one embodiment, the formulation is a frozen formulation. In another embodiment, the liquid formulation is stored at about 5°C. In yet another embodiment, the formulation is stored frozen at about -20°C or lower. In yet another embodiment, the formulation is stored frozen at about -70°C or lower. In another embodiment, the liquid formulation is a reconstitution solution from a lyophilized formulation.

[0039] In certain embodiments, after the formulation is stored at about 3°C to about 5°C (e.g., 3°C, 4°C, 5°C) for up to 6 months, (i) the % monomer of the anti-ILT4 antibody or its antigen-binding fragment is at least about 99%, as determined by ultra-high performance size exclusion chromatography; (ii) the turbidity of the formulation is at most about 0.135, as measured by OD 350-500 ; (iii) the % main peak of the anti-ILT4 antibody or its antigen-binding fragment is at least about 63%, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23%, and / or the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14%, as determined by high performance ion exchange chromatography; (iv) the subvisible particle count of particles of at least 2 μm in size is at most about 3500, as determined by microflow imaging; and / or (v) the % oxidation of one or more amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%, as determined by reduced peptide mapping analysis.

[0040] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the % monomer of the anti-ILT4 antibody or its antigen-binding fragment is at least about 99%, as determined by ultra-high performance size exclusion chromatography.

[0041] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the turbidity of the formulation is at most about 0.135, as measured by OD 350-500 .

[0042] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by high performance ion exchange chromatography, the % main peak of the anti-ILT4 antibody or its antigen-binding fragment is at least about 63%. In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by high performance ion exchange chromatography, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23%. In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by high performance ion exchange chromatography, the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14%. In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by high performance ion exchange chromatography, the % main peak of the anti-ILT4 antibody or its antigen-binding fragment is at least about 63%, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23%, and the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14%.

[0043] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by microflow imaging, the subvisible particle count of particles with a size of at least 2 μm is at most about 3500.

[0044] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of one or more amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, the amino acid residue is W7 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80. In another embodiment, the amino acid residue is W102 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80. In yet another embodiment, the amino acid residue is M253 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80. In another embodiment, the amino acid residue is M359 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80. In yet another embodiment, the amino acid residue is M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80.

[0045] In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of one amino acid residue selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of two amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of three amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of four amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of all five amino acid residues from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%.

[0046] In yet another aspect, provided herein is a method of treating cancer in a human patient in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical formulation described herein.

[0047] In another aspect, provided herein is the use of the pharmaceutical formulation described herein for the preparation of a medicament for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figures 1A-1I An early stability study of various anti-ILT4 formulations as described in Example 4 is shown, including turbidity (OD Figure 1A , 1D , 1G), % HMW (high molecular weight) ( Figure 1B , 1E , 1H), or % monomer ( Figure 1C , 1F , 1I) under storage conditions of 5°C ( 350-500 ), Figures 1A-1C ), 25°C ( Figures 1D-1F ), or 40°C ( Figures 1G-1I ).

[0049] Figures 2A-2I Shows the early stability studies of various anti-ILT4 formulations as described in Example 4, including % acidic variants ( Figure 2A , 2D , 2G), % main peak ( Figure 2B , 2E , 2H), or % basic variants ( Figure 2C , 2F , 2I) under storage conditions of 5 °C ( Figures 2A-2C ), Figures 2D-2F ), or Figures 2G-2I ).

[0050] Figures 3A-3I Shows the pH range stability studies of various anti-ILT4 formulations as described in Example 5, including turbidity (OD Figure 3A , 3D , 3G), % HMW ( Figure 3B , 3E , 3H), or % monomer ( Figure 3C , 3F , 3I) under storage conditions of 5 °C ( 350-500 )( Figures 3A-3C ), Figures 3D-3F ), or Figures 3G-3I ).

[0051] Figures 4A-4I Shows the pH range stability studies of various anti-ILT4 formulations as described in Example 5, including % acidic variants ( Figure 4A , 4D , 4G), % main peak ( Figure 4B , 4E , 4H), or % basic variants ( Figure 4C , 4F , 4I) under storage conditions of 5 °C ( Figures 4A-4C ), Figures 4D-4F ), or Figures 4G-4I ).

[0052] Figures 5A-5G Shows the surfactant range stability studies of various anti-ILT4 formulations as described in Example 6, including turbidity (OD Figure 5D ) before the start of agitation, Figure 5E ) after 3 days of agitation, Figure 5F ) after 7 days of agitation, or Figure 5G ) after 7 days of environmental control ( 350-500 )( Figure 5A ), % HMW ( Figure 5B ), % monomer ( Figure 5C ), subvisible particle count.

[0053] Figures 6A-6K Shows the photo-stress stability study of various anti-ILT4 formulations as described in Example 7, including turbidity (OD 350-500 )( Figure 6A ), % HMW( Figure 6B ), % monomer( Figure 6C ), % acidic variant( Figure 6D ), % main peak( Figure 6E ), % basic variant( Figure 6F ), M359( Figure 6G ), M253( Figure 6H ), M429( Figure 6I ), W102( Figure 6J ), or % oxidation of W7( Figure 6K ).

[0054] Figure 7 shows the results of initial and 3-day agitation from the surfactant range study as described in Example 6.

[0055] Figure 8 shows the results of 7-day agitation and 7-day environmental control from the surfactant range study as described in Example 6.

[0056] Figure 9 shows the results of the photo-stress study using antioxidant L-methionine as described in Example 7. Detailed Description

[0057] Definitions and Abbreviations

[0058] As used throughout the specification and the appended claims, the following abbreviations apply:

[0059] API Active Pharmaceutical Ingredient

[0060] CDR Complementary Determining Region in the immunoglobulin variable region defined by the Kabat numbering system, unless otherwise specified

[0061] CE-SDS Capillary Electrophoresis Sodium Dodecyl Sulfate

[0062] CHO Chinese Hamster Ovary

[0063] cIEF Capillary Isoelectric Focusing

[0064] DLS Dynamic Light Scattering Test

[0065] DSC Differential Scanning Calorimetry

[0066] DSF Differential Scanning Fluorimetry

[0067] ELISA Enzyme-Linked Immunosorbent Assay

[0068] FR framework region

[0069] HC heavy chain

[0070] HMW high molecular weight

[0071] HP-IEX high performance ion exchange chromatography

[0072] ICH International Council on Harmonization

[0073] IEX ion exchange

[0074] IgG immunoglobulin G

[0075] LC liquid chromatography

[0076] LC-MS liquid chromatography - mass spectrometry

[0077] LMW low molecular weight

[0078] mAb monoclonal antibody

[0079] NR-CE-SDS non-reducing capillary electrophoresis sodium dodecyl sulfate

[0080] PS20 (or PS-20) polysorbate 20

[0081] PS80 (or PS-80) polysorbate 80

[0082] R-CE-SDS reducing capillary electrophoresis sodium dodecyl sulfate

[0083] RH relative humidity

[0084] UP-SEC ultra performance size exclusion chromatography

[0085] V H variable region of immunoglobulin heavy chain

[0086] V L variable region of immunoglobulin light chain

[0087] v / v volume / volume

[0088] WFI water for injection

[0089] w / v weight / volume

[0090] Unless otherwise specifically defined elsewhere in this document, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0091] As used throughout the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0092] References to "or" indicate either or both possibilities, unless the context clearly dictates one of the indicated possibilities. In some instances, "and / or" is used to emphasize either or both possibilities.

[0093] As used herein, "acidic variant" refers to an anti-ILT4 antibody that is more acidic than the major species of the anti-ILT4 antibody (e.g., as determined by cation exchange chromatography). Such acidic variants are detected by various chromatographic purification methods that separate molecular variants by charge, such as ion exchange, e.g., cation exchange chromatography or WCX-10 HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. Generally, acidic variants have a lower isoelectric point (pI) than the major species and can have more acidic characteristics due to, for example, methionine oxidation, sialylation of asparagine residues, or deamidated variants of the antibody or combinations thereof. In one embodiment, the anti-ILT4 antibody acidic variant is an anti-ILT4 antibody species identified by the acidic variant peak in Figure 6D and elutes according to the cation exchange method described in Example 10. In an ion exchange chromatography method, "% acidic variant species" refers to the total area of the acidic variant peak divided by the total area of all peaks in the elution chromatogram. In one embodiment, the anti-ILT4 antibody acidic variant is identified by a peak that elutes before the main peak according to the cation exchange method. In another embodiment, the anti-ILT4 antibody acidic variant is identified by a peak that elutes before the main peak according to the weak cation exchange method. In an ion exchange chromatography method, "% acidic variant" refers to the total area of the acidic species peak divided by the total area of all peaks in the elution chromatogram.

[0094] "Affinity" refers to the total strength of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of the binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including KinExA and Biacore.

[0095] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, and chimeric antibodies.

[0096] As used herein, unless otherwise specified, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to bind to an antigen bound by the full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, and individual antibody heavy or light chains, as well as individual heavy or light chain variable regions.

[0097] As used herein, "basic variant" refers to an anti-ILT4 antibody that is more basic than the major species of the anti-ILT4 antibody (e.g., as determined by cation exchange chromatography). Such basic variants are detected by various chromatographic purification methods that separate molecular variants by charge, such as ion exchange, e.g., cation exchange chromatography (e.g., the method described in Example 10 or WCX-10 HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. Generally, the basic species has a higher pH than the major species and can have a more basic character due to modifications or differences from the major species. In one embodiment, the anti-ILT4 antibody basic species is identified by a peak eluting after the main peak according to the cation exchange method. In another embodiment, the anti-ILT4 antibody basic species is identified by a peak eluting after the main peak according to the weak cation exchange method. In an ion exchange chromatography method, "% basic species" refers to the total area of the basic species peak divided by the total area of all peaks in the elution chromatogram.

[0098] A "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

[0099] The "Fc" region contains two heavy chain fragments that include the CH1 and CH2 domains of the antibody. The two heavy chain fragments are joined together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0100] A "Fab’ fragment" contains a light chain and a portion or fragment of a heavy chain that contains the V H domain and the CH1 domain and the region between the CH1 and CH2 domains such that an interchain disulfide bond can form between the two heavy chains of two Fab’ fragments to form an F(ab’)2 molecule.

[0101] The "F(ab')2 fragment" contains two light chains and two heavy chains, which contain a portion of the constant region between the CH1 and CH2 domains such that an inter-chain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments joined together by a disulfide bond between the two heavy chains. The "F(ab')2 fragment" can be the product of pepsin digestion of an antibody.

[0102] The "Fv region" contains the variable regions from both the heavy and light chains but lacks the constant regions.

[0103] An "isolated antibody" refers to a purified state, and in such context, means that the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth medium. Generally, the term "isolated" is not intended to mean the complete absence of such materials or the absence of water, buffer, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compound as described herein.

[0104] As used herein, "major species" or "major" refers to an anti-ILT4 antibody species that is identified as the most abundant antibody species in a mixture having one or more acidic or basic species thereof. Such major species are detected by various chromatographic purification methods that separate molecular variants by charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 10) or WCX-10 HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. The mixture can be, for example, the result of antibody preparations from mammalian cells and their post-translational modifications, upstream and downstream processing, or storage. In one embodiment, the major species is identified as the main peak according to the cation exchange method. In the ion exchange method, "% major" refers to the total area of the main peak divided by the total area of all peaks in the elution chromatogram. In one aspect of measuring major species, acidic species, or basic species, a Thermo Scientific ProPac WCX-10 column is used for the cation exchange method. In another embodiment, a Thermo Scientific ProPac WCX-10 column is used, with mobile phase (A) being 24 mM MES pH 6.1 with 4% acetonitrile and mobile phase (B) being 20 mM sodium phosphate, 95 mM NaCl pH 8.0 with 4% acetonitrile, and a column temperature of 35°C. In one embodiment, a non-linear gradient is used having: 22%–22% B for 0–0.6 minutes; 22%–29% B for 0.6–15.0 minutes; 29%–70% B for 15.0–30.0 minutes; 70%–100% B for 30.0–30.5 minutes; and 100%–100% B for 30.5–33.0 minutes. In a further embodiment, the cation exchange method is described in Example 10.

[0105] As used herein, the term "monoclonal antibody" refers to a population of antibodies that are substantially homogeneous, i.e., the amino acid sequences of the antibody molecules that make up the population are the same, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations generally include a large number of different antibodies that have different amino acid sequences in their variable domains, which are usually specific for different epitopes. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies, and it is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0106] The term "fully human antibody" or "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in mouse cells, or in a hybridoma derived from mouse cells, a fully human antibody can contain murine carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only murine immunoglobulin sequences. Alternatively, if produced in a rat, in rat cells, or in a hybridoma derived from rat cells, a fully human antibody can contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0107] Typically, the basic "antibody" structural unit comprises a tetramer. In a monospecific antibody, each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a "variable region" or "variable domain" of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain can define the constant region that is primarily responsible for effector functions.

[0108] Typically, human constant light chains are classified as kappa and lambda light chains. In addition, human constant heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and the isotypes of the antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Subtypes of these IgG include, for example, IgG1 and IgG4. The present invention includes anti-ILT4 antibodies and antigen-binding fragments that contain any of these light and / or heavy constant chains.

[0109] As used herein, "variable region", "variable domain", "V region" or "V chain" refers to the segment of an IgG chain that is variable in sequence between different antibodies. The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy chain variable region may be referred to as "V H ". The light chain variable region may be referred to as "V L ". Generally, the variable regions of both the heavy and light chains contain three hypervariable regions, also known as complementarity determining regions (CDRs), which are located in relatively conserved framework regions (FRs). The CDRs are typically arranged by the framework regions such that specific epitopes can be bound. Generally, from the N-terminus to the C-terminus, the variable domains of the light and heavy chains each contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acids are typically assigned to the individual domains according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0110] "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the antibody V H β-sheet framework, or in the antibody V LOne of three hypervariable regions (L1, L2, or L3) within the non-framework region of the β-sheet framework. Thus, CDRs are variable region sequences that are interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined, for example by Kabat, as the most variable regions within the antibody variable domain. Chothia has also structurally defined CDR region sequences as those residues that are not part of the conserved β-sheet framework and are thus capable of adopting different conformations. Both terms are well known in the art. AbM, Contact, and IMGT have also defined CDR region sequences. The positions of CDRs within the canonical antibody variable region have also been determined by comparing a large number of structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Since the number of residues within the hypervariable regions varies between different antibodies, other residues relative to the canonical positions are usually numbered with a, b, c, etc. next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra). Similarly, this nomenclature is well known to those skilled in the art. The relationships between numbering systems, including for example the Kabat numbering and the IMGT unique numbering system, are well known to those skilled in the art and are shown in Table 1 below. In some embodiments, the CDRs are defined by the Kabat numbering system. In other embodiments, the CDRs are defined by the IMGT numbering system. In still other embodiments, the CDRs are defined by the AbM numbering system. In still other embodiments, the CDRs are defined by the Chothia numbering system. In still other embodiments, the CDRs are defined by the Contact numbering system.

[0111] Table 1. Correspondence between CDR numbering systems

[0112] IMGT Kabat AbM Chothia Contact <![CDATA[V H CDR1]]> 27-38 31-35 26-35 26-32 30-35 <![CDATA[V H CDR2]]> 56-65 50-65 50-58 52-56 47-58 <![CDATA[V H CDR3]]> 105-117 95-102 95-102 95-102 93-101 <![CDATA[V L CDR1]]> 27-38 24-34 24-34 24-34 30-36 <![CDATA[V L CDR2]]> 56-65 50-56 50-56 50-56 46-55 <![CDATA[V L CDR3]]> 105-117 89-97 89-97 89-97 89-96

[0113] Sequence identity refers to the degree to which two polypeptides have the same amino acids at equivalent positions when the two sequences are optimally aligned.

[0114] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids that share similar properties and are interchangeable are discussed above.

[0115] "Conservative modification variant" or "conservative substitution" refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.), such that the change can be frequently made without altering the biological activity of the protein. Those skilled in the art will recognize that, generally, a single amino acid substitution in a non-critical region of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.)). In addition, substitution of amino acids that are structurally or functionally similar hardly disrupts biological activity. Exemplary conservative substitutions are set forth in Table 2.

[0116] Table 2. Exemplary conservative amino acid substitutions

[0117] Original Residue Conservative Substitution Ala(A) Gly; Ser Arg(R) Lys; His Asn(N) Gln; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0118] As used herein, the term "epitope" refers to the extent or region on an antigen to which an antibody or antigen-binding fragment binds. Binding of an antibody or antigen-binding fragment disclosed herein to an epitope means that the antibody or antigen-binding fragment binds to one or more amino acid residues within the epitope.

[0119] As used herein, "Treat" or "treating" a cancer or an infectious condition means administering a formulation of the present invention to a subject having an immune condition or a cancerous condition or diagnosed with cancer or a pathogen infection (e.g., virus, bacterium, fungus) to achieve at least one positive therapeutic effect, such as, for example, a reduction in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration of peripheral organs, or a decrease in tumor metastasis or tumor growth rate. "Treatment" may include one or more of the following: inducing / increasing an anti-tumor immune response, stimulating an immune response against a pathogen, toxin, and / or autoantigen, stimulating an immune response against a viral infection, reducing the amount of one or more tumor markers, inhibiting the growth or survival of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors, reducing the level of one or more tumor markers, improving, reducing the severity or duration of cancer, and prolonging patient survival relative to the expected survival in untreated similar patients.

[0120] The terms "cancer", "cancerous", or "malignant" refer to or describe a physiological condition in a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0121] The term "patient" (which may alternatively be referred to herein as "subject" or "individual") refers to a mammal (e.g., rat, mouse, dog, cat, rabbit) that can be treated with the formulations of the present invention, most preferably a human. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient. A "patient in need of treatment" is an individual who has been diagnosed with, suspected of having, or is predisposed to a disease or condition (e.g., an ILT4-related disease such as cancer) for which the formulations of the present invention are intended to treat or ameliorate, or a patient for whom prevention of such a condition is desired.

[0122] The term "therapeutically effective amount" or "effective amount" means an amount of a therapeutic composition or formulation that, when introduced into a patient, will be sufficient to treat a disease or condition. Those skilled in the art will recognize that this level may vary depending on the characteristics of the patient such as age, weight, etc.

[0123] When modifying a numerical quantity of a substance or composition (e.g., mM or M), a percentage of a formulation component (v / v or w / v), the pH of a solution / formulation, a parameter value of a step in a characterization method, etc., the term "about" refers to a variation in the numerical amount that may occur, for example, by virtue of: typical measurement, handling, and sampling procedures involved in the preparation, characterization, and / or use of the substance or composition; instrumental error in these procedures; differences in the manufacture, source, or purity of the ingredients used to prepare or use the composition or perform the procedure; etc. In certain embodiments, "about" may mean a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

[0124] As used herein, "x%(w / v)" is equivalent to x g / 100 ml (e.g., 5% w / v is equal to 50 mg / ml).

[0125] As used throughout the specification and claims, the phrase "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" indicates the inclusion of any stated element or group of elements, and the optional inclusion of other elements having similar or different properties, which other elements do not materially alter the basic or novel properties of the specified dosage regimen, method, or composition. As a non-limiting example, a binding compound consisting essentially of the amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, which substitutions do not materially affect the properties of the binding compound.

[0126] "Comprising" or variations such as "comprise" or "comprises" is used in an inclusive sense throughout the specification and claims, i.e., to specify the presence of the stated feature, but not to exclude the presence or addition of further features that may enhance the operation or utility of any embodiment of the invention, unless the context requires otherwise by express language or necessary implication.

[0127] The term "buffer" encompasses those reagents that maintain the solution pH of a formulation within an acceptable range, or for a lyophilized formulation of the present invention, those reagents that provide an acceptable solution pH prior to lyophilization.

[0128] The terms "lyophilize", "lyophilized", and "freeze-dried" refer to the process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Excipients may be included in the pre-lyophilized formulation to enhance the stability of the lyophilized product upon storage.

[0129] The term "pharmaceutical formulation" refers to a formulation that is in such form as to permit the active ingredient to be effective and that contains no additional components that are toxic to the subject to which the formulation is to be administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout.

[0130] "Pharmaceutically acceptable" refers to such excipients (vehicles, additives) and compositions that can be reasonably administered to a subject to provide an effective dose of the active ingredient employed and are "generally regarded as safe", e.g., when administered to humans, are physiologically tolerable and generally do not produce allergic or similar untoward reactions, such as gastric upset and the like. In another embodiment, the term refers to molecular entities and compositions that are approved by a regulatory agency of the Federal or State government or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals and more particularly in humans.

[0131] A "reconstituted" formulation is a formulation that has been prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration, e.g., parenteral administration), and may optionally be suitable for subcutaneous administration.

[0132] A "stable" formulation is one in which the protein substantially retains its physical stability and / or chemical stability and / or biological activity upon storage or under stress conditions. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For example, in one embodiment, a stable formulation is one in which no significant change is observed for at least 6 months at refrigerated temperature (2-8°C). In one embodiment, a stable formulation is one in which no significant change is observed for at least 12 months at refrigerated temperature (2-8°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 24 months at refrigerated temperature (2-8°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 3 months at room temperature (23-27°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 6 months at room temperature (23-27°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 12 months at room temperature (23-27°C). In another embodiment, a stable formulation is one in which no significant change is observed for at least 18 months at room temperature (23-27°C). The criteria for the stability of antibody formulations are as follows. Generally, no more than 10%, preferably 5% of the antibody monomer degrades, as measured by SEC-HPLC. Generally, the formulation is colorless or transparent to slightly opalescent by visual analysis. Generally, the concentration, pH, and osmotic pressure of the formulation have a change of no more than + / -10%. Potency is generally within 60-140%, preferably 80-120% of the control or reference. Generally, no more than 10%, preferably no more than 5% of the antibody is cleaved, i.e., the % low molecular weight species as determined, for example, by HP-SEC. Generally, no more than 10%, preferably no more than 5% of the antibody aggregates, i.e., the % high molecular weight species as determined, for example, by HP-SEC.

[0133] An antibody "retains its physical stability" in a pharmaceutical formulation if, upon visual inspection of color and / or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering, it does not show a significant increase in aggregation, precipitation, and / or denaturation. Changes in protein conformation can be evaluated by fluorescence spectroscopy, which determines protein tertiary structure, and FTIR spectroscopy, which determines protein secondary structure.

[0134] If the antibody does not show significant chemical changes, it "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be evaluated by detecting and quantifying the chemically altered forms of the protein. Degradation processes that often alter the chemical structure of proteins include hydrolysis or cleavage (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, measurement of isoaspartic acid), and isomerization (evaluated by measuring the content of isoaspartic acid, peptide mapping, etc.).

[0135] If the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited during the preparation of the pharmaceutical formulation, the antibody "retains its biological activity" in the pharmaceutical formulation. The biological activity of the antibody can be determined, for example, by antigen binding assays.

[0136] The term "isotonic" means that the formulation of interest has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 270 - 328 mOsm. Slightly hypotonic is 250 - 269, while slightly hypertonic is 328 - 350 mOsm. The osmotic pressure can be measured, for example, using a vapor pressure or freezing point osmometer.

[0137] Anti-ILT4 Antibodies and Their Antigen-Binding Fragments

[0138] The formulations disclosed herein can be used with any antibody that binds to ILT4 and its antigen-binding fragments (e.g., fully human antibodies). In one embodiment, the anti-ILT4 antibody or its antigen-binding fragment is an antagonist. In another embodiment, the anti-ILT4 antibody or its antigen-binding fragment is the anti-ILT4 antibody or its antigen-binding fragment described herein (e.g., 1E1, 2A6, 3G7, or 2C1).

[0139] In one aspect, the formulations can be used with anti-ILT4 antibodies and their antigen-binding fragments as described herein having one or more of the following properties:

[0140] ●Binds to human ILT4 at one or more amino acid residues in LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61), and / or HTGRYGCQ (SEQ ID NO:62), and / or protects LYREKKSASW (SEQ ID NO:59), TRIRPEL (SEQ ID NO:60), NGQF (SEQ ID NO:61), and / or HTGRYGCQ (SEQ ID NO:62) from deuterium (e.g., D2O) exchange as determined, for example, by hydrogen-deuterium exchange mass spectrometry;

[0141] ●Binds to human ILT4 at domain 1 (see Wilcox et al. BMC Structural Biology 2:6 (2002));

[0142] ●Binds to the extracellular domain of human ILT4 or the TM form of ILT4 expressed on the cell surface, such as pre-B cells, Chinese hamster ovary cells, U937 cells, or Jurkat JE6 cells.

[0143] ●Calculated pI ~ 7.29 (e.g., 7.29 or 7.30);

[0144] ●Experimentally determined pI ~ 7.2;

[0145] ●Characterized by a thermal profile with Tm onset > 60 °C, Tm1 ~ 65.2 °C, and Tm2 ~ 78.8 °C;

[0146] ●Binds to human ILT4 with a K -8 of approximately 1.7X10 D M (e.g., as determined by surface plasmon resonance, such as the binding of anti-ILT4 to human ILT4 with a polyhistidine tag);

[0147] ●Ka = 5.5X10 5 M -1 s -1 (e.g., as determined by surface plasmon resonance, such as the binding of anti-ILT4 to human ILT4 with a polyhistidine tag);

[0148] ●Kd = 9X10 -3 s -1 (e.g., as determined by surface plasmon resonance, such as the binding of anti-ILT4 to human ILT4 with a polyhistidine tag);

[0149] ●For example, as determined by surface plasmon resonance, at approximately 0.25 micrograms / ml ( +IC of 0.06 μg / ml 50 Block the binding of HLA-G (e.g., Fc-fused HLA-G) to human ILT4 (e.g., ILT4 on mouse 3A9T cells transfected with and expressing ILT4);

[0150] ● For example, as determined by surface plasmon resonance, block the binding of HLA-A, HLA-B (e.g., dexamers of fluorescent dye-labeled HLA-A such as HLA*A2:01 or dexamers of HLA-B such as HLA*B7:02) and / or HLA-F (e.g., fluorescent dye-labeled HLA-F tetramer) to ILT4 (e.g., ILT4 on mouse 3A9 T cells transfected with and expressing ILT4);

[0151] ● For example, as determined by surface plasmon resonance, block the binding of ILT4 (e.g., ILT4 on mouse 3A9 T cells transfected with and expressing ILT4) to ANGPTL1, ANGPTL4, and / or ANGPTL7 (e.g., biotinylated ANGPTL protein);

[0152] ● Do not bind to ILT2, ILT3, ILT5, LILRB5, LILRA1, LILRA2, ILT7, ILT8, and / or ILT11;

[0153] ● For example, with an EC of 0.43 μg / ml ( + 0.14 μg / ml) 50 Reverse ILT4-mediated IL2 inhibition in ILT4-transfected 3A9 cells;

[0154] ● Rescue ILT4:HLA-G-induced inhibition of mast cell degranulation (e.g., in the presence of plate-bound HLA-G tetramer), e.g., wherein the mast cells express ILT4 and CD200RLa and are stimulated, for example, with antibody-mediated CD200RLa crosslinking;

[0155] ● Enhance the expression of pro-inflammatory myeloid cytokines such as GM-CSF and / or TNFα induced by lipopolysaccharide (LPS) from peripheral blood mononuclear cells (PBMC);

[0156] ● Enhance the expression of pro-inflammatory myeloid cytokines such as GM-CSF and / or TNFα induced by anti-CD3 from peripheral blood mononuclear cells (PBMC);

[0157] ● Inhibit tumor growth in humans or other mammals such as mice (e.g., immunodeficient NSG mice), which are reconstituted with human hematopoietic stem cells having, for example, peripheral human CD45+ immune cells. For example, wherein the tumor is a human cutaneous melanoma tumor, such as from the cell line SKMEL5;

[0158] ● Alleviate MDSC-mediated tumor tolerance in a subject (e.g., a human subject) having a tumor;

[0159] ● Do not bind to cynomolgus monkey ILT4 and / or mouse pirB;

[0160] ● Stain CD14+ human monocytes and / or CD11B+ human granulocytes; and / or

[0161] ● Bind to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10) of the human ILT4 haplotypes.

[0162] Antibody 1E1 (Q1E) heavy chain (IgG4)

[0163] Heavy chain

[0164]

[0165] (SEQ ID NO:1; variable domain underlined; CDR double underlined)

[0166] Heavy chain variable domain

[0167] EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSS

[0168] (SEQ ID NO:63)

[0169] Antibody 1E1 (Q1E, S54A) heavy chain (IgG4)

[0170] Heavy chain

[0171]

[0172] (SEQ ID NO:2; variable domain underlined; CDR double underlined)

[0173] Heavy chain variable domain

[0174] EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHAGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSS

[0175] (SEQ ID NO:57)

[0176] Antibody 1E1 heavy chain (IgG1)

[0177] Heavy chain

[0178]

[0179] (SEQ ID NO:44; variable domain underlined; CDR double underlined)

[0180] Heavy chain variable domain

[0181] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSS

[0182] (SEQ ID NO:69)

[0183] 1E1 heavy chain CDR

[0184] CDR-H1: GYYWS (SEQ ID NO:16)

[0185] CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO:17) (e.g., EINHSGSTNYNPSLKS (SEQ ID NO:47) or EINHAGSTNYNPSLKS (SEQ ID NO:48))

[0186] CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18)

[0187] Antibody 1E1 (Q1E) light chain (λ)

[0188] Light chain

[0189]

[0190] (SEQ ID NO:3; variable domain underlined; CDR double underlined)

[0191] Light chain variable domain

[0192] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0193] (SEQ ID NO:70)

[0194] Antibody 1E1 (Q1E, S54A) light chain (λ)

[0195] Light chain

[0196]

[0197] (SEQ ID NO:4; variable domain underlined; CDRs double underlined)

[0198] Light chain variable domain

[0199] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNANRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0200] (SEQ ID NO:71)

[0201] Antibody 1E1 (Q1E, N53Q) light chain (λ)

[0202] Light chain

[0203]

[0204] (SEQ ID NO:5; variable domain underlined; CDRs double underlined)

[0205] Light chain variable domain

[0206] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGQSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0207] (SEQ ID NO:72)

[0208] Antibody 1E1 (Q1E, N53E) light chain (λ)

[0209] Light chain

[0210]

[0211] (SEQ ID NO:6; variable domain underlined; CDRs double underlined)

[0212] Light chain variable domain

[0213] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGESNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0214] (SEQ ID NO:73)

[0215] Antibody 1E1 (Q1E, N53D) light chain (λ)

[0216] Light chain

[0217]

[0218] (SEQ ID NO:7; variable domain underlined; CDRs double underlined)

[0219] Light chain variable domain

[0220] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0221] (SEQ ID NO:58)

[0222] Antibody 1E1 light chain (λ)

[0223] Light chain

[0224]

[0225] (SEQ ID NO:45; variable domain underlined; CDRs double underlined)

[0226] Light chain variable domain

[0227] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0228] (SEQ ID NO:77)

[0229] 1E1 light chain CDR

[0230] CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19)

[0231] CDR-L2: GX1X2NRPS; wherein X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20) (for example, GNSNRPS (SEQ ID NO:49), GQSNRPS (SEQ ID NO:50), GESNRPS (SEQ ID NO:51), GDSNRPS (SEQ ID NO:52), GNANRPS (SEQ ID NO:53), GQANRPS (SEQ ID NO:54), GEANRPS (SEQ ID NO:55), or GDANRPS (SEQ ID NO:56))

[0232] CDR-L3: QSFDNSLSAYV (SEQ ID NO:21)

[0233] Comprising 1E1 heavy and light chain CDRs or 1E1 V H and V L Antibodies and antigen-binding fragments thereof comprising 1E1 heavy and light chains (or variants thereof, such as described herein) may be referred to as "1E1".

[0234] Antibody 2A6 (Q1E) heavy chain (IgG4)

[0235] Heavy chain

[0236]

[0237] (SEQ ID NO:8; variable domain underlined; CDRs double underlined)

[0238] Heavy chain variable domain

[0239] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDSSGWYKGGAFDIWGQGTMVTVSS

[0240] (SEQ ID NO:64)

[0241] Antibody 2A6 (Q1E, S102A, M119L) heavy chain (IgG4)

[0242] Heavy chain

[0243]

[0244] (SEQ ID NO:9; variable domain underlined; CDR double underlined)

[0245] Heavy chain variable domain

[0246] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDASGWYKGGAFDIWGQGTLVTVSS

[0247] (SEQ ID NO:65)

[0248] Antibody 2A6 (Q1E, D101S, M119L) heavy chain (IgG4)

[0249] Heavy chain

[0250]

[0251] (SEQ ID NO:10; variable domain underlined; CDR double underlined)

[0252] Heavy chain variable domain

[0253] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFSSSGWYKGGAFDIWGQGTLVTVSS

[0254] (SEQ ID NO:66)

[0255] The formulations disclosed herein can be used in combination with antibodies and antigen-binding fragments thereof that comprise SEQ ID NO:8, 9, 10, 64, 65 or 66, wherein residue 1 of SEQ ID NO:8, 9, 10, 64, 65 or 66 is Q rather than E.

[0256] 2A6 heavy chain CDR

[0257] CDR-H1: SYAIS (SEQ ID NO:22)

[0258] CDR-H2: GIIPIFGTANYAQKFQG (SEQ ID NO:23)

[0259] CDR-H3: YFX1X2SGWYKGGAFDI; wherein X1 is D or S, and X2 is S or A (SEQ ID NO:24) (e.g., YFDSSGWYKGGAFDI (SEQ ID NO:91), YFSSSGWYKGGAFDI (SEQ ID NO:92), YFDASGWYKGGAFDI (SEQ ID NO:93), or YFSASGWYKGGAFDI (SEQ ID NO:94))

[0260] Antibody 2A6 light chain (λ)

[0261] Light chain

[0262]

[0263] (SEQ ID NO:11; variable domain underlined; CDRs double underlined)

[0264] Light chain variable domain

[0265] QSVLTQPSSLSASPGASASLTCTLRSGINVDTYRIHWYQQKPGSPPQYLLRYKSDSDKHQGSGVPSRFSGSKDPSANAGILLISGLQSEDEA

[0266] DYYCAIWYSSTWVFGGGTQLTVL

[0267] (SEQ ID NO:74)

[0268] The formulations disclosed herein can be used in combination with antibodies and antigen-binding fragments thereof that comprise SEQ ID NO:11 or 74, wherein residue 1 of SEQ ID NO:11 or 74 is E rather than Q.

[0269] 2A6 light chain CDR

[0270] CDR-L1: TLRSGINVDTYRIH (SEQ ID NO:25)

[0271] CDR-L2: YKSDSDKHQGS (SEQ ID NO:26)

[0272] CDR-L3: AIWYSSTWV (SEQ ID NO:27)

[0273] Comprising the 2A6 heavy and light chain CDRs or 2A6 V H and V L Antibodies and antigen-binding fragments thereof comprising the 2A6 heavy and light chains (or variants thereof, e.g., as described herein) may be referred to as "2A6".

[0274] Antibody 3G7 (Q1E) heavy chain (IgG4)

[0275] Heavy chain

[0276]

[0277]

[0278] (SEQ ID NO:12; variable domain underlined; CDRs double underlined).

[0279] Heavy chain variable domain

[0280] EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGEWIQLWSPFDYWGQGTLVTVSS

[0281] (SEQ ID NO:67)

[0282] The formulations disclosed herein may be used in combination with antibodies and antigen-binding fragments thereof comprising SEQ ID NO:12 or 67, wherein residue 1 of SEQ ID NO:12 or 67 is Q rather than E.

[0283] 3G7 heavy chain CDRs

[0284] CDR-H1: SYAMH (SEQ ID NO:28)

[0285] CDR-H2: VISYDGSNKYYADSVKG (SEQ ID NO:29)

[0286] CDR-H3: VGEWIQLWSPFDY (SEQ ID NO:30)

[0287] Antibody 3G7 light chain (κ)

[0288] Light chain

[0289]

[0290] (SEQ ID NO:13; variable domain underlined; CDRs double underlined)

[0291] Variable domain of light chain

[0292] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKFLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK

[0293] (SEQ ID NO:75)

[0294] 3G7 light chain CDRs

[0295] CDR-L1: RASQGISSWLA (SEQ ID NO:31)

[0296] CDR-L2: AASSLQS (SEQ ID NO:32)

[0297] CDR-L3: QQYNSYPPT (SEQ ID NO:33)

[0298] Antibodies and antigen-binding fragments thereof that include the 3G7 heavy and light chain CDRs or 3G7 V H and V L or the 3G7 heavy and light chains (or variants thereof, e.g., as described herein) may be referred to as "3G7".

[0299] Antibody 2C1 (Q1E) heavy chain (IgG4)

[0300] Heavy chain

[0301]

[0302] (SEQ ID NO:14; variable domain underlined; CDRs double underlined)

[0303] Variable domain of heavy chain

[0304] EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLR

[0305] SEDTAVYYCARAGPLYTIFGVVIIPDNWFDPWGQGTLVTVSS

[0306] (SEQ ID NO:68)

[0307] The formulations disclosed herein can be used in combination with antibodies and antigen-binding fragments thereof comprising SEQ ID NO:14 or 68, wherein residue 1 of SEQ ID NO:14 or 68 is Q instead of E.

[0308] 2C1 heavy chain CDR

[0309] CDR-H1: ELSMH (SEQ ID NO:34)

[0310] CDR-H2: GFDPEDGETIYAQKFQG (SEQ ID NO:35)

[0311] CDR-H3: AGPLYTIFGVVIIPDNWFDP (SEQ ID NO:36)

[0312] Antibody 2C1 light chain (Q1E) (λ)

[0313] Light chain

[0314]

[0315]

[0316] (SEQ ID NO:15; variable domain underlined; CDR double underlined)

[0317] Light chain variable domain

[0318] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGSGVVFGGGTQLIIL

[0319] (SEQ ID NO:76)

[0320] The formulations disclosed herein can be used in combination with antibodies and antigen-binding fragments thereof comprising SEQ ID NO:15 or 76, wherein residue 1 of SEQ ID NO:15 or 76 is Q instead of E.

[0321] 2C1 light chain CDRs

[0322] CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:37)

[0323] CDR-L2: GNSNRPS (SEQ ID NO:38)

[0324] CDR-L3: QSYDSSLSGSGVV (SEQ ID NO:39)

[0325] Antibodies and antigen-binding fragments thereof comprising the 2C1 heavy and light chain CDRs or 2C1 V H and V L or the 2C1 heavy and light chains (or variants thereof, e.g., as described herein) may be referred to as "2C1".

[0326] In various embodiments of the antibody or antigen-binding fragment thereof, the C-terminal lysine of the heavy chain immunoglobulin is absent.

[0327] Thus, in some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light chain and a heavy chain immunoglobulin, wherein the light chain immunoglobulin comprises the amino acid sequence shown in SEQ ID NO:3, 4, 5, 6, 7, 11, 13, 15, or 45; and / or the heavy chain immunoglobulin comprises the amino acid sequence shown in SEQ ID NO:1, 2, 8, 9, 10, 12, 14, 44, 79, 80, 81, 82, 83, 84, 85, or 86.

[0328] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:1 or 79 and a light chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:3.

[0329] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:2 or 80 and a light chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:4.

[0330] In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:2 or 80 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:5.

[0331] In still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:2 or 80 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:6.

[0332] In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:2 or 80 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:7.

[0333] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:2 or 80 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:3.

[0334] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:8 or 82 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:11.

[0335] In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:9 or 83 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:11.

[0336] In still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:10 or 84 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:11.

[0337] In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:12 or 85 and a light-chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:13.

[0338] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 14 or 86 and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0339] In certain other embodiments, the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin, a heavy chain immunoglobulin, or both a light chain and a heavy chain immunoglobulin, wherein the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 70, 71, 72, 73, 58, 74, 75, 76, or 77, and / or the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 63, 57, 64, 65, 66, 67, 68, or 69.

[0340] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 63 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0341] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 71.

[0342] In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 72.

[0343] In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 73.

[0344] In yet other additional embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 58.

[0345] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0346] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:64 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:74.

[0347] In other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:65 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:74.

[0348] In still other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:66 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:74.

[0349] In yet other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:67 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:75.

[0350] In yet further other embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:68 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:76.

[0351] In a further embodiment, the antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L ) domain comprising the CDR-L1, CDR-L2, and CDR-L3 of 1E1 (e.g., SEQ ID NOs: 19-21); and an immunoglobulin heavy chain variable (V H ) domain comprising the CDR-H1, CDR-H2, and CDR-H3 of 1E1 (e.g., SEQ ID NOs: 16-18).

[0352] In a further embodiment, the antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L ) domain comprising the CDR-L1, CDR-L2, and CDR-L3 of 2A6 (e.g., SEQ ID NOs: 25-27); and an immunoglobulin heavy chain variable (V H ) domain comprising the CDR-H1, CDR-H2, and CDR-H3 of 2A6 (e.g., SEQ ID NOs: 22-24).

[0353] In a further embodiment, an antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L ) domain comprising CDR-L1, CDR-L2, and CDR-L3 of 3G7 (e.g., SEQ ID NOs: 31-33); and an immunoglobulin heavy chain variable (V H ) domain comprising CDR-H1, CDR-H2, and CDR-H3 of 3G7 (e.g., SEQ ID NOs: 28-30).

[0354] In a further embodiment, an antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L ) domain comprising CDR-L1, CDR-L2, and CDR-L3 of 2C1 (e.g., SEQ ID NOs: 37-39); and an immunoglobulin heavy chain variable (V H ) domain comprising CDR-H1, CDR-H2, and CDR-H3 of 2C1 (e.g., SEQ ID NOs: 34-36).

[0355] In one embodiment, the antibody or antigen-binding fragment comprises a V H domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and / or a V L domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQID NO: 19), CDR-L2: GNSNRPS (SEQ ID NO: 49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0356] In another embodiment, the antibody or antigen-binding fragment comprises a V H domain comprising: CDR-H1: GYYWS (SEQ ID NO: 16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO: 47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18); and / or a V L domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQID NO: 19), CDR-L2: GQSNRPS (SEQ ID NO: 50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0357] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V H domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GESNRPS (SEQ ID NO:51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0358] In another embodiment, the antibody or antigen-binding fragment thereof comprises a V H domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0359] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V H domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GNANRPS (SEQ ID NO:53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0360] In another embodiment, the antibody or antigen-binding fragment thereof comprises a V HDomain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or V comprising the following L Domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GQANRPS (SEQ ID NO:54) and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0361] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising the following H Domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or V comprising the following L Domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GEANRPS (SEQ ID NO:55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0362] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising the following H Domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or V comprising the following L Domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GDANRPS (SEQ ID NO:56) and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0363] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising the following H Domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or V comprising the followingL Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GNSNRPS (SEQ ID NO:49), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0364] In another embodiment, the antibody or antigen-binding fragment thereof comprises a V H Domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L Domain: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GQSNRPS (SEQ ID NO:50), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0365] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V H Domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L Domain: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GESNRPS (SEQ ID NO:51), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0366] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V H Domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L Domain: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0367] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V H domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GNANRPS (SEQ ID NO:53), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0368] In another embodiment, the antibody or antigen-binding fragment thereof comprises a V H domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GQANRPS (SEQ ID NO:54), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0369] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V H domain: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or a V L domain: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GEANRPS (SEQ ID NO:55), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0370] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V HDomains: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48) and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and / or V domains comprising the following L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQID NO:19), CDR-L2: GDANRPS (SEQ ID NO:56) and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0371] The formulations disclosed herein can be used in combination with an antibody or an antigen-binding fragment thereof that binds to ILT4, said antibody or antigen-binding fragment thereof comprising the V L domains of antibody 1E1 (e.g., SEQ ID NO:70, 71, 72, 73, 58 or 77) and / or the V H domains of antibody 1E1 (e.g., SEQ ID NO:63, 57 or 69).

[0372] The formulations disclosed herein can be used in combination with an antibody or an antigen-binding fragment thereof that binds to ILT4, said antibody or antigen-binding fragment thereof comprising the V L domains of antibody 2A6 (e.g., SEQ ID NO:74) and / or the V H domains of antibody 2A6 (e.g., SEQ ID NO:64, 65 or 66).

[0373] The formulations disclosed herein can be used in combination with an antibody or an antigen-binding fragment thereof that binds to ILT4, said antibody or antigen-binding fragment thereof comprising the V L domains of antibody 3G7 (e.g., SEQ ID NO:75) and / or the V H domains of antibody 3G7 (e.g., SEQ ID NO:67).

[0374] The formulations disclosed herein can be used in combination with an antibody or an antigen-binding fragment thereof that binds to ILT4, said antibody or antigen-binding fragment thereof comprising the V L domains of antibody 2C1 (e.g., SEQ ID NO:76) and / or the V H domains of antibody 2C1 (e.g., SEQ ID NO:68).

[0375] The formulations disclosed herein can be used in combination with an antibody that binds to ILT4 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising the light chain immunoglobulin of antibody 1E1 (e.g., SEQ ID NO: 3, 4, 5, 6, 7, or 45) and / or the heavy chain immunoglobulin of antibody 1E1 (e.g., SEQ ID NO: 1, 2, 44, 79, 80, or 81).

[0376] The formulations disclosed herein can be used in combination with an antibody that binds to ILT4 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising the light chain immunoglobulin of antibody 2A6 (e.g., SEQ ID NO: 11) and / or the heavy chain immunoglobulin of antibody 2A6 (e.g., SEQ ID NO: 8, 9, 10, 82, 83, or 84).

[0377] The formulations disclosed herein can be used in combination with an antibody that binds to ILT4 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising the light chain immunoglobulin of antibody 3G7 (e.g., SEQ ID NO: 13) and / or the heavy chain immunoglobulin of antibody 3G7 (e.g., SEQ ID NO: 12 or 85).

[0378] The formulations disclosed herein can be used in combination with an antibody that binds to ILT4 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising the light chain immunoglobulin of antibody 2C1 (e.g., SEQ ID NO: 15) and / or the heavy chain immunoglobulin of antibody 2C1 (e.g., SEQ ID NO: 14 or 86).

[0379] The formulations disclosed herein can be used in combination with an antibody consisting of two heavy chains and two light chains, wherein each light chain comprises the V L or light chain immunoglobulin of antibody 1E1, 2A6, 3G7, or 2C1, and each heavy chain comprises the V H or heavy chain immunoglobulin of antibody 1E1, 2A6, 3G7, or 2C1.

[0380] In one embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence shown in SEQ ID NO: 58, and each heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57.

[0381] In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence shown in SEQ ID NO: 58, and each heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57, wherein the light chain further comprises the amino acid sequence shown in SEQ ID NO: 90.

[0382] In yet another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence shown in SEQ ID NO:58, and each heavy chain comprises the amino acid sequence shown in SEQ ID NO:57, wherein the heavy chain further comprises the amino acid sequence shown in SEQ ID NO:89.

[0383] In yet another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence shown in SEQ ID NO:58, and each heavy chain comprises the amino acid sequence shown in SEQ ID NO:57, wherein the light chain further comprises the amino acid sequence shown in SEQ ID NO:90, and the heavy chain further comprises the amino acid sequence shown in SEQ ID NO:89.

[0384] In one embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain comprises the amino acid sequence shown in SEQ ID NO:7, and each heavy chain comprises the amino acid sequence shown in SEQ ID NO:2.

[0385] In another embodiment, the antibody consists of two heavy chains and two light chains, wherein each light chain consists of the amino acid sequence shown in SEQ ID NO:7, and each heavy chain consists of the amino acid sequence shown in SEQ ID NO:2.

[0386] In one embodiment, the antibody or an antigen-binding fragment thereof comprises V L (with or without a signal sequence), e.g., V in any one of SEQ ID NO:58 or 70 - 77 L , which has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions; and / or V H (with or without a signal sequence), e.g., V in any one of SEQ ID NO:57 or 63 - 69 H , which has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions while still binding to ILT4.

[0387] The formulations disclosed herein can be used together with polypeptides comprising the amino acid sequences disclosed herein, such as SEQ ID NO:1 - 39, 44, 45, 47 - 58, 63 - 77 or 79 - 86, and polypeptides comprising such amino acid sequences with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative or non-conservative amino acid substitutions therein.

[0388] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light-chain immunoglobulin, a heavy-chain immunoglobulin, or both a light-chain and a heavy-chain immunoglobulin, wherein the light-chain immunoglobulin has at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 11, 13, 15, or 45, and / or the heavy-chain immunoglobulin has at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 8, 9, 10, 12, 14, 44, 79, 80, 81, 82, 83, 84, 85, or 86.

[0389] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light-chain immunoglobulin, a heavy-chain immunoglobulin, or both a light-chain and a heavy-chain immunoglobulin, wherein the light-chain immunoglobulin comprises a light-chain variable domain having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 70, 71, 72, 73, 58, 74, 75, 76, or 77, and / or the heavy-chain immunoglobulin comprises a heavy-chain variable domain having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 63, 57, 64, 65, 66, 67, 68, or 69.

[0390] In one embodiment, the immunoglobulin heavy chain of the anti-ILT4 antibody or antigen-binding fragment of the invention is operably linked to a signal sequence such as comprising the amino acid sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 41), and / or the immunoglobulin light chain of the anti-ILT4 antibody or antigen-binding fragment of the invention is operably linked to a signal sequence such as comprising the amino acid sequence MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 42).

[0391] In one embodiment, the N-terminal glutamine (Q) of the immunoglobulin chain (e.g., heavy chain and / or light chain) described herein is replaced with pyroglutamic acid. In one embodiment, the N-terminal Q of the heavy-chain immunoglobulin is replaced with pyroglutamic acid. In another embodiment, the N-terminal Q of the light-chain immunoglobulin is replaced with pyroglutamic acid. In yet another embodiment, the N-terminal Q of the heavy-chain immunoglobulin and the N-terminal Q of the heavy-chain immunoglobulin are replaced with pyroglutamic acid.

[0392] The formulations disclosed herein can also be used in combination with such an antibody or antigen-binding fragment that binds to the same ILT4 (e.g., human ILT4) epitope as any of the anti-ILT4 antibodies or antigen-binding fragments thereof disclosed herein (e.g., 1E1, 2A6, 3G7, or 2C1). In one embodiment, the epitope is LYREKKSASW (SEQ ID NO:59). In another embodiment, the epitope is TRIRPEL (SEQ ID NO:60). In yet another embodiment, the epitope is NGQF (SEQ ID NO:61). In still another embodiment, the epitope is HTGRYGCQ (SEQ ID NO:62). In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same human ILT4 epitope as an antibody or antigen-binding fragment thereof that respectively comprises the heavy chain and light chain amino acid sequences shown in any of the following: SEQ ID NO:1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and 3; 8 and 11; 9 and 11; 10 and 11; 12 and 13; 14 and 15; 79 and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 11; 83 and 11; 84 and 11; 85 and 13; and 86 and 15. In some embodiments, the antibody or antigen-binding fragment thereof binds to the same human ILT4 epitope as an antibody or antigen-binding fragment thereof that respectively comprises the heavy chain variable domain and light chain variable domain amino acid sequences shown in any of the following: SEQ ID NO:63 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and 58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76.

[0393] The formulations disclosed herein can be used with antibodies and antigen-binding fragments that cross-block the binding of any anti-ILT4 antibody or antigen-binding fragment disclosed herein (e.g., 1E1, 2A6, 3G7, or 2C1) to ILT4 (e.g., human ILT4), or compete with any anti-ILT4 antibody or antigen-binding fragment disclosed herein (e.g., 1E1, 2A6, 3G7, or 2C1) for ILT4 (e.g., human ILT4). The cross-blocking antibodies and antigen-binding fragments discussed herein can be identified based on their ability to block the binding of any antibody or fragment specifically set forth herein to ILT4 in a binding assay (e.g., Biolayer Interferometry (BLI; e.g., FORTEBIO OCTET binding assay; Pall ForteBio Corp; Menlo Park, CA), Surface Plasmon Resonance (SPR), BIACore, ELISA, flow cytometry). For example, in one embodiment of the invention, when using BLI, the tip of the fiber optic probe is coated with a ligand (e.g., ILT4) and serves as a biosensor, wherein the binding of an anti-ILT4 antibody or antigen-binding fragment to ILT4 alters the interference pattern of white light reflected from the probe layer bound to ILT4 and an internal reference layer. The shift indicates ILT4 / anti-ILT4 binding. In one embodiment of the invention, the ILT4-coated tip is dipped into an analyte solution containing an antibody or antigen-binding fragment, e.g., in a well of a 96- or 384-well plate. In one embodiment of the invention, the plate is oscillated during the reading process to create an orbital flow. To read the assay, white light is directed along the length of the fiber. As mentioned above, the interference between the light reflected from the reference layer and the ILT4-containing immobilized surface of the tip creates a unique light pattern that returns to the fiber. When molecules bind to the immobilized sensor surface, this pattern changes in proportion to the degree of binding. For example, an assay can be used in which an ILT4 (e.g., human ILT4) protein is immobilized on a BLI probe or plate, a reference anti-ILT4 antibody or fragment is bound to ILT4 (e.g., at a saturating concentration), and a test anti-ILT4 antibody or fragment is added. The ability of the test antibody to compete with the reference antibody for ILT4 binding is then determined. In the BLI format, the light interference of the ILT4 complex is monitored to determine whether the test antibody competes effectively with the reference antibody, e.g., by monitoring the nanometers of wavelength shift over time, wherein the shift indicates the lack of additional binding and cross-blocking by the test antibody. In one embodiment of the invention, in the BLI format, if no additional binding of the test antibody is observed, cross-blocking is considered to have occurred qualitatively between the antibodies. In one embodiment of the invention, as a control, cross-blocking of the reference antibody with itself is confirmed; wherein if the reference antibody can cross-block its own binding to ILT4, the assay is determined to be operating correctly.Testing the ability of a test antibody to inhibit the binding of an anti-ILT4 antibody or fragment 1E1, 2A6, 3G7, or 2C1 to ILT4 (e.g., human ILT4) confirms that the test antibody can cross-block the binding of the antibody or fragment to ILT4 (e.g., human ILT4), and thus, in some cases, may bind to the same epitope on ILT4 (e.g., human ILT4) as 1E1, 2A6, 3G7, and / or 2C1. As stated above, antibodies and fragments that bind to the same epitope as any anti-ILT4 antibody or fragment of the invention also form part of the invention. In one embodiment of the invention, BLI is performed in a sandwich format, where a reference anti-ILT4 antibody or antigen-binding fragment is immobilized on a probe and then bound to ILT4. The test anti-ILT4 antibody or antigen-binding fragment is then tested for its ability to block the binding of the reference antibody or fragment.

[0394] In certain embodiments, the antibody or its antigen-binding fragment competes for binding to human ILT4 with an antibody or fragment comprising the heavy and light chain amino acid sequences shown, respectively, in: SEQ ID NO:1 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and 3; 8 and 11; 9 and 11; 10 and 11; 12 and 13; 14 and 15; 79 and 3; 80 and 4; 80 and 5; 80 and 6; 80 and 7; 80 and 3; 82 and 11; 83 and 11; 84 and 11; 85 and 13; and 86 and 15. In some embodiments, the antibody or its antigen-binding fragment competes for binding to human ILT4 with an antibody or fragment comprising the heavy chain variable domain and light chain variable domain amino acid sequences shown, respectively, in: SEQ ID NO:63 and 70; 57 and 71; 57 and 72; 57 and 73; 57 and 58; 57 and 70; 64 and 74; 65 and 74; 66 and 74; 67 and 75; 68 and 76.

[0395] The formulations disclosed herein can be used in conjunction with anti-ILT4 antibodies and antigen-binding fragments thereof that contain N-linked glycans, which are typically incorporated into immunoglobulins produced in Chinese hamster ovary cells (CHO N-linked glycans) or engineered yeast cells (engineered yeast N-linked glycans) such as, for example, Pichia pastoris. For example, in one embodiment, the anti-ILT4 antibody and antigen-binding fragments thereof contain one or more “engineered yeast N-linked glycans” or “CHO N-linked glycans” (e.g., G0 and / or G0-F and / or G1 and / or G1-F and / or G2-F and / or Man5). In one embodiment, the anti-ILT4 antibody and antigen-binding fragments thereof contain engineered yeast N-linked glycans, i.e., G0 and / or G1 and / or G2, optionally further including Man5. In one embodiment, the anti-ILT4 antibody and antigen-binding fragments thereof contain CHO N-linked glycans, i.e., G0-F, G1-F, and G2-F, optionally further including G0 and / or G1 and / or G2 and / or Man5. In one embodiment, about 80% to about 95% (e.g., about 80 - 90%, about 85%, about 90%, or about 95%) of all the N-linked glycans on the anti-ILT4 antibody and antigen-binding fragments thereof are engineered yeast N-linked glycans or CHO N-linked glycans. See Nett et al., Yeast. 28(3):237 - 252 (2011); Hamilton et al., Science. 313(5792):1441 - 1443 (2006); Hamilton et al., Curr Opin Biotechnol. 18(5):387 - 392 (2007). For example, in one embodiment, the engineered yeast cells are GFI5.0 or YGLY8316 or strains described in U.S. Patent No. 7,795,002 or Zha et al., Methods Mol Biol. 988:31 - 43 (2013). See also International Patent Application Publication No. WO2013 / 066765.

[0396] Anti-ILT4 antibodies and antigen-binding fragments thereof that can be used in conjunction with the various formulations disclosed herein (e.g., 1E1, 2A6, 3G7, and / or 2C1) can also be engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or effector function (e.g., antigen-dependent cellular cytotoxicity). In addition, the antibodies and antigen-binding fragments thereof that can be used in conjunction with the various formulations disclosed herein (e.g., 1E1, 2A6, 3G7, and / or 2C1) can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody, such as a labeled antibody or an antibody-drug conjugate) or modified to alter their glycosylation, again to alter one or more functional properties of the antibody.

[0397] In some embodiments of the various formulations disclosed herein, the antibody or antigen-binding fragment is a monoclonal antibody.

[0398] Pharmaceutical Formulations

[0399] The pharmaceutical formulations described herein can maintain the physical, chemical, and / or biological stability of the anti-ILT4 antibody (e.g., 1E1, 2A6, 3G7, or 2C1) or its antigen-binding fragment during storage (e.g., at about 5°C for at least 6 months, 12 months, 24 months, or 36 months, at about -20°C for at least 6 months, 12 months, 24 months, or 36 months, or at about -70°C for at least 6 months, 12 months, 24 months, or 36 months) and / or under various stress conditions (e.g., agitation, freeze-thaw cycles, or light exposure).

[0400] In one aspect, the present disclosure provides various formulations of anti-ILT4 monoclonal antibodies (e.g., 1E1, 2A6, 3G7, or 2C1) or their antigen-binding fragments, which comprise (i) an anti-ILT4 antibody or its antigen-binding fragment (e.g., 1E1, 2A6, 3G7, or 2C1); (ii) a buffer (e.g., L-histidine buffer or acetate buffer); (iii) a non-reducing sugar (e.g., sucrose); (iv) a non-ionic surfactant (e.g., PS-80); and (v) an antioxidant (e.g., L-methionine).

[0401] Buffers that can be used in the pharmaceutical formulations disclosed herein include, but are not limited to, succinate (sodium or potassium), L-histidine, phosphate (sodium or potassium), Tris (tris(hydroxymethyl)aminomethane), diethanolamine, citrate (sodium), acetate (sodium), and the like. In some embodiments of the formulation, the buffer is present in the formulation at a concentration of about 1 - 20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM).

[0402] The buffers of the various formulations described herein have a pH in the range of from about 4.5 to about 7.0, from about 4.5 to about 6.8, from about 5.0 to about 6.8, from about 5.0 to about 6.5, 5.0 to about 6.0, from about 5.5 to about 6.0. In obtaining the exemplary formulations, the suitability of L-histidine and acetate buffers in the pH range of 5.0 - 6.8 was explored. When reciting a range of pH values, such as "a pH of from about pH 5.5 to about 6.0", the range is intended to include the recited values therein. For example, the range of from about 5.0 to about 6.0 includes 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0. In cases where the anti-ILT4 formulations described herein are lyophilized formulations, the pH refers to the pH after reconstitution of the lyophilized formulation. The pH is typically measured at 25 °C using a standard glass electrode pH meter. As used herein, a solution containing "histidine buffer at pH X" refers to a solution at pH X and containing a histidine buffer, i.e., the pH is expected to refer to the pH of the solution.

[0403] In some embodiments, the anti-ILT4 formulation comprises a non-reducing sugar. As used herein, a "non-reducing sugar" is a sugar that cannot act as a reducing agent because it does not contain or cannot be converted to contain a free aldehyde group or a free ketone group. Examples of non-reducing sugars include, but are not limited to, disaccharides such as sucrose and trehalose. In one embodiment of the invention, the non-reducing sugar is present in an amount of from about 1% (w / v) to about 10% (w / v) (about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w / v)). In another embodiment, the non-reducing sugar is present in an amount of from about 6% (w / v) to about 8% (w / v) (about 6, 7, or 8% (w / v)). In a further embodiment, the non-reducing sugar is present in an amount of about 6% (w / v). In a further embodiment, the non-reducing sugar is present in an amount of about 7% (w / v). In a further embodiment, the non-reducing sugar is present in an amount of about 8% (w / v). In one embodiment, the non-reducing sugar is sucrose, trehalose, or raffinose. In a further embodiment, sucrose is present in an amount of from about 6% (w / v) to about 8% (w / v). In one embodiment, sucrose is present in an amount of about 6% (w / v). In one embodiment, sucrose is present in an amount of about 7% (w / v). In one embodiment, sucrose is present in an amount of about 8% (w / v).

[0404] In certain embodiments, the anti-ILT4 agent further comprises a surfactant. As used herein, a surfactant is a surface-active agent that is amphiphilic in nature. Surfactants can be added to the formulations herein to provide stability, reduce and / or prevent aggregation, or prevent and / or inhibit protein damage during processing conditions (such as purification, filtration, lyophilization, transportation, storage, and delivery). In some embodiments, surfactants can be used to provide additional stability to the active ingredient.

[0405] Nonionic surfactants that can be used in the formulations of the present invention include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (polysorbates, sold under the trade name (Uniquema Americas LLC, Wilmington, DE)), including polysorbate-20 (PS-20, polyoxyethylene sorbitan monolaurate), polysorbate-40 (PS-40, polyoxyethylene sorbitan monopalmitate), polysorbate-60 (PS-60, polyoxyethylene sorbitan monostearate), and polysorbate-80 (PS-80, polyoxyethylene sorbitan monooleate); polyoxyethylene alkyl ethers such as 58 (Uniquema Americas LLC, Wilmington, DE) and 35; poloxamers (such as poloxamer 188); X-100 (Union Carbide Corp., Houston, TX) and X-114; NP40; Span 20, Span40, Span 60, Span 65, Span 80, and Span 85; copolymers of ethylene glycol and propylene glycol (e.g., series of nonionic surfactants, such as F68, 10R5, F108, F127, F38, L44, L62 (BASF Corp., Ludwigshafen, Germany); and sodium dodecyl sulfate (SDS). In one embodiment, the nonionic surfactant is PS-80 or PS-20. In one embodiment, the nonionic surfactant is PS-20. In another embodiment, the nonionic surfactant is PS-80.

[0406] The amount of non-ionic surfactant to be included in the formulation is an amount sufficient to perform the desired function, i.e., the minimum amount required to stabilize the active pharmaceutical ingredient in the formulation (i.e., the anti-ILT4 antibody or its antigen-binding fragment (e.g., 1E1, 2A6, 3G7, or 2C1)). All percentages of non-ionic surfactant are listed as %(w / v). Generally, the surfactant is present at a concentration of from about 0.008% to about 0.1% (w / v). In some embodiments of this aspect of the invention, the surfactant is present in the formulation in an amount of: from about 0.01% to about 0.1%, from about 0.01% to about 0.09%, from about 0.01% to about 0.08%, from about 0.01% to about 0.07%, from about 0.01% to about 0.06%, from about 0.01% to about 0.05%, from about 0.01% to about 0.04%, from about 0.01% to about 0.03%, from about 0.01% to about 0.02%, from about 0.015% to about 0.04%, from about 0.015% to about 0.03%, from about 0.015% to about 0.02%, from about 0.02% to about 0.04%, from about 0.02% to about 0.035%, or from about 0.02% to about 0.03%. In alternative embodiments, the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, or about 0.04%.

[0407] In a specific embodiment, the formulation described herein contains from about 0.01% to about 0.04% (w / v) PS-80. In one embodiment, the formulation described herein contains PS-80 in an amount of about 0.01%. In one embodiment, the amount of PS-80 is about 0.015%. In another embodiment, the amount of PS-80 is about 0.02%. In a further embodiment, the amount of PS-80 is about 0.025%. In another embodiment, the amount of PS-80 is about 0.03%. In a further embodiment, the amount of PS-80 is about 0.035%. In another embodiment, the amount of PS-80 is about 0.04%. In a further embodiment, the amount of PS-80 is about 0.045%.

[0408] The formulations described herein also contain methionine or a pharmaceutically acceptable salt thereof as an antioxidant. In one embodiment, the methionine is L-methionine. In another embodiment, the methionine is a pharmaceutically acceptable salt of L-methionine, for example, methionine HCl. In one embodiment of the present invention, methionine is present in the formulation at a concentration of about 1-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 mM). In another embodiment, there is about 5 mM to about 10 mM (5, 6, 7, 8, 9, and 10 mM) of methionine. In another embodiment, methionine is present at about 10 mM.

[0409] Thus, in one aspect, the present invention provides a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) about 10 mg / mL to about 200 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 5 mM to about 20 mM of a buffer; (iii) about 6% to about 8% weight / volume (w / v) of a non-reducing sugar; (iv) about 0.01% to about 0.10% (w / v) of a non-ionic surfactant; and (v) about 1 mM to about 20 mM of an antioxidant.

[0410] In certain embodiments, the buffer is selected from L-histidine buffer, acetate buffer, and citrate buffer. In one embodiment, the buffer is L-histidine buffer. In another embodiment, the buffer is acetate buffer. In yet another embodiment, the buffer is citrate buffer.

[0411] In some embodiments, the non-reducing sugar is sucrose.

[0412] In certain embodiments, the non-ionic surfactant is PS-80 or PS-20. In one embodiment, the non-ionic surfactant is PS-80. In another embodiment, the non-ionic surfactant is PS-20.

[0413] In some embodiments, the antioxidant is L-methionine.

[0414] Thus, in another aspect, the present invention provides a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) about 10 mg / mL to about 200 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 5 mM to about 20 mM of L-histidine buffer; (iii) about 6% to about 8% (w / v) sucrose; (iv) about 0.01% to about 0.10% (w / v) PS-80; and (v) about 1 mM to about 20 mM of L-methionine.

[0415] In some embodiments, the formulation comprises from about 8 mM to about 12 mM L - histidine buffer.

[0416] In certain embodiments, the formulation comprises from about 5 mM to about 10 mM L - methionine.

[0417] In other embodiments, the formulation comprises from about 0.01% to about 0.05% (w / v) PS - 80.

[0418] In still other embodiments, the formulation comprises from about 10 mg / mL to about 150 mg / mL of an anti - ILT4 antibody or an antigen - binding fragment thereof. In yet other embodiments, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 10 mg / mL, about 12.5 mg / mL, about 15 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL, or about 150 mg / mL. In one embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 10 mg / mL. In another embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 12.5 mg / mL. In still another embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 15 mg / mL. In yet another embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 25 mg / mL. In one embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 50 mg / mL. In another embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 75 mg / mL. In still another embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 100 mg / mL. In yet another embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 125 mg / mL. In still yet another embodiment, the concentration of the anti - ILT4 antibody or an antigen - binding fragment thereof is about 150 mg / mL.

[0419] Thus, in one specific embodiment, the formulation comprises about 25 mg / mL of an anti - ILT4 antibody or an antigen - binding fragment thereof, about 10 mM L - histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L - methionine.

[0420] In another specific embodiment, the formulation comprises about 50 mg / mL of an anti - ILT4 antibody or an antigen - binding fragment thereof, about 10 mM L - histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L - methionine.

[0421] In yet another specific embodiment, the formulation comprises about 75 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0422] In another specific embodiment, the formulation comprises about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0423] In yet another specific embodiment, the formulation comprises about 125 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0424] In certain embodiments of the various formulations provided herein, the formulation has a pH of about 5.0 to about 6.8. In some embodiments, the formulation has a pH of about 5.5 to about 6.0. In other embodiments, the formulation has a pH of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0. In one embodiment, the formulation has a pH of about 5.5. In another embodiment, the formulation has a pH of about 5.6. In yet another embodiment, the formulation has a pH of about 5.7. In another embodiment, the formulation has a pH of about 5.8. In another embodiment, the formulation has a pH of about 5.9. In yet another embodiment, the formulation has a pH of about 6.0.

[0425] Thus, in one specific embodiment, a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof comprises: (i) about 50 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer, pH about 5.5; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) polysorbate 80; and (v) about 10 mM L-methionine.

[0426] In certain embodiments of the various formulations disclosed herein, the anti-ILT4 antibody or antigen-binding fragment thereof is the anti-ILT4 antibody or antigen-binding fragment thereof described herein (e.g., 1E1, 2A6, 3G7, or 2C1). In some embodiments of the formulation, the anti-ILT4 antibody or antigen-binding fragment thereof is 1E1 or a variant thereof. In some embodiments of the formulation, the anti-ILT4 antibody or antigen-binding fragment thereof is 2A6 or a variant thereof. In some embodiments of the formulation, the anti-ILT4 antibody or antigen-binding fragment thereof is 3G7 or a variant thereof. In some embodiments of the formulation, the anti-ILT4 antibody or antigen-binding fragment thereof is 2C1 or a variant thereof.

[0427] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, where X1 is N, Q, E, or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0428] In some embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0429] In other embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:58.

[0430] In yet other embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:7.

[0431] In yet additional other embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:80 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:7.

[0432] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO:57 and a light chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO:58.

[0433] In some embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:7.

[0434] In other embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:80 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:7.

[0435] Accordingly, in some embodiments, the present disclosure provides a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, where X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0436] In certain embodiments, the present disclosure provides a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0437] In other embodiments, provided herein are pharmaceutical formulations of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:58.

[0438] In still other embodiments, provided herein are pharmaceutical formulations of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:2 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0439] In yet other embodiments, provided herein are pharmaceutical formulations of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:80 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0440] In some embodiments, provided herein are pharmaceutical formulations of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:58.

[0441] In some embodiments, provided herein are pharmaceutical formulations of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:7.

[0442] In some embodiments, provided herein are pharmaceutical formulations of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) from about 25 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer; (iii) about 7% (w / v) sucrose; (iv) about 0.025% (w / v) PS-80; and (v) about 10 mM L-methionine; wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:80 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:7.

[0443] In one embodiment, the formulation is a liquid formulation. In another embodiment, the liquid formulation is stored at about 3°C - 5°C. In yet another embodiment, the liquid formulation is frozen to at least -70°C or lower. In a further embodiment, the liquid formulation is a reconstitution solution from a lyophilized formulation.

[0444] In certain embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, (i) the % monomer of the anti-ILT4 antibody or an antigen-binding fragment thereof is at least about 99%, as determined by ultra-high performance size exclusion chromatography; (ii) as determined by OD 350-500Measured, the turbidity of the formulation is at most about 0.135; (iii) the % main peak of the anti-ILT4 antibody or its antigen-binding fragment is at least about 63%, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23%, and / or the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14% as determined by high performance ion exchange chromatography; (iv) the subvisible particle count of particles having a size of at least 2 μm is at most about 3500 as determined by microflow imaging; and / or (v) the % oxidation of one or more amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4% as determined by reduced peptide mapping analysis.

[0445] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the % monomer of the anti-ILT4 antibody or its antigen-binding fragment is at least about 99% as determined by ultra performance size exclusion chromatography.

[0446] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by OD 350-500 Measured, the turbidity of the formulation is at most about 0.135.

[0447] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the % main peak of the anti-ILT4 antibody or its antigen-binding fragment is at least about 63% as determined by high performance ion exchange chromatography. In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23% as determined by high performance ion exchange chromatography. In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14% as determined by high performance ion exchange chromatography. In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the % main peak of the anti-ILT4 antibody or its antigen-binding fragment is at least about 63%, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23%, and the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14% as determined by high performance ion exchange chromatography.

[0448] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, the subvisible particle count of particles having a size of at least 2 μm is at most about 3500 as determined by microflow imaging.

[0449] In some embodiments, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of one or more amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of one amino acid residue selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of two amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of three amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of four amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%. In one embodiment, after the formulation is stored at about 3°C to about 5°C for 6 months, as determined by reduced peptide mapping analysis, the % oxidation of all five amino acid residues from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%.

[0450] The formulations described herein can be prepared as liquid formulations. A liquid antibody formulation can be prepared by obtaining a drug substance (e.g., an anti-ILT4 monoclonal antibody) that is in liquid form (e.g., an anti-ILT4 monoclonal antibody in an aqueous pharmaceutical formulation) and buffer-exchanging it into the desired buffer as the final step of the purification process. There is no lyophilization step in this embodiment. The drug substance in the final buffer is concentrated to the desired concentration. Excipients such as sucrose and PS80 are added to the drug substance, and it is diluted to the final protein concentration using an appropriate buffer. The final formulated drug substance is filtered through a 0.22 μm filter and filled into final containers (e.g., glass vials).

[0451] The formulations described herein can also be reconstituted from lyophilized formulations. Lyophilized formulations of therapeutic proteins offer several advantages. Lyophilized formulations generally provide better chemical stability than solution formulations and, as a result, an increased half-life. Lyophilized formulations can also be reconstituted at different concentrations depending on clinical factors such as the route of administration or the dose. For example, a lyophilized formulation can be reconstituted at a high concentration (i.e., in a small volume) for subcutaneous administration (if necessary), or at a lower concentration if administered intravenously. A high concentration may also be necessary if a high dose is required for a particular subject, especially if administered subcutaneously (where the injection volume must be minimized). U.S. Patent No. 6,267,958 discloses one such lyophilized antibody formulation, which is hereby incorporated by reference in its entirety. U.S. Patent No. 7,247,707 discloses another lyophilized formulation of a therapeutic protein, which is hereby incorporated by reference in its entirety.

[0452] Typically, lyophilized formulations are prepared anticipating reconstitution at a high concentration of the drug product (DP, in the exemplary embodiment an anti-ILT4 antibody or an antigen-binding fragment thereof), i.e., reconstitution in a low volume of water. Subsequent dilution with water or an isotonic buffer can be readily used to dilute the DP to a lower concentration. Typically, excipients are included in the lyophilized formulations of the invention at levels that will result in a formulation that is approximately isotonic when reconstituted at a high DP concentration, e.g., for subcutaneous administration. Reconstitution in a larger volume of water to give a lower DP concentration necessarily reduces the osmotic pressure of the reconstituted solution, but such a reduction may be of little significance in non-subcutaneous, e.g., intravenous, administration. If isotonicity is desired at a lower DP concentration, the lyophilized powder can be reconstituted in a standard low volume of water and then further diluted with an isotonic diluent such as 0.9% sodium chloride.

[0453] The lyophilized preparation of the present invention is formed by lyophilizing (freeze-drying) a pre-lyophilized solution. Freeze-drying is achieved by freezing the preparation and then sublimating water at a temperature suitable for primary drying. Under such conditions, the product temperature is below the eutectic point or collapse temperature of the preparation. Generally, the shelf temperature for primary drying ranges from about -30 to 25 °C at a suitable stress in the general range of about 50 to 250 mTorr (provided that the product remains frozen during primary drying). The size and type of the preparation, the container (such as a glass vial) containing the sample, and the volume of the liquid will determine the time required for drying, and the drying time can range from several hours to several days (such as 40 - 60 hours). The secondary drying stage can be carried out at about 0 - 40 °C, mainly depending on the type and size of the container and the type of protein employed. The secondary drying time is determined by the level of residual moisture required in the product and generally requires at least about 5 hours. Generally, the water content of the lyophilized preparation is less than about 5%, and preferably less than about 3%. The stress can be the same as that employed during the primary drying step. The freeze-drying conditions can vary depending on the preparation and vial size.

[0454] In some cases, it may be desirable to lyophilize the protein preparation in the container in which protein reconstitution is to be carried out in order to avoid the transfer step. In such cases, the container can be, for example, a 3, 5, 10, 20, 50 or 100 cc vial.

[0455] The lyophilized formulation is reconstituted prior to administration. The protein can be reconstituted at a concentration of about 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg / mL, or at a higher concentration such as 150 mg / mL, 200 mg / mL, 250 mg / mL or 300 mg / mL up to about 500 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 10 - 300 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 20 - 250 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 150 - 250 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 180 - 220 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 50 - 150 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 150 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 125 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 100 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 75 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 50 mg / mL. In one embodiment, the concentration of the reconstituted protein is about 25 mg / mL. High protein concentrations are particularly useful when subcutaneous delivery of the reconstituted formulation is anticipated. However, for other routes of administration, such as intravenous administration, lower protein concentrations (e.g., about 5 - 50 mg / mL) may be required.

[0456] Reconstitution is generally carried out at a temperature of about 25°C to ensure complete hydration, although other temperatures can be employed if needed. The time required for reconstitution will depend on, for example, the type of diluent, excipients and amount of protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution or dextrose solutions.

[0457] Methods of Use

[0458] In another aspect, provided herein are methods of treating cancer in a subject, which comprise administering to the subject a therapeutically effective amount of any of the formulations described herein. In some specific embodiments of the method, the formulation is administered to the subject via intravenous administration. In other embodiments, the formulation is administered to the subject by subcutaneous administration. In one embodiment, the invention includes a method of treating cancer in a human patient, which comprises administering to the patient any of the formulations described herein.

[0459] In any method of the present invention, the cancer can be selected from the following: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, kidney cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary cancer, prostate cancer (e.g., hormone-refractory prostatic adenocarcinoma), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma, and other neoplastic malignancies.

[0460] In some embodiments, the lung cancer is non-small cell lung cancer.

[0461] In alternative embodiments, the lung cancer is small cell lung cancer.

[0462] In some embodiments, the lymphoma is Hodgkin lymphoma.

[0463] In other embodiments, the lymphoma is non-Hodgkin lymphoma. In certain embodiments, the lymphoma is mediastinal large B-cell lymphoma.

[0464] In some embodiments, the breast cancer is triple-negative breast cancer.

[0465] In further embodiments, the breast cancer is ER+ / HER2- breast cancer.

[0466] In some embodiments, the bladder cancer is urothelial carcinoma.

[0467] In some embodiments, the head and neck cancer is nasopharyngeal cancer. In some embodiments, the cancer is thyroid cancer. In other embodiments, the cancer is salivary cancer. In other embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0468] In some embodiments, the cancer is a solid tumor with high levels of microsatellite instability (MSI-H).

[0469] In some embodiments, the cancer is a solid tumor with a high mutational burden.

[0470] In some embodiments, the cancer is metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

[0471] In some embodiments, the cancer is selected from colorectal cancer, esophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, and small cell lung cancer.

[0472] In other embodiments of the above-described treatment methods, the cancer is a hematological malignancy. In certain embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL).

[0473] The methods and treatments described herein encompass malignancies in which improved disease-free survival and overall survival have been demonstrated to be associated with the presence of tumor-infiltrating lymphocytes in biopsy or surgical material, such as melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, breast cancer, pancreatic cancer, and ovarian cancer. Such cancer subtypes are known to be susceptible to immune control by T lymphocytes. Additionally, refractory or recurrent malignancies are included, the growth of which can be inhibited using the antibodies described herein.

[0474] Additional cancers that may benefit from treatment with the formulations described herein include those associated with persistent viral infections, such as human immunodeficiency virus, hepatitis A virus, hepatitis B virus, and hepatitis C virus, Epstein-Barr virus, and human papillomavirus, which is known to be causally related to, for example, Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer.

[0475] The formulations can also be used to prevent or treat infections and infectious diseases. Accordingly, the present invention provides a method for treating chronic infections in a mammalian subject, which comprises administering to the subject an effective amount of the formulation of the present invention. In some specific embodiments of the method, the formulation is administered to the subject via intravenous administration. In other embodiments, the formulation is administered to the subject by subcutaneous administration.

[0476] These agents can be used alone or in combination with a vaccine to stimulate an immune response against pathogens, toxins, and autoantigens. Antibodies or antigen-binding fragments thereof can be used to stimulate an immune response against viruses that are infectious to humans, including but not limited to: human immunodeficiency virus, hepatitis A virus, hepatitis B virus, and hepatitis C virus, Epstein-Barr virus, human cytomegalovirus, human papillomavirus, and herpes virus. Antagonistic anti-PD-1 antibodies or antibody fragments can be used to stimulate an immune response against infections by bacterial or fungal parasites and other pathogens. Viral infections by hepatitis B virus, hepatitis C virus, and HIV are among those viral infections considered to be chronic viral infections.

[0477] The formulations of the present invention can be administered to a patient in combination with one or more "additional therapeutic agents". The additional therapeutic agents can be biotherapeutic agents (including but not limited to antibodies against VEGF, EGFR, Her2 / neu, VEGF receptor, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB, and ICOS), immunogenic agents (e.g., attenuated cancer cells, tumor antigens, antigen-presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), and cells transfected with a gene encoding an immunostimulatory cytokine such as but not limited to GM-CSF).

[0478] As noted above, in some embodiments of the methods of the present invention, the method further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-LAG3 antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-GITR antibody or an antigen-binding fragment thereof, an anti-CTL4 antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease virus vector expressing IL-12. In a further embodiment, the additional therapeutic agent is dinaciclib. In yet a further embodiment, the additional therapeutic agent is a STING agonist.

[0479] Suitable routes of administration can include, for example, parenteral delivery, including intramuscular, subcutaneous, as well as intrathecal, direct intraventricular, intravenous, and intraperitoneal. The drug can be administered in various conventional ways, such as by intraperitoneal, parenteral, intraarterial, or intravenous injection. Administration modes where the solution volume must be restricted (e.g., subcutaneous administration) require that the lyophilized formulation be able to be reconstituted at a high concentration.

[0480] The selection of the dosage of an additional therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs in the individual to be treated. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Thus, the dosage and frequency of administration of each additional therapeutic agent (e.g., a biotherapeutic or a chemotherapeutic agent) will depend in part on the particular therapeutic agent, the severity of the cancer to be treated, and patient characteristics. Guidance is available in selecting appropriate dosages of antibodies, cytokines, and small molecules. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th ed.); Medical Economics Company; ISBN: 1563634457; 57th ed. (November 2002). The determination of an appropriate dosage regimen can be made by a clinician, e.g., using parameters or factors known or suspected to affect or predicted to affect treatment in the art, and will depend on, e.g., the patient's clinical history (e.g., prior treatments), the type and stage of the cancer to be treated, and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0481] A variety of literature references can be used to facilitate the selection of pharmaceutically acceptable carriers or excipients for additional therapeutic agents. See, e.g., Remington’s Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0482] The pharmaceutical antibody preparation can be administered by continuous infusion or by doses at intervals such as once a day, 1 - 7 times a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, etc. The preferred dosage regimen is a dosage regimen that involves the maximum dose or dose frequency that avoids significant undesirable side effects. The total weekly dose is usually at least 0.05 μg / kg, 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.2 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg body weight or more. See, e.g., Yang et al. (2003) New Engl. J. Med. 349:427 - 434; Herold et al. (2002) New Engl. J. Med. 346:1692 - 1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451 - 456; Portielji et al. (20003) Cancer Immunol. Immunother. 52:133 - 144. The required dose of small molecule therapeutic agents such as peptidomimetics, natural products or organic chemicals is roughly the same as that of antibodies or polypeptides on a molar / kg basis.

[0483] Embodiments of the invention also include one or more of the biological agents described herein, which are (i) for use in, (ii) used as an agent or composition for, or (iii) for the preparation of an agent for: (a) treatment (e.g., of the human body); (b) medicine; (c) inducing or increasing an anti - tumor immune response; (d) reducing the number of one or more tumor markers in a patient; (e) stopping or delaying the growth of a tumor or blood cancer; (f) stopping or delaying the progression of a PD - 1 - related disease or an ILT4 - related disease; (g) stabilizing a PD - 1 - related disease or an ILT4 - related disease; (h) inhibiting the growth or survival of tumor cells; (i) eliminating or reducing the size of one or more cancerous lesions or tumors; (j) reducing the progression, onset or severity of a PD - 1 - related disease or an ILT4 - related disease; (k) reducing the severity or duration of the clinical symptoms of a PD - 1 - related disease or an ILT4 - related disease; (l) prolonging the survival of a patient relative to the expected survival in untreated similar patients; (m) inducing a complete or partial remission of a cancerous condition or other PD - 1 - related disease or ILT4 - related disease; (n) treating cancer; or (o) treating a chronic infection.

[0484] General Methods

[0485] Standard methods in molecular biology are described in: Sambrook, Fritsch and Maniatis (1982 & 1989 2nd ed., 2001 3rd ed.) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA. Standard methods also appear in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0486] Methods for protein purification are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins are described (see, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, ibid). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0487] Monoclonal antibodies, polyclonal antibodies, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter et al. (2000) J. Immunol. 165:6205; He et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Patent No. 6,329,511).

[0488] Humanized alternatives use human antibody libraries displayed on phage or in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

[0489] Purification of the antigen is not necessary for antibody production. Animals can be immunized with cells bearing the antigen of interest. Spleen cells can then be isolated from the immunized animals and the spleen cells can be fused with a myeloma cell line to produce hybridomas (see, for example, Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., ibid.; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

[0490] Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies can be used for therapeutic, diagnostic, kit or other purposes and include antibodies conjugated to, for example, dyes, radioisotopes, enzymes or metals such as colloidal gold (see, for example, Le Doussal et al. (1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

[0491] Methods for flow cytometry, including fluorescence-activated cell sorting (FACS), are available (see, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, which are used as, for example, diagnostic reagents, are available (Molecular Probesy (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0492] Standard methods for the histology of the immune system are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0493] Software packages and databases for determining, for example, antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available (see, e.g., GenBank, Vector Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16:741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0494] Analytical Methods

[0495] Analytical methods suitable for assessing product stability include size exclusion chromatography (SEC), dynamic light scattering (DLS), differential scanning calorimetry (DSC), isoaspartic acid quantification, potency, UV at 340 nm, UV spectroscopy, and FTIR. SEC (J. Pharm. Sci., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm. Bio. Anal., 14:1133-1140 (1986)) measures the percentage monomer in the product and gives information on the amount of soluble aggregates. DSC (Pharm. Res., 15:200 (1998); Pharm. Res., 9:109 (1982)) gives information on the protein denaturation temperature and the glass transition temperature. DLS (American Lab., November (1991)) measures the average diffusion coefficient and gives information on the amount of soluble and insoluble aggregates. UV at 340 nm measures the scattered light intensity at 340 nm and gives information on the amount of soluble and insoluble aggregates. UV spectroscopy measures the absorbance at 278 nm and gives information on the protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45:231 (1998); Pharm. Res., 12:1250 (1995); J. Pharm. Sci., 85:1290 (1996); J. Pharm. Sci., 87:1069 (1998)) measures the IR spectrum in the amide I region and gives information on the protein secondary structure.

[0496] The isopeptide content in the sample was measured using the Isoquant Isoaspartate Detection System (Promega). This kit uses the enzyme protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) to specifically detect the presence of isoaspartate residues in the target protein. PIMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the isoaspartate at the α-carboxyl position, generating S-adenosyl-L-homocysteine (SAH) in the process. This is a relatively small molecule and can generally be separated and quantified by reverse-phase HPLC using the SAH HPLC standard provided in the kit.

[0497] The potency or bioidentity of an antibody can be measured by its ability to bind to its antigen. Specific binding of an antibody to its antigen can be quantified by any method known to those skilled in the art, such methods as immunoassays, such as ELISA (enzyme-linked immunosorbent assay).

[0498] All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the methods and materials that may be used in connection with the present invention.

[0499] The different embodiments of the present invention herein have been described with reference to the accompanying drawings, but it should be understood that the present invention is not limited to those exact embodiments, and various changes and modifications can be made therein by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

[0500] Embodiment 1 provides a formulation of an anti-human immunoglobulin-like transcript 4 (anti-ILT4) antibody or an antigen-binding fragment thereof, comprising: (i) from about 10 mg / mL to about 200 mg / mL of the anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) from about 5 mM to about 20 mM of a buffer; (iii) from about 6% to about 8% weight / volume (w / v) of a non-reducing sugar; (iv) from about 0.01% to about 0.10% (w / v) of a non-ionic surfactant; and (v) from about 1 mM to about 20 mM of an antioxidant, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, wherein X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21), wherein the buffer is an L-histidine buffer, an acetate buffer or a citrate buffer, wherein the non-reducing sugar is a disaccharide, wherein the non-ionic surfactant is polysorbate 20 or polysorbate 80, and wherein the antioxidant is methionine or a pharmaceutically acceptable salt thereof.

[0501] Embodiment 2 provides the formulation of Embodiment 1, wherein the buffer is an L-histidine buffer.

[0502] Embodiment 3 provides the formulation of Embodiment 1 or 2, wherein the non-reducing sugar is sucrose.

[0503] Embodiment 4 provides the formulation of any one of Embodiments 1-3, wherein the non-ionic surfactant is polysorbate 80.

[0504] Embodiment 5 provides the formulation of any one of Embodiments 1-4, wherein the antioxidant is L-methionine.

[0505] Embodiment 6 provides a formulation of an anti-human immunoglobulin-like transcript 4 (“anti-ILT4”) antibody or an antigen-binding fragment thereof, comprising: (i) from about 10 mg / mL to about 200 mg / mL of the anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) from about 5 mM to about 20 mM L-histidine buffer; (iii) from about 6% to about 8% weight / volume (w / v) sucrose; (iv) from about 0.01% to about 0.10% (w / v) polysorbate 80; and (v) from about 1 mM to about 20 mM L-methionine, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, wherein X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0506] Embodiment 7 provides the formulation of Embodiment 6, which comprises from about 8 mM to about 12 mM L-histidine buffer.

[0507] Embodiment 8 provides the formulation of Embodiment 6 or 7, which comprises from about 5 mM to about 10 mM L-methionine.

[0508] Embodiment 9 provides the formulation of any one of Embodiments 6-8, which comprises from about 0.01% to about 0.05% (w / v) polysorbate 80.

[0509] Embodiment 10 provides the formulation of any one of Embodiments 1-9, which comprises from about 10 mg / mL to about 150 mg / mL of the anti-ILT4 antibody or an antigen-binding fragment thereof.

[0510] Embodiment 11 provides the formulation of Embodiment 10, wherein the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 10 mg / mL, about 12.5 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL or about 150 mg / mL.

[0511] Embodiment 12 provides a formulation of any one of Embodiments 1-11, which comprises an anti-ILT4 antibody or an antigen-binding fragment thereof at about 25 mg / mL, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0512] Embodiment 13 provides a formulation of any one of Embodiments 1-11, which comprises an anti-ILT4 antibody or an antigen-binding fragment thereof at about 50 mg / mL, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0513] Embodiment 14 provides a formulation of any one of Embodiments 1-11, which comprises an anti-ILT4 antibody or an antigen-binding fragment thereof at about 75 mg / mL, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0514] Embodiment 15 provides a formulation of any one of Embodiments 1-11, which comprises an anti-ILT4 antibody or an antigen-binding fragment thereof at about 100 mg / mL, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0515] Embodiment 16 provides a formulation of any one of Embodiments 1-11, which comprises an anti-ILT4 antibody or an antigen-binding fragment thereof at about 125 mg / mL, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

[0516] Embodiment 17 provides a formulation of any one of Embodiments 1-16, wherein the formulation has a pH of about 5.0 to about 6.8.

[0517] Embodiment 18 provides a formulation of any one of Embodiments 1-16, wherein the formulation has a pH of about 5.5 to about 6.0.

[0518] Embodiment 19 provides a formulation of any one of Embodiments 1-16, wherein the formulation has a pH of about 5.5.

[0519] Embodiment 20 provides a formulation of an anti-human immunoglobulin-like transcript 4 (“anti-ILT4”) antibody or an antigen-binding fragment thereof, comprising: (i) from about 50 mg / mL to about 100 mg / mL of the anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mM L-histidine buffer, pH about 5.5; (iii) about 7% weight / volume (w / v) sucrose; (iv) about 0.025% (w / v) polysorbate 80; and (v) about 10 mM L-methionine, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, where X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0520] Embodiment 21 provides the formulation of any one of Embodiments 1-20, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, where X1 is N, Q, E or D and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0521] Embodiment 22 provides a formulation of any one of Embodiments 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0522] Embodiment 23 provides a formulation of any one of Embodiments 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:58.

[0523] Embodiment 24 provides a formulation of any one of Embodiments 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO:2 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0524] Embodiment 25 provides a formulation of any embodiment of Claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO:80 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0525] Embodiment 26 provides a formulation of any one of Embodiments 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:58.

[0526] Embodiment 27 provides a formulation of any one of Embodiments 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:7.

[0527] Embodiment 28 provides a formulation of any one of Embodiments 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:80 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:7.

[0528] Embodiment 29 provides a formulation of any one of Embodiments 1-28, wherein the formulation is a liquid formulation stored at about 3°C to about 5°C, frozen to -20°C or lower, frozen to -70°C or lower, or is a reconstitution solution from a lyophilized formulation.

[0529] Embodiment 30 provides a formulation of any one of Embodiments 1-27, wherein after storage at about 3°C to about 5°C for 6 months: (i) the % monomer of the anti-ILT4 antibody or antigen-binding fragment thereof is at least about 99% as determined by ultra-high performance size exclusion chromatography; (ii) the turbidity of the formulation is at most about 0.135 as measured by OD350-500; (iii) the % main peak of the anti-ILT4 antibody or antigen-binding fragment thereof is at least about 63%, the % acidic variant of the anti-ILT4 antibody or antigen-binding fragment thereof is at most about 23%, and / or the basic variant of the anti-ILT4 antibody or antigen-binding fragment thereof is at most about 14% as determined by high performance ion exchange chromatography; (iv) the subvisible particle count of particles having a size of at least 2 μm is at most about 3500 as determined by microflow imaging; and / or (v) the % oxidation of one or more amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody shown in SEQ ID NO:2 or 80 is less than about 4% as determined by reduced peptide mapping analysis.

[0530] Embodiment 31 provides a formulation of any one of Embodiments 1-30, wherein the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0531] Embodiment 32 provides a method of treating cancer in a human patient in need thereof, the method comprising administering an effective amount of a formulation of any one of Embodiments 1-31.

[0532] Embodiment 33 provides the method of Embodiment 32, wherein the cancer is selected from colorectal cancer, esophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, renal cell cancer, and small cell lung cancer.

[0533] Embodiment 34 provides the use of a formulation of any one of Embodiments 1-31 for the preparation of a medicament for treating cancer.

[0534] Embodiment 35 provides the use of a formulation of any one of Embodiments 1-31 for treating cancer in a human patient.

[0535] Embodiment 36 provides the use of Embodiment 34, wherein the cancer is selected from colorectal cancer, esophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, and small cell lung cancer.

[0536] Example 1

[0537] Anti-ILT4 formulation analysis

[0538] Table 3 summarizes the formulation developability plan. The fully human anti-ILT4 monoclonal antibody 1E1 ("Antibody 1") with a human lambda light chain constant domain and a human IgG4-S228P heavy chain constant domain was tested. All formulations were prepared in 96-well Axygen polypropylene deep well plates (product #P-DW-11-C) using a JANUS automated liquid handling system equipped with a Parisian pipetting arm. Each well had a 500 μL aliquot with a final concentration of 1 mg / mL of Antibody 1.

[0539] Table 3. Anti-ILT4 formulation developability plan

[0540]

[0541] A 200 μL aliquot was removed from the plate for preliminary sample analysis (20 μL for differential scanning fluorimetry (DSF) thermal transition analysis and 180 μL for subvisible particle analysis using an easyCyte flow cytometer). The remaining 300 μL of sample in the 96-well Axygen plate was covered with a Nunc TM microwell plate sealing gasket (product #276000) and tightly sealed. The plate was double-packed in SCSDri- 2000 Series Moisture Barrier Bags (product #700810) to prevent evaporation. The sealed plate was subjected to stress at 50 °C for 10 days in a thermal chamber. Protein concentration was monitored before and after thermal stress to account for any increase in protein concentration due to evaporation. The increase in protein concentration across the plate was less than 5%. All standard precautions were observed during the handling of the protein solution (e.g., equilibrating the solution to room temperature, minimizing agitation and exposure to room light).

[0542] After thermal stress, equilibrate the plates at room temperature for 30 minutes. Then carefully open the plates in a laminar flow hood. Using a 200 μL multi-channel pipette, mix the stressed samples up to 5 times. Then take out 200 μL of the sample and dispense it into a 96-well Costar clear plate (product #3635), and measure the absorbance at wavelengths 280, 320, 350, and 500 nm. After UV analysis, carefully transfer 180 μL of the 200 μL aliquot to a 96-well clear V-bottom microplate (product #3894) for subvisible particle analysis. The remaining 20 μL is used for quantitative protein purity analysis using the Caliper's LabChip GXII system with reduced high-throughput CE-SDS. On the same day, transfer the remaining 100 μL of the stressed samples in the Axygen deep well plate together with the initial (unstressed) samples to an ABgene 96-well PCR plate (product #AB-2800) for UP-SEC and cIEF analysis.

[0543] Thermal stability by DSF

[0544] The thermal stability of antibody 1 formulations was evaluated by DSF under different buffer, pH, and ionic strength conditions. Tm1 (the first unfolding midpoint) for most of the test conditions fell within the range of 60 - 66 °C (data not shown). Overall, the results from this assay confirmed no defects in thermal stability.

[0545] Soluble aggregates by UP-SEC

[0546] UP-SEC analysis of the stressed samples of antibody 1 across different formulation conditions showed similar colloidal stability profiles (data not shown). Antibody 1 formulations showed resistance to fragmentation and > 3% soluble aggregates in formulations with pH < 5.5 (data not shown).

[0547] By Subvisible particles by easyCyte flow cytometer

[0548] By Subvisible particle (from 0.22 μm to 10 μm) testing for antibody 1 was performed by easyCyte flow cytometer. In most formulation compositions, antibody 1 did not show significant formation of subvisible aggregates after stress (data not shown).

[0549] Fragmentation by CE-SDS

[0550] To obtain a comprehensive understanding of the proteolytic cleavage of the heavy or light chain of the anti-ILT4 monoclonal antibody, stressed samples were also analyzed by reducing capillary electrophoresis sodium dodecyl sulfate (R-CE-SDS). Fragmentation of antibody 1 was minimal in 10 mM histidine at a pH range of 5.6 - 6.8 or 10 mM acetate at a pH range of 5.0 - 5.6.

[0551] Chemical stability by cIEF

[0552] The chemical stability of antibody 1 formulations was evaluated by cIEF under different buffer, pH, and ionic strength conditions. The cIEF profiles showed a decrease in the relative main peak area of the acidic variants and a significant increase in the relative peak area after 10 days at 50 °C. The addition of salt did not result in any significant improvement in the stability of the protein across the studied compositions. The formulation that conferred the maximum stability to antibody 1 was identified as 10 mM histidine in the pH range of 5.5 - 6.0 or 10 mM acetate at pH 5.6.

[0553] Stressed samples were also analyzed by UV / Vis spectrophotometry for turbidity (A 350-500 ) to evaluate colloidal instability. The results did not vary according to any of the evaluated parameters.

[0554] The above results showed that antibody 1 formulations had favorable colloidal (as measured by turbidity and subvisible particle analysis) and thermal stability profiles, while the unformulated antibody exhibited chemical instability and a tendency to aggregate. Therefore, 10 mM histidine or 10 mM acetate buffer in the pH range of approximately 5.5 - 6.0 was selected for further formulation development.

[0555] Example 2

[0556] Early feasibility evaluation of anti-ILT4 formulations

[0557] Five anti-ILT4 monoclonal antibody formulations at a concentration of 50 mg / mL of antibody 1 were prepared in the following matrices: 10 mM acetate pH 5.4, 10 mM acetate pH 5.8, 10 mM L-histidine pH 5.4, 10 mM L-histidine pH 5.8, and 10 mM L-histidine pH 5.8 with 9% (w / v) sucrose. The concentrateability of antibody 1 formulations was further evaluated by concentrating antibody 1 to 100 mg / mL in each of the five formulation matrices. The formulations were tested for visual appearance, turbidity (OD 350-500 ), viscosity, concentration, and pH. The self-association properties of antibody 1 in each of the five formulations were determined based on the diffusion interaction parameter (k D ) and the relative solubility value.

[0558] The data are summarized in Table 4. The data indicate that, in both 10 mM acetate and 10 mM histidine buffers, the colloidal stability (OD 350-500 ) of antibody 1 is better at low pH (5.4) than at high pH (5.8), with improved stability in the presence of 9% sucrose as a stabilizer (10 mM histidine buffer, pH 5.8). Overall, the k D value is negative, indicating the inherent property of antibody self-association. The k D value is less negative at low pH (5.4) for both buffers, indicating lower self-association at this pH. The presence of the stabilizer sucrose was found to reduce the crowding effect in 10 mM histidine buffer at pH 5.8. Overall, the relative solubility of antibody 1 in 10 mM acetate buffer (pH 5.4 and pH 5.8) and 10 mM histidine buffer (pH 5.4) is comparable. Solubility is relatively lower at higher pH (5.8) in 10 mM histidine buffer. There is no effect of pH or formulation buffer on the viscosity of five formulations (similar values) at a concentration of 100 mg / mL, confirming the ability of the molecule to concentrate above 50 mg / mL.

[0559]

[0560]

[0561] Example 3

[0562] Forced Degradation Studies of Anti-ILT4 Formulations

[0563] Forced degradation studies were performed on formulations containing antibody 1. The tested antibody 1 formulations contained 50 mg / mL antibody, 10 mM L-histidine buffer at pH 6.0, 7% (w / v) sucrose, and 0.02% (w / v) PS-80. The antibody 1 formulations were exposed to various stress conditions, including heat (50 °C, up to 7 days), high pH (pH 10, 25 °C, up to 7 days), low pH (pH 3.5, 25 °C, up to 7 days), light (25 °C, up to 2X ICH level), or peroxide (0.1% tBHP, 25 °C, up to 24 hours).

[0564] The stressed samples were visually inspected for color and particulates and tested by UV, UP-SEC, NR-CE-SDS, R-CE-SDS, HP-IEX, DLS, and LC-MS (intact and reduced mass as well as reduced peptide mapping analysis).

[0565] Major degradation (>10% change at the stress end point compared to the starting point) included an increase in acidic variants under heat (50 °C), high pH (pH 10.0), and light exposure stress; an increase in deamidation and isomerization under high pH stress; an increase in HMW species, basic variants, and Trp oxidation under light exposure (2X) stress; and an increase in Met oxidation under light exposure and peroxide (0.1% tBHP) stress. There was no major degradation pathway under low pH (pH 3.5) stress.

[0566] Minor degradation (≤10% change at the stress end point compared to the starting point) included an increase in HMW species under heat, high pH, and low pH stress; an increase in LMW species under heat, high pH, low pH, and light exposure stress; an increase in turbidity under light exposure stress; and an increase in basic variants under peroxide stress.

[0567] The results of the light exposure stress study are summarized in Table 5 below. Detailed results of the other stress studies are not shown.

[0568] Table 5. Analytical summary of anti-ILT4 formulations subjected to light stress

[0569]

[0570] After light exposure at 2X ICH level, the effect on potency was examined by ELISA. The GeoMean values were 106 and 28 for the dark control and the 2X exposed stressed samples, respectively, indicating that light exposure decreased the potency of Antibody 1.

[0571] Example 4

[0572] Early stability study of anti-ILT4 formulations

[0573] Early feasibility data confirmed the preference for a lower pH (5.4 ± 0.2) (Example 2) based on better colloidal, biophysical, and chemical stability. Based on forced degradation studies (Example 3), photooxidation is known to be one of the major drawbacks of the anti-ILT4 monoclonal antibody. Four different formulations of Antibody 1 were staged for early thermal stability at a target pH of 5.5 at an antibody concentration of 50 mg / mL. Two formulation matrices consisted of 10 mM histidine (with or without 5 mM L-methionine as an antioxidant), and the other two consisted of 10 mM acetate (with or without 5 mM L-methionine as an antioxidant); each of the four formulations was prepared at pH 5.5 with 7% (w / v) sucrose as a stabilizer and 0.025% w / v polysorbate 80 (PS-80) as a surfactant. The 10 mM L-histidine buffer consisted of 0.29 mg / mL L-histidine and 1.71 mg / mL L-histidine monohydrochloride monohydrate to obtain a target formulation pH of 5.5. 10 mM L-methionine in the formulation was equivalent to 1.49 mg / mL L-methionine, while 7% (w / v) sucrose and 0.025% (w / v) polysorbate 80 were equivalent to 70 mg / mL sucrose and 0.25 mg / mL PS-80 in the formulation, respectively. The formulations were staged for stability up to 6 months under ICH conditions: 5°C (5°C ± 3°C), 25°C (25°C, 60% RH), and 40°C (40°C, 75% RH). The stability plan is provided in Table 6 below. The four formulations were tested for: visual appearance, pH, protein concentration (UV 280 ) and colloidal stability measured by turbidity (OD 350-500 ), as well as purity by ultra-performance size exclusion chromatography (UP-SEC), charge variants by high-performance ion exchange chromatography (HP-IEX), subvisible particle count by microflow imaging (MFI), and % change in oxidation.

[0574] Table 6. Early Stability Plan

[0575]

[0576] Early stability data for the four anti-ILT4 monoclonal antibody formulations are summarized in Tables 7 - 10. The stability trends for the four formulations are shown in Figures 1A-1I and Figures 2A-2IProvided in these figures. As can be seen in these figures, under all three stability conditions (5 °C, 25 °C, and 40 °C), in the presence of 7% (w / v) sucrose as a stabilizer and 0.025% (w / v) polysorbate 80 as a surfactant, antibody 1 showed a similar stability profile in both acetate and L-histidine buffers at pH 5.5. For all assays, the degradation rate was high under the accelerated conditions at 25 °C and even higher under the stressed conditions at 40 °C. According to the stability plan, the antibody 1 formulation was stable for up to 6 months when stored at 5 °C. For the anti-ILT4 monoclonal antibody formulations in both buffer matrices, the presence of 5 mM L-methionine as an antioxidant in the formulation was found to improve the colloidal stability (OD 350-500 ) and reduce the soluble aggregates (%HMW) to a certain extent ( Figures 1A-1I ). The absence of 5 mM L-methionine had no effect on the charge profile by HP-IEX ( Figures 2A-2I ). L-histidine was selected as the primary buffer, and acetate was selected as the alternative buffer. As can be seen in the results of the reduced peptide map analysis in Tables 8-11, especially for the L-histidine formulation, the presence of 5 mM L-methionine in the acetate or L-histidine formulation was found to reduce the oxidation levels of methionine residues (M359, M253, and M429) under thermal stress and, to a certain extent, reduce the oxidation levels of tryptophan residues (W102 and W7).

[0577] Table 7. Early stability (L-histidine formulation at pH 5.5)

[0578]

[0579] Table 8. Early stability (L-histidine + L-methionine formulation at pH 5.5)

[0580]

[0581] Table 9. Early stability (acetate formulation at pH 5.5)

[0582]

[0583] Table 10. Early stability (acetate + L-methionine formulation at pH 5.5)

[0584]

[0585] Example 5

[0586] pH Range Stability Study of Anti-ILT4 Formulations

[0587] A pH range study was conducted to determine the stability of the Antibody 1 drug product within ±0.5 pH units of the target formulation pH of 5.5, i.e., at pH 5.0 or pH 6.0, and to help define the pH specification regarding form stability. Four Antibody 1 pH range formulations were staged for stability up to 6 months under ICH conditions: 5°C (5°C ± 3°C), 25°C (25°C, 60% RH), and 40°C (40°C, 75% RH). Two formulations were prepared in 10 mM histidine buffer with 7% (w / v) sucrose as a stabilizer and 0.025% (w / v) polysorbate 80 as a surfactant; one formulation at pH 5.0 and the other at pH 6.0. Two formulations were prepared in 10 mM acetate buffer with 7% (w / v) sucrose as a stabilizer and 0.025% (w / v) polysorbate 80 as a surfactant; one formulation at pH 5.0 and the other at pH 6.0. Purified Antibody 1 was dialyzed against each buffer, the pH of the final formulation was measured after dialysis, and no pH adjustment was made after this step. The final formulated drug substance was filtered to obtain the drug product bulk, which was subsequently filled into 2 mL glass vials, stoppered with rubber stoppers and sealed. The stability protocol was the same as that shown in Table 6. The four formulations were tested for visual appearance, pH, protein concentration (UV 280 ), colloidal stability measured by turbidity (OD 350-500 ), purity by UP-SEC, charge variants by HP-IEX, and subvisible particle count by MFI.

[0588] The data are summarized in Tables 11 - 14. The stability trends for the four Antibody 1 pH range formulations are provided in Figures 3A-3I and Figures 4A-4I and include data from earlier stability studies at pH 5.5 to allow comparison. As can be seen in these figures, there were no significant changes at any extreme pH conditions (pH 5.0 and pH 6.0) compared to the target pH 5.5 of the anti-ILT4 monoclonal antibody formulation in acetate or histidine buffer matrices stored at 5°C. The anti-ILT4 monoclonal antibody was found to be stable within ±0.5 units of the target pH 5.5. Under accelerated conditions at 25°C and stressed conditions at 40°C, the anti-ILT4 monoclonal antibody showed better colloidal (OD 350-500 ), physical (% HMW and % monomer), and chemical stability at the lower pH (5.0), followed by the target pH (5.5) and the higher pH (6.0). Based on these data sets, pH 5.5 was selected as the pH for the anti-ILT4 monoclonal antibody formulation.

[0589] Table 11. pH Range Study (L-Histidine Formulation pH 5.0)

[0590]

[0591] Table 12. pH Range Study (L-Histidine Formulation at pH 6.0)

[0592]

[0593] Table 13. pH Range Study (Acetate Formulation at pH 5.0)

[0594]

[0595] Table 14. pH Range Study (Acetate Formulation at pH 6.0)

[0596]

[0597] Example 6

[0598] Surfactant Range Study of Anti-ILT4 Formulations

[0599] A surfactant range study of an anti-ILT4 monoclonal antibody formulation containing antibody 1 was conducted using polysorbate 80 (PS-80) to analyze the sensitivity of the antibody to agitation stress in the presence or absence of surfactant. The anti-ILT4 monoclonal antibody formulation consisted of 50 mg / mL antibody 1 in 10 mM histidine buffer at pH 5.5 and 7% (w / v) sucrose. Five different formulations were prepared, each covering a range of polysorbate 80, namely 0% (w / v) or 0 mg / mL PS-80, 0.01% (w / v) or 0.1 mg / mL PS-80, 0.025% (w / v) or 0.25 mg / mL PS-80, 0.05% (w / v) or 0.5 mg / mL PS-80, and 0.1% (w / v) or 1 mg / mL PS-80. 2 mL glass vials were filled with 2.2 mL of filtered bulk anti-ILT4 drug product, stoppered with rubber stoppers and sealed. The anti-ILT4 drug product vials were agitated on a Thermo Scientific shaker at 300 rpm for up to 3 days, or placed horizontally for up to 7 days (under ambient conditions). The same five anti-ILT4 formulations (PS-80 range) were placed at ambient room temperature for up to 7 days without agitation and used as separate study controls to eliminate temperature effects. All vials (stressed and ambient controls) were covered with aluminum foil to eliminate light effects. Samples were analyzed for visual appearance, protein concentration (UV 280 ), turbidity (OD 350-500 ), subvisible particles by MFI, purity by UP-SEC, and charge variants by HP-IEX.

[0600] The data are summarized in Figures 7 and 8. The stability trends for five anti-ILT4 monoclonal antibody surfactant-range formulations are provided in Figures 5A-5G . As can be seen in these figures, the absence of surfactant resulted in an increasing amount of visible particle formation from 3 days of agitation up to 7 days of agitation. A small amount of visible particles was also visible in the 7-day environmental control samples. There was an increase in turbidity (OD 350-500 ), an increase in subvisible particle counts (≥2 μm, ≥5 μm, ≥10 μm, and ≥25 μm), and an increase in the soluble aggregate level (%HMW). In the presence of 0.1 mg / mL [0.01% (w / v)], 0.25 mg / mL [0.025% (w / v)], 0.5 mg / mL [0.5% (w / v)], or 1.0 mg / mL [0.1% (w / v)] surfactant, there were no significant differences in visual appearance, turbidity (OD 350-500 ) or soluble aggregate level (%HMW). After agitation for up to 7 days, a certain degree of increase in subvisible particles was visible for ≥0.5 mg / mL PS-80 concentration.

[0601] The data suggest that surfactant plays a key role in preventing agitation-induced aggregation. After the addition of surfactant, all stability-indicating properties became comparable. Overall, 0.25 mg / mL PS-80 seems to minimize the particle content compared to 0.5 mg / mL or 1 mg / mL PS-80. Therefore, adding higher levels of surfactant does not confer any significant advantage. Therefore, a PS-80 concentration of 0.25 mg / mL was selected for the L-histidine formulation containing 50 mg / mL anti-ILT4 monoclonal antibody.

[0602] Example 7

[0603] Photo-stress study of anti-ILT4 formulation using 10 mM L-methionine

[0604] During the forced degradation study, the major degradation pathway of the anti-ILT4 monoclonal antibody was identified as photo-induced oxidation, which affected the potency of the anti-ILT4 monoclonal antibody (Example 3). Early stability data (Example 4) suggested the benefit of 5 mM L-methionine in reducing the oxidation of L-methionine as well as L-tryptophan residues under heat stress. In an attempt to further explore the effect of a higher (10 mM) L-methionine content in the anti-ILT4 monoclonal antibody L-histidine formulation, a photo-stress study was conducted.

[0605] Based on early stability data, 10 mM L-histidine at pH 5.5 was finally determined as the buffer-pH matrix for formulation development activities. Two L-histidine formulations were tested. One formulation (H) contained 50 mg / mL anti-ILT4 monoclonal antibody, 10 mM L-histidine at pH 5.5, 7% (w / v) sucrose, and 0.025% (w / v) polysorbate 80; the other formulation (H+M) contained 50 mg / mL anti-ILT4 monoclonal antibody, 10 mM L-histidine at pH 5.5, 10 mM L-methionine, 7% (w / v) sucrose, and 0.025% (w / v) polysorbate 80. A third formulation (HC) consisted of 50 mg / mL anti-ILT4 monoclonal antibody, 10 mM L-histidine at pH 5.5, 7% (w / v) sucrose, and 0.025% (w / v) polysorbate 80 sucrose to act as a dark control (non-light-exposed) at 0.2 ICH, 0.5 ICH, or 1 ICH light stress (a combination of ultraviolet - UV and cool white light - CWL or visible light) in a Caron Photostability Chamber at ambient room temperature. The dark control samples were covered with aluminum foil and placed in the same chamber as the light-exposed samples to eliminate temperature effects. The samples were exposed to a cumulative amount of light exposure and removed when the exposure limit was reached. The samples were analyzed for the following: visual appearance, protein concentration (UV 280 ), turbidity (OD 350-500 ), purity by UP-SEC, charge variants by HP-IEX, oxidation level by reduced peptide mapping (anti-ILT4 monoclonal antibody oxidation hotspots M359, W7, M253, W102, M429), and potency by binding ELISA.

[0606] As Figures 6A-6K and visible in Figure 9, it was found that 10 mM L-methionine improved colloidal stability (OD 350-500 ), reduced the soluble aggregate level, maintained the monomer content (% monomer), minimized the change in charged species, and minimized (to some extent) the oxidation of L-methionine and L-tryptophan residues in the anti-ILT4 monoclonal antibody. In addition, as shown in Figure 9, after exposure to extreme light conditions (1 ICH), it was found that the potency was improved in the presence of 10 mM L-methionine. Therefore, the leading anti-ILT4 monoclonal antibody formulation was selected as follows: 50 mg / mL anti-ILT4 antibody, 0.29 mg / mL L-histidine, 1.71 mg / mL L-histidine monohydrochloride monohydrate, 1.49 mg / mL L-methionine, 70 mg / mL sucrose, and 0.25 mg / mL polysorbate 80.

[0607] Example 8

[0608] Freeze-Thaw Study of Anti-ILT4 Formulation

[0609] The stability of the leading anti-ILT4 monoclonal antibody formulation (50 mg / mL anti-ILT4, 0.29 mg / mL L-histidine, 1.71 mg / mL L-histidine monohydrochloride monohydrate, 1.49 mg / mL L-methionine, 70 mg / mL sucrose, 0.25 mg / mL polysorbate 80, pH 5.5) was evaluated under freeze-thaw stress. Using a 2.2 mL volume, the anti-ILT4 drug product was filled into 2 mL Type 1 glass vials, stoppered with rubber stoppers and sealed. The drug product vials were exposed to up to five freeze-thaw cycles (i.e., 1XF / T, 3XF / T, and 5XF / T); each cycle consisted of freezing at -80 °C followed by thawing at 25 °C (25 °C, 60% RH chamber), with a 5 °C control. Samples were analyzed for visual appearance, protein concentration (UV 280 ), turbidity (OD 350-500 ), purity by UP-SEC, and charge variants by HP-IEX.

[0610] As shown in Table 15, up to 5X freeze-thaw of the leading anti-ILT4 L-histidine formulation did not show any changes. Visual appearance, turbidity (OD 350-500 ), subvisible particle count, and biochemical profile (UP-SEC and HP-IEX) were comparable to up to 5X freeze-thaw stress. Overall, the freeze-thaw cycles had no measurable effect on the aggregation or biochemical properties of the leading anti-ILT4 formulation.

[0611] Example 9

[0612] Properties of Anti-ILT4 Formulation

[0613] The properties of the anti-ILT4 monoclonal antibody formulation, such as density, viscosity, osmotic pressure, and apparent glass transition temperature (T g ’ ) were determined.

[0614] Apparent glass transition temperature (T g ’ )

[0615] Modulated differential scanning microcalorimetry (mDSC) was used to determine T g ’ . The following parameters were used during the run: the temperature was equilibrated at -90.0 °C; adjusted by ±0.5 °C every 60 seconds, held at constant temperature for 5.0 minutes, followed by heating at 2.0 °C / minute to 25.0 °C.

[0616] Osmotic Pressure

[0617] The Advanced Instruments, Inc. Osmometer was calibrated using two calibration standards (100 mOsm / kg and 500 mOsm / kg) provided by Advanced Instruments, Inc., which were measured as 100 mOsm / kg and 500 mOsm / kg respectively (within acceptable limits). A 250 μL sample was taken in the sample dispenser and the osmotic pressure was measured in triplicate.

[0618] Density

[0619] The Anton Paar Densitometer was used for density measurement. The oscillating U-tube of the densitometer was rinsed with water and then with ethanol, and air-dried using the built-in pump. Before measuring the sample density, the air density in the oscillating U-tube was measured as 0.00126 g / cm 3 . For density measurement, 1 mL of the sample was filled into a sterile 1 mL syringe. The sample was checked for air bubbles and gently inserted into the oscillating U-tube via the sample port. The density was measured at 5 °C, 20 °C, and 30 °C.

[0620] Viscosity

[0621] The Rheosense mVROC viscometer was used for viscosity measurement. The water bath was stabilized at 20 °C and a system check was performed using an aqueous solution. Approximately 400 μL of the sample was placed into a clean 0.5 mL syringe and locked into the system for measurement. The viscosity was measured at 20 °C with a flow rate of 50 μL / min and a measurement time of 60 seconds. The viscosity value with an Rsqrd value having a slope fit of at least 0.98 was used for recording.

[0622] Properties of the anti-ILT4 monoclonal antibody formulation at 100 mg / vial (50 mg / mL), such as the apparent glass transition temperature (Tg’), osmotic pressure, density, viscosity, and extractable volume, are summarized in Table 16.

[0623] Table 16. Properties of the anti-ILT4 monoclonal antibody formulation

[0624]

[0625] Example 10: Ion Exchange (IEX) Method for Measuring Acidic Species of Anti-ILT4 Antibody

[0626] For the IEX method, a Waters Alliance LC system (Milford, MA, U.S.A.) was used, and ProPac WCX-10 from Thermo Scientific (p / n: 054993, particle size 10 μm, diameter 4 mm, length 250 mm) was selected, with a loading of 80 μg of sample. Mobile phase (A) was 24 mM MES pH 6.1 with 4% acetonitrile, and mobile phase (B) was 20 mM sodium phosphate, 95 mM NaCl pH 8.0 with 4% acetonitrile and was used as a non-linear S-shaped pH gradient, and the separation was monitored at a flow rate of 0.5 mL min–1 over 34 minutes, with the column temperature at 35 °C. The gradient used was: 22%–22% B for 0–0.6 minutes; 22%–29% B for 0.6–15.0 minutes; 29%–70% B for 15.0–30.0 minutes; 70%–100% B for 30.0–30.5 minutes; and 100%–100% B for 30.5–33.0 minutes. Mobile phase (C) 10 mM CHES pH 8.0, 40 mM Tris, 15 mM EDTA, 200 mM NaCl and 4% acetonitrile was used to strip the column at 0.5 mL min–1 from 33.1–34.0 minutes, followed by re-equilibration with 22% B at 1.0 mL min–1 from 34.5–44.5 minutes. From 44.5–45 minutes, the flow rate was reduced to 0.5 mL min–1. Elution was monitored at 280 nm for peak detection. The assay variability was determined to be within 1%.

[0627] Table 15. Freeze-Thaw Studies of Drug Products

[0628]

Claims

1. A preparation of an anti-human immunoglobulin-like transcript 4 (anti-ILT4) antibody or its antigen-binding fragment, comprising: (i) an anti-ILT4 antibody or its antigen-binding fragment at about 10 mg / mL to about 200 mg / mL; (ii) a buffer at about 5 mM to about 20 mM; (iii) a non-reducing sugar at about 6% to about 8% weight / volume (w / v); (iv) a non-ionic surfactant at about 0.01% to about 0.10% (w / v); and (v) an antioxidant at about 1 mM to about 20 mM, wherein the anti-ILT4 antibody or its antigen-binding fragment comprises: a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, where X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21), wherein the buffer is an L-histidine buffer, an acetate buffer or a citrate buffer, wherein the non-reducing sugar is a disaccharide, wherein the non-ionic surfactant is polysorbate 20 or polysorbate 80, and wherein the antioxidant is methionine.

2. The preparation according to claim 1, wherein the buffer is an L-histidine buffer.

3. The preparation according to claim 1 or 2, wherein the non-reducing sugar is sucrose.

4. The preparation according to any one of claims 1-3, wherein the non-ionic surfactant is polysorbate 80.

5. The preparation according to any one of claims 1-4, wherein the antioxidant is L-methionine.

6. A preparation of an anti-human immunoglobulin-like transcript 4 ("anti-ILT4") antibody or its antigen-binding fragment, comprising: (i) an anti-ILT4 antibody or its antigen-binding fragment at about 10 mg / mL to about 200 mg / mL; (ii) an L-histidine buffer at about 5 mM to about 20 mM; (iii) sucrose at about 6% to about 8% weight / volume (w / v); (iv) polysorbate 80 at about 0.01% to about 0.10% (w / v); and (v) L-methionine at about 1 mM to about 20 mM, wherein the anti-ILT4 antibody or its antigen-binding fragment comprises: A heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, where X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and A light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, where X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

7. The preparation according to claim 6, which comprises from about 8 mM to about 12 mM L-histidine buffer.

8. The preparation according to claim 6 or 7, which comprises from about 5 mM to about 10 mM L-methionine.

9. The preparation according to any one of claims 6-8, which comprises from about 0.01% to about 0.05% (w / v) polysorbate 80.

10. The preparation according to any one of claims 1-9, which comprises from about 10 mg / mL to about 150 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof.

11. The preparation according to claim 10, wherein the concentration of the anti-ILT4 antibody or an antigen-binding fragment thereof is about 10 mg / mL, about 12.5 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 125 mg / mL or about 150 mg / mL.

12. The preparation according to any one of claims 1-11, which comprises about 25 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80 and about 10 mM L-methionine.

13. The preparation according to any one of claims 1-11, which comprises about 50 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80 and about 10 mM L-methionine.

14. The preparation according to any one of claims 1-11, which comprises about 75 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80 and about 10 mM L-methionine.

15. The preparation according to any one of claims 1-11, which comprises about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80 and about 10 mM L-methionine.

16. The preparation according to any one of claims 1-11, which comprises an anti-ILT4 antibody or an antigen-binding fragment thereof at about 125 mg / mL, about 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.025% polysorbate 80, and about 10 mM L-methionine.

17. The preparation according to any one of claims 1-16, wherein the preparation has a pH of about 5.0 to about 6.

8.

18. The preparation according to any one of claims 1-16, wherein the preparation has a pH of about 5.5 to about 6.

0.

19. The preparation according to any one of claims 1-16, wherein the preparation has a pH of about 5.

5.

20. A preparation of an anti-human immunoglobulin-like transcript 4 ("anti-ILT4") antibody or an antigen-binding fragment thereof, which comprises: (i) an anti-ILT4 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 100 mg / mL; (ii) about 10 mM L-histidine buffer, pH about 5.5; (iii) about 7% weight / volume (w / v) sucrose; (iv) about 0.025% (w / v) polysorbate 80; and (v) about 10 mM L-methionine, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises: a heavy chain variable domain, which comprises: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain, which comprises: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, wherein X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

21. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or an antigen-binding fragment thereof comprises: a heavy chain variable domain, which comprises: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHSGSTNYNPSLKS (SEQ ID NO:47), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain, which comprises: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GX1X2NRPS, wherein X1 is N, Q, E or D and X2 is S or A (SEQ ID NO:20), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

22. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising: CDR-H1: GYYWS (SEQ ID NO:16), CDR-H2: EINHAGSTNYNPSLKS (SEQ ID NO:48), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

23. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:

58.

24. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO:2 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

7.

25. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO:80 and a light chain comprising the amino acid sequence shown in SEQ ID NO:

7.

26. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:57 and a light chain variable domain consisting of the amino acid sequence shown in SEQ ID NO:

58.

27. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:

7.

28. The preparation according to any one of claims 1-20, wherein the anti-ILT4 antibody or antigen-binding fragment thereof comprises: a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:80 and a light chain consisting of the amino acid sequence shown in SEQ ID NO:

7.

29. The preparation according to any one of claims 1-28, wherein the preparation is a liquid preparation stored at about 3°C to about 5°C, frozen to -20°C or lower, frozen to -70°C or lower, or is a reconstitution solution from a lyophilized preparation.

30. The formulation according to any one of claims 1-27, wherein after storage at about 3°C to about 5°C for 6 months: (i) the % monomer of the anti-ILT4 antibody or antigen-binding fragment thereof is at least about 99%, as determined by ultra-high performance size exclusion chromatography; (ii) such as by OD 350-500 the turbidity of the formulation is at most about 0.135 as measured (iii) the % main peak of the anti-ILT4 antibody or antigen-binding fragment thereof is at least about 63%, the % acidic variant of the anti-ILT4 antibody or antigen-binding fragment thereof is at most about 23%, and / or the basic variant of the anti-ILT4 antibody or antigen-binding fragment thereof is at most about 14%, as determined by high performance ion exchange chromatography; (iv) the sub-visible particle count of particles with a size of at least 2 μm is at most about 3500, as determined by microflow imaging; and / or (v) the % oxidation of one or more amino acid residues selected from W7, W102, M253, M359, and M429 in the heavy chain of the anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4%, as determined by reduced peptide mapping analysis.

31. The formulation according to any one of claims 1-30, wherein the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody.

32. A method of treating cancer in a human patient in need thereof, the method comprising administering an effective amount of the formulation according to any one of claims 1-31.

33. The method according to claim 32, wherein the cancer is selected from colorectal cancer, esophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, and small cell lung cancer.

34. Use of the formulation according to any one of claims 1-31 for the preparation of a medicament for treating cancer.

35. Use of the formulation according to any one of claims 1-31 for treating cancer in a human patient.

36. The use according to claim 34, wherein the cancer is selected from colorectal cancer, esophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, and small cell lung cancer.

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