Stable formulations of anti-ILT4 antibodies or antigen-binding fragments thereof in combination with anti-PD-1 antibodies and methods of use thereof

Abstract

The present invention relates to stable co-formulations of antibodies or antigen-binding fragments thereof that bind to human immunoglobulin-like transcript 4 (ILT4) and PD-1. Also provided are methods of treating various cancers using the formulations disclosed herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 336,839, filed on April 29, 2022, the disclosure of which is incorporated herein in its entirety.

[0003] Reference to electronically submitted sequence listings

[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 October 17, 2022 is named 25400-WO-PCT_SL.xml and is 99,833 bytes in size. Field of the Invention

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

[0007] A common strategy used by tumor cells to evade innate and adaptive immune responses is 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 impairment 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 Loumagne 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 (MDSCs) (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, thereby weakening the innate immune anti-tumor response. The interaction between HLA-G and monocytes due to ILT4 inhibits the maturation of human monocyte-derived antigen presenting cells (APCs), 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 (DCs) and ILT4 transgenic mice, HLA-G was shown to induce the development of tolerogenic APCs, accompanied by maturation / activation arrest of myeloid DCs, and that the induction of tolerogenic DCs by HLA-G was via disruption of the MHC class II presentation pathway (Ristich et al. Eur J Immunol. 2005, 35: 1133-1142).

[0008] PD-1 is considered to be an important molecule in immunoregulation and maintenance of peripheral tolerance. PD-1 is moderately expressed on initial T, B and NKT cells, and is upregulated by T / B cell receptor signaling on lymphocytes, monocytes and myeloid cells. Two known ligands of PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers that occur in various tissues. In a large sample collection with, for example, ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer and melanoma, PD-L1 expression was shown to be associated with poor prognosis, and overall survival was reduced regardless of subsequent treatment. Similarly, it was found that PD-1 expression on tumor-infiltrating lymphocytes marked dysfunctional T cells in breast cancer and melanoma, and was associated with a poor prognosis of renal cancer. Therefore, it has been proposed that tumor cells expressing PD-L1 interact with T cells expressing PD-1 to weaken T cell activation and escape immune surveillance, thereby contributing to weakening the immune response against tumors.

[0009] Several monoclonal antibodies that inhibit the interaction between PD-1 and one or both of its ligands, PD-L1 and PD-L2, have been approved by the FDA for the treatment of cancer. It has been proposed that the efficacy of such antibodies may be enhanced if administered in combination with other approved or experimental cancer therapies, such as radiation, surgery, chemotherapeutic agents, targeted therapies, agents that inhibit other signaling pathways that are deregulated in tumors, and other immune enhancers.

[0010] Therefore, there is a need for stable co-formulations of anti-ILT4 antibodies and anti-PD-1 antibodies. Such stable formulations preferably exhibit stability over months to years under conditions typically used to store drugs for self-administration (i.e., in syringes at refrigerated temperatures), resulting in a long shelf life for the corresponding drug products. Summary of the invention

[0011] In one aspect, the present invention provides a co-formulation of an anti-human ILT4 (anti-ILT4) antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof. In one formulation, the co-formulation comprises: (i) about 10 mg / mL to about 200 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) about 5 mM to about 20 mM buffer; (iv) about 6% to about 8% weight / volume (w / v) of a non-reducing sugar; (v) about 0.01% to about 0.10% (w / v) of a non-ionic surfactant; and (vi) about 1 mM to about 20 mM of an antioxidant, 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: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO: 17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18). NO: 18); and a light chain variable domain comprising the following: 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 anti-PD-1 antibody or its antigen-binding fragment comprises a variable light chain region comprising the following: CDRL1 of SEQ ID NO: 195, CDRL2 of SEQ ID NO: 296, and CDRL3 of SEQ ID NO: 397, and a variable heavy chain region comprising the following: CDRH1 of SEQ ID NO: 6100, CDRH2 of SEQ ID NO: 7101, and CDRH3 of SEQ ID NO: 8113. NO: CDRH3 of 8102, wherein the buffer is L-histidine buffer, acetate buffer or 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.

[0012] In some embodiments, the non-reducing sugar is sucrose. In some embodiments, the buffer is an L-histidine buffer. In some embodiments, the buffer is an L-histidine buffer with a pH of about 5.0-6.5. In certain embodiments, the nonionic surfactant is polysorbate 80 (PS-80). In some embodiments, the antioxidant is L-methionine.

[0013] Thus, in another aspect, provided herein is a 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 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) about 5 mM to about 20 mM L-histidine buffer; (iv) about 6% to about 8% (w / v) sucrose; (v) about 0.01% to about 0.10% (w / v) PS-80; and (vi) about 1 mM to about 20 mM L-methionine, 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: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO: 17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18). NO:18); and a light chain variable domain comprising the following: 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), and wherein the anti-PD-1 antibody or its antigen-binding fragment includes a variable light chain region comprising the following: CDRL1 of SEQ ID NO:95, CDRL2 of SEQ ID NO:96, and CDRL3 of SEQ ID NO:97, and a variable heavy chain region comprising the following: CDRH1 of SEQ ID NO:100, CDRH2 of SEQ ID NO:101, and CDRH3 of SEQ ID NO:102.

[0014] In some embodiments, the formulation comprises about 8 mM to about 12 mM L-histidine buffer.

[0015] In certain embodiments, the formulation comprises about 5 mM to about 10 mM L-methionine.

[0016] In other embodiments, the formulation comprises from about 0.01% to about 0.05% (w / v) PS-80.

[0017] In yet other embodiments, the formulation comprises an anti-ILT4 antibody or antigen-binding fragment thereof of about 10 mg / mL to about 150 mg / mL. In yet other embodiments, the concentration of the anti-ILT4 antibody or 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 antigen-binding fragment thereof is about 10 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 12.5 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 15 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 25 mg / mL. In one embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 50 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 75 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 100 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 125 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 150 mg / mL.

[0018] Thus, in a specific embodiment, the formulation comprises about 25 mg / mL of an anti-ILT4 antibody or 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.

[0019] In another specific embodiment, the formulation comprises about 50 mg / mL of an anti-ILT4 antibody or 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 yet another specific embodiment, the formulation comprises about 75 mg / mL of an anti-ILT4 antibody or 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 another specific embodiment, the formulation comprises about 100 mg / mL of an anti-ILT4 antibody or 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 yet another specific embodiment, the formulation comprises about 125 mg / mL of an anti-ILT4 antibody or 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 certain embodiments of the various preparations provided herein, the preparation has a pH range of about 5.0 to about 6.8. In some embodiments, the preparation has a pH range of about 5.5 to about 6.0. In other embodiments, the preparation 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 preparation has a pH of about 5.5. In another embodiment, the preparation has a pH of about 5.6. In yet another embodiment, the preparation has a pH of about 5.7. In yet another embodiment, the preparation has a pH of about 5.8. In yet another embodiment, the preparation has a pH of about 5.9. In yet another embodiment, the preparation has a pH of about 6.0.

[0024] Thus, in a specific embodiment, a pharmaceutical formulation for 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 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) about 10 mM L-histidine buffer, pH about 5.5; (iv) about 7% (w / v) sucrose; (v) about 0.025% (w / v) polysorbate 80; and (vi) 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). NO:18); and a light chain variable domain comprising the following: 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), and wherein the anti-PD-1 antibody or its antigen-binding fragment includes a variable light chain region comprising the following: CDRL1 of SEQ ID NO:95, CDRL2 of SEQ ID NO:96, and CDRL3 of SEQ ID NO:97, and a variable heavy chain region comprising the following: CDRH1 of SEQ ID NO:100, CDRH2 of SEQ ID NO:101, and CDRH3 of SEQ ID NO:10.

[0025] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof includes 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 some embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof includes 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).

[0027] 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.

[0028] 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 shown in SEQ ID NO:2 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0029] 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 shown in SEQ ID NO:80 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0030] 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.

[0031] 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.

[0032] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody comprising: 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.

[0033] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody comprising: 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.

[0034] 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.

[0035] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody comprising: 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.

[0036] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof is a monoclonal antibody comprising: 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.

[0037] 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 below. In yet another embodiment, the formulation is stored frozen at about -70°C or below. In yet another embodiment, the liquid formulation is a reconstituted solution from a lyophilized formulation.

[0038] 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 9 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 % monomer of the anti-PD-1 antibody or antigen-binding fragment thereof is at least about 98% as determined by ultra-high performance size exclusion chromatography; (iii) the % monomer of the anti-PD-1 antibody or antigen-binding fragment thereof is at least about 98% as determined by ultra-high performance size exclusion chromatography; 350-500The turbidity of the formulation is at most about 0.135 as measured by high performance ion exchange chromatography; (iv) the % main peak of the anti-ILT4 antibody or its antigen-binding fragment is at least about 52%, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 15%, and / or the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 12% as determined by high performance ion exchange chromatography; (v) the % main peak of the anti-PD-1 antibody or its antigen-binding fragment is at least about 10%, the % acidic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 5%, and / or the basic variant of the anti-ILT4 antibody or its antigen-binding fragment is at most about 4% as determined by high performance ion exchange chromatography; (vi) the subvisible particle count of particles with a size of at least 2 μm is at most about 3500 as determined by microfluidic imaging; and / or (vii) as determined by reduced peptide mapping analysis, such as SEQ ID 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 set forth in NO: 2 or 80 is less than about 4%.

[0039] In some embodiments, the % monomer of the anti-ILT4 antibody or antigen-binding fragment thereof is at least about 99% as determined by ultra-performance size exclusion chromatography after storage of the formulation at about 3°C ​​to about 5°C for 6 months.

[0040] In some embodiments, after the formulation has been stored at about 3°C ​​to about 5°C for 6 months, the 350-500 The turbidity of the formulation was measured to be at most about 0.135.

[0041] 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 Variants 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 Variants 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 Variants of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23%, and the Basic Variants of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14% as determined by high performance ion exchange chromatography.

[0042] In some embodiments, after the formulation has been stored at about 3°C ​​to about 5°C for 6 months, the subvisible particle count for particles having a size of at least 2 μm as determined by microfluidic imaging is at most about 3500.

[0043] In some embodiments, after the formulation is stored at about 3°C ​​to about 5°C for 6 months, 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. 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 yet 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.

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

[0045] In yet another aspect, provided herein are methods of treating cancer in a human patient in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical formulation described herein.

[0046] In another aspect, provided herein is a use of the pharmaceutical formulation described herein for the preparation of a medicament for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Shown are high molecular weight (HMW) species as determined by UP-SEC in anti-ILT4 / anti-PD-1 co-formulations, anti-ILT4 SE, and pembrolizumab SE samples at different concentrations of L-methionine and for different levels of light exposure and residual metals as described in Example 6.

[0048] Figure 2Shown are PS80 content as determined by reverse phase chromatography in anti-ILT4 / anti-PD-1 co-formulations, anti-ILT4 SE, and pembrolizumab SE samples at different concentrations of L-methionine and for different levels of light exposure and residual metals as described in Example 6.

[0049] Figure 3 Shown is M105 oxidation as determined by reduced peptide mapping analysis for anti-ILT4 / anti-PD-1 co-formulation samples with varying L-methionine concentrations (7-15 mM) at 40°C for 4 weeks, as well as a function of metal concentration and light exposure, as described in Example 6.

[0050] Figure 4 Shown is W102 oxidation as determined by reduced peptide mapping for anti-ILT4 / anti-PD-1 co-formulations and anti-ILT4 SE samples with varying L-methionine concentrations (7-15 mM) at 40°C for 4 weeks, as well as a function of metal concentration and light exposure, as described in Example 6.

[0051] Figure 5 Shown is W7 oxidation as determined by reduced peptide mapping for anti-ILT4 / anti-PD-1 co-formulations and anti-ILT4 SE samples with varying L-methionine concentrations (7-15 mM) at 40°C for 4 weeks, as well as a function of metal concentration and light exposure, as described in Example 6.

[0052] Figure 6 Shown is the total oxidation of methionine residues as determined by hydrophobic interaction chromatography for pembrolizumab SE samples with different L-methionine concentrations (7-15 mM) at 40°C for 4 weeks as described in Example 6, and as a function of metal concentration and light exposure.

[0053] Figure 7 Contour plots showing predicted values ​​for HMW co-formulations at different factor levels at 40°C as described in Example 7.

[0054] Figure 8 Contour plots of predicted values ​​of HMW for anti-ILT4 SE at different factor levels at 40°C as described in Example 7 are shown.

[0055] Fig. 9 Contour plots of predicted values ​​of HMW for pembrolizumab SE at different factor levels at 40°C as described in Example 7 are shown.

[0056] Fig.10 Shown are contour plots with predicted values ​​for % acidic variants of anti-ILT4 in co-formulations at different factor levels at 40°C for 4 weeks as described in Example 7.

[0057] Fig.11 Shown is a contour plot of the % major for anti-ILT4 in the co-formulations according to different factors (L-methionine concentration, light exposure and metal content). As described in Example 7, this figure illustrates that an increase in light stress reduces the % major for anti-ILT4.

[0058] Fig.12 Shown is a contour plot of the % basic variants for anti-ILT4 in co-formulations according to different factors (L-methionine concentration, light exposure and metal content). As described in Example 7, this figure illustrates that an increase in light stress increases the % basic variants for anti-ILT4.

[0059] Fig.13 Contour plots with predicted values ​​from the model fit over time at different settings of three parameters (L-methionine concentration, light exposure, metal content) as described in Example 7 are shown.

[0060] Fig.14 Shown are the main contour plots for the % of pembrolizumab in the co-formulations according to different factors (L-methionine concentration, light exposure and metal content) as described in Example 7.

[0061] Fig.15 Shown are contour plots for the % basic variants of pembrolizumab in co-formulations according to different factors (L-methionine concentration, light exposure, and metal content) as described in Example 7.

[0062] Fig.16 Contour plots with predicted values ​​from the model fit over time at different settings of three parameters (L-methionine concentration, light exposure, metal content) as described in Example 7 are shown.

[0063] Fig.17 Shown is a primary contour plot of the % anti-ILT4 in the anti-ILT4 SE formulations according to different factors (L-methionine concentration, light exposure and metal content) as described in Example 7.

[0064] Fig.18 Shown is a contour plot of the % basic variant of anti-ILT4 in anti-ILT4 SE formulations according to different factors (L-methionine concentration, light exposure and metal content) as described in Example 7.

[0065] Fig.19 The L-methionine concentrations at all stability time points for all three formulations as described in Example 7 are shown.

[0066] Fig. 20A graph of turbidity values ​​for all three formulations at 5°C and 40°C as described in Example 8 is shown.

[0067] Figure 21-25 Raw data from peptide mapping analysis for SE and co-formulations at initial and 40°C conditions as described in Example 8 are shown. Fig.21 Depicts M253 (shared), Fig. 22 Depicts M429 (shared), Fig.23 W7 (anti-ILT4) is depicted, Fig.24 W102 (anti-ILT4) is depicted, while Fig.25 M105 (pembrolizumab) is depicted.

[0068] Fig.26 Shown are the high molecular weight (HMW) and low molecular weight (LMW) species in co-formulation 1 (CF1) and co-formulation 2 (CF2) as determined by UP-SEC when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles) and 40°C (downward triangles) as described in Example 8.

[0069] Fig. 27 Shown are anti-ILT4 and pembrolizumab charge variants in CF 1 and CF 2 as determined by IEX when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles) and 40°C (downward triangles) as described in Example 8.

[0070] Fig.28 Shown are the total L-methionine contents in CF 1 and CF 2 when stored for 12 months at 5°C (circles), 6 months at 25°C (downward triangles) and 40°C (upward triangles) as described in Example 8.

[0071] Fig.29 Shown are the total PS-80 contents in CF1 and CF2 when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles) and 40°C (downward triangles) as described in Example 8.

[0072] Fig.30 Shown are subvisible particle counts in CF1 and CF2 when stored for 12 months at 5°C (light grey), 6 months at 25°C (dark grey) and 40°C (black) as described in Example 8.

[0073] Fig.31Shown is the oxidation of M105 (anti-PD-1), M429 (shared residues) and M253 (shared residues) as determined by reduced peptide mapping of CF1 and CF2 when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles) and 40°C (downward triangles) as described in Example 8.

[0074] Fig.32 Shown is the oxidation of W102 (anti-ILT4 monoclonal antibody) and W7 (anti-ILT4 monoclonal antibody) as determined by reduced peptide mapping of CF 1 and CF 2 when stored at 5°C (circles) for 12 months, 25°C (upward triangles) and 40°C (downward triangles) for 6 months as described in Example 8. DETAILED DESCRIPTION

[0075] Definitions and Abbreviations

[0076] As used throughout the specification and appended claims, the following abbreviations apply:

[0077] AAPH 2,2'-Azobis(2-methylpropionamidine) dihydrochloride

[0078] API Active Pharmaceutical Ingredient

[0079] CDR Complementarity determining regions in immunoglobulin variable regions are defined using the Kabat numbering system unless otherwise specified.

[0080] CE-SDS capillary electrophoresis sodium dodecyl sulfate

[0081] CHO Chinese Hamster Ovary

[0082] cIEF Capillary isoelectric focusing

[0083] DLS Dynamic Light Scattering Test

[0084] DSC Differential Scanning Calorimetry

[0085] DSF Differential Scanning Fluorescence

[0086] ELISA enzyme-linked immunosorbent assay

[0087] FR Framework Region

[0088] HC Heavy Chain

[0089] HMW High Molecular Weight

[0090] HP-IEX High Performance Ion Exchange Chromatography

[0091] ICH International Council on Harmonization

[0092] IgG Immunoglobulin G

[0093] LC Liquid Chromatography

[0094] LC-MS liquid chromatography-mass spectrometry

[0095] LMW Low Molecular Weight

[0096] mAb monoclonal antibody

[0097] NR-CE-SDS Non-reducing capillary electrophoresis Sodium dodecyl sulfate

[0098] PS20 (or PS-20) Polysorbate 20

[0099] PS80 (or PS-80) Polysorbate 80

[0100] R-CE-SDS reduced capillary electrophoresis sodium dodecyl sulfate

[0101] RH Relative humidity

[0102] SE Single Entity

[0103] t-BHP tert-butyl hydroperoxide

[0104] UP-SEC Ultra-Performance Size Exclusion Chromatography

[0105] V H Immunoglobulin heavy chain variable region

[0106] V L Immunoglobulin light chain variable region

[0107] v / v volume / volume

[0108] WFI Water for Injection

[0109] w / v weight / volume

[0110] Unless specifically defined elsewhere in this document, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0111] 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.

[0112] References to "or" indicate either or both possibilities, unless the context clearly states one of the indicated possibilities. In some cases, "and / or" is used to emphasize either or both possibilities.

[0113] As used herein, "acidic variant" refers to an anti-ILT4 antibody that is more acidic (e.g., as determined by cation exchange chromatography) than the main species of the anti-ILT4 antibody. Such acidic variants are detected by various chromatographic purification methods used to separate molecular variants by charge, such as ion exchange, such as cation exchange chromatography or WCX-10 HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. Typically, acidic variants have a lower isoelectric point (pI) than the main species and may have more acidic properties due to, for example, methionine oxidation, sialylation of asparagine residues, or deamidation variants of the antibody, or a combination thereof. In one embodiment, an anti-ILT4 antibody acidic variant is as determined by Figure 6 D, and eluted according to the cation exchange method described in Example 11. In the ion exchange chromatography method, "% acidic variant species" refers to the total area of ​​the acidic variant peaks divided by the total area of ​​all peaks in the elution chromatogram. In one embodiment, the anti-ILT4 antibody acidic variants are identified by the peak eluting before the main peak according to the cation exchange method. In another embodiment, the anti-ILT4 antibody acidic variants are identified by the peak eluting before the main peak according to the weak cation exchange method. In the ion exchange chromatography method, "% acidic variants" refers to the total area of ​​the acidic species peaks divided by the total area of ​​all peaks in the elution chromatogram.

[0114] "Affinity" refers to the summed strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D Affinity can be measured by common methods known in the art including KinExA and Biacore.

[0115] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), fully human antibodies, and chimeric antibodies.

[0116] As used herein, unless otherwise indicated, "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 a 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, and individual heavy or light chain variable regions.

[0117] As used herein, "basic variant" refers to an anti-ILT4 antibody that is more basic (e.g., as determined by cation exchange chromatography) than the main species of the anti-ILT4 antibody. Such basic variants are detected by various chromatographic purification methods used to separate molecular variants by charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 11 or WCX-10 HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. Typically, basic species have a higher pH than the main species and may have more basic properties due to modifications or differences from the main species. In one embodiment, the anti-ILT4 antibody basic species are identified by peaks eluting after the main peak according to the cation exchange method. In another embodiment, the anti-ILT4 antibody basic species are identified by peaks eluting after the main peak according to the weak cation exchange method. In the ion exchange chromatography method, "% basic species" refers to the total area of ​​the basic species peaks divided by the total area of ​​all peaks in the elution chromatogram.

[0118] "Fab fragment" is composed of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. "Fab fragment" can be the product of papain cleavage of an antibody.

[0119] The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0120] A "Fab' fragment" contains a portion or fragment of a light chain and a heavy chain, wherein the portion or fragment contains V H The CH1 domain and the region between the CH1 and CH2 domains allow for the formation of interchain disulfide bonds between the two heavy chains of the two Fab' fragments to form F(ab')2 molecules.

[0121] A "F(ab')2 fragment" contains two light chains and two heavy chains, which contains a portion of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments bound together by a disulfide bond between the two heavy chains. A "F(ab')2 fragment" may be a product of pepsin cleavage of an antibody.

[0122] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0123] An "isolated antibody" refers to a purified state, and in such contexts, means that the molecule is substantially free of other biomolecules, 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 refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compounds as described herein.

[0124] As used herein, "major species" or "mainly" refers to the anti-ILT4 antibody species that is identified as the majority of antibody species in a mixture with one or more of its acidic or basic species. Such major species are detected by various chromatographic purification methods used to separate molecular variants by charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 11) or WCX-10HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. The mixture can be, for example, the result of an antibody preparation from a mammalian cell and its post-translational modification, 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, "% main" 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 the 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 a mobile phase (A) of 24 mM MES pH 6.1 with 4% acetonitrile and a mobile phase (B) of 20 mM sodium phosphate, 95 mM NaCl pH 8.0 with 4% acetonitrile, and a column temperature of 35°C. In one embodiment, a nonlinear gradient is used with: 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 11.

[0125] As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies having different amino acid sequences in their variable domains (which are typically specific for different epitopes). The modifier "monoclonal" indicates the characteristic that an antibody is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of antibodies by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may 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 in, for example, 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.

[0126] 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 a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat carbohydrate chains if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0127] Typically, the basic "antibody" structural unit comprises a tetramer. In a monospecific antibody, each tetramer comprises 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 that is primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain may define a constant region primarily responsible for effector function.

[0128] Generally, human constant light chains are classified as kappa and lambda light chains. In addition, human constant heavy chains are generally classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. The subtypes of these IgGs include, for example, IgG1 and IgG4. The present invention includes anti-ILT4 antibodies and antigen-binding fragments comprising any of these light and / or heavy constant chains.

[0129] As used herein, "variable region", "variable domain", "V region" or "V chain" means a segment of an IgG chain whose sequence is variable between different antibodies. The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The heavy chain variable region may be referred to as the "V H The light chain variable region may be referred to as the "V L ". Typically, 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 to enable binding to specific epitopes. Typically, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acids are typically assigned to each domain 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.

[0130] "CDR" refers to the V H One of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the β-sheet framework, or in the antibody V LOne of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the β-pleated sheet framework. Therefore, the CDR is a variable region sequence interspersed within the framework region sequence. The CDR region is well known to those skilled in the art and has been defined, for example, by Kabat as the most highly variable region within the antibody variable domain. Chothia also structurally defines the CDR region sequence as those residues that are not part of the conserved β-pleated sheet framework and are therefore able to adapt to different conformations. Both terms are well known in the art. AbM, Contact and IMGT have also defined CDR region sequences. The position of the CDR within the canonical antibody variable region has 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 in the hypervariable region in different antibodies is different, other residues relative to the standard position are usually numbered with a, b, c, etc. next to the residue number in the standard variable region numbering scheme (Al-Lazikani et al., supra). Similarly, this nomenclature is well known to those skilled in the art. The relationship between numbering systems including, for example, Kabat numbering and IMGT unique numbering systems is well known to those skilled in the art, and is shown in Table 1 below. In some embodiments, CDRs are defined by the Kabat numbering system. In other embodiments, CDRs are defined by the IMGT numbering system. In yet other embodiments, CDRs are defined by the AbM numbering system. In yet other embodiments, CDRs are defined by the Chothia numbering system. In yet other embodiments, CDRs are defined by the Contact numbering system.

[0131] Table 1. Correspondence between CDR numbering systems relation

[0132] 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

[0133] Sequence identity refers to the extent to which amino acids in equivalent positions in two polypeptides are identical when the two sequences are optimally aligned.

[0134] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids that share similar properties and can be interchanged are discussed above.

[0135] "Conservatively modified variants" or "conservative substitutions" refer to the use of other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) to replace amino acids in proteins, so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in non-critical regions of polypeptides do not substantially change biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition)). In addition, substitutions of amino acids with similar structure or function hardly destroy biological activity. Exemplary conservative substitutions are set forth in Table 2.

[0136] Table 2. Exemplary conservative amino acid substitutions

[0137]

[0138]

[0139] As used herein, the term "epitope" refers to the area or region on an antigen to which an antibody or antigen-binding fragment binds. The binding of an antibody or antigen-binding fragment thereof disclosed herein to an epitope means that the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within the epitope.

[0140] As used herein, "treating" or "treating" a cancer or infectious condition means administering a formulation of the invention to a subject suffering from an immune condition or cancerous condition or diagnosed with a cancer or pathogen infection (e.g., viral, bacterial, fungal) to achieve at least one positive therapeutic effect, such as, for example, a decrease 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 the rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the following: inducing / increasing an anti-tumor immune response, stimulating an immune response against pathogens, toxins and / or self-antigens, stimulating an immune response against viral infection, reducing the number 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 a similar untreated patient.

[0141] The terms "cancer", "cancerous" or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer 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, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0142] The term "patient" (alternatively referred to herein as "subject" or "individual") refers to a mammal (e.g., rat, mouse, dog, cat, rabbit), most preferably a human, that can be treated with the formulation of the present invention. 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 diagnosed with, suspected of having, or susceptible to a disease or condition (e.g., an ILT4-related disease such as cancer) that the formulation of the present invention is intended to treat or improve, or a patient for whom it is desired to prevent such a condition.

[0143] The term "therapeutically effective amount" or "effective amount" means an amount whereby sufficient therapeutic composition or formulation is introduced into a patient to treat a disease or condition. Those skilled in the art recognize that this level may vary depending on the patient's characteristics such as age, weight, etc.

[0144] The term "about" when modifying an amount 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, or a parameter value of a step in a characterization method, etc., refers to variations in the numerical amount that may occur, for example, by: typical measurements, handling, and sampling procedures involved in the preparation, characterization, and / or use of the substance or composition; instrumental errors in these procedures; differences in the manufacture, source, or purity of ingredients used to prepare or use the composition or perform the procedure; etc. In certain embodiments, "about" may mean variations of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

[0145] As used herein, "x% (w / v)" is equivalent to x g / 100 ml (eg, 5% w / v is equal to 50 mg / ml).

[0146] 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 recited element or group of elements, and the optional inclusion of other elements having similar or different properties from the recited elements, which do not substantially 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 recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, which substitutions do not substantially affect the properties of the binding compound.

[0147] “Comprising” or variations such as “comprise” or “comprises” are used throughout the specification and claims in an inclusive sense, that is, to specify the presence of recited features but not to exclude the presence or addition of further features that may materially enhance the operation or utility of any embodiment of the invention, unless the context requires otherwise by explicit language or necessary implication.

[0148] The term "buffer" encompasses those agents that maintain the solution pH of the formulation within an acceptable range or, for the lyophilized formulations of the invention, provide an acceptable solution pH prior to lyophilization.

[0149] The terms "lyophilization," "freeze-dried," and "freeze-dried" refer to a 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 pre-lyophilized formulations to enhance the stability of the lyophilized product during storage.

[0150] The term "pharmaceutical formulation" refers to a preparation that is in such form as to allow the active ingredients to be effective, and 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.

[0151] "Pharmaceutically acceptable" refers to excipients (vehicles, additives) and compositions that can reasonably be administered to a subject to provide an effective dosage of the active ingredient employed, and are "generally regarded as safe", e.g., are physiologically tolerated and do not generally produce allergic or similar adverse reactions, such as stomach upset, etc., when administered to humans. In another embodiment, the term refers to molecular entities and compositions approved by a regulatory agency of the Federal or state government or listed in the US Pharmacopeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0152] A "reconstituted" formulation is one 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.

[0153] A "stable" formulation is one in which the protein substantially maintains its physical stability and / or chemical stability and / or biological activity when stored or under stress conditions. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For example, in one embodiment, a stable formulation is a formulation in which no significant changes are observed at refrigerated temperatures (2-8° C.) for at least 6 months. In one embodiment, a stable formulation is a formulation in which no significant changes are observed at refrigerated temperatures (2-8° C.) for at least 12 months. In another embodiment, a stable formulation is a formulation in which no significant changes are observed at refrigerated temperatures (2-8° C.) for at least 24 months. In another embodiment, a stable formulation is a formulation in which no significant changes are observed for at least 3 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation in which no significant changes are observed for at least 6 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation in which no significant changes are observed for at least 12 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation in which no significant changes are observed for at least 18 months at room temperature (23-27°C). The standards for the stability of antibody formulations are as follows. Typically, no more than 10%, preferably 5%, of antibody monomer degradation as measured by SEC-HPLC. Typically, the formulation is colorless or transparent to slightly opalescent by visual analysis. Typically, the concentration, pH, and osmotic pressure of the formulation have no more than + / -10% variation. The efficacy is typically within 60-140%, preferably 80-120%, of the control or reference. Typically, no more than 10%, preferably 5% of antibody shearing, i.e., % low molecular weight species as determined, for example, by HP-SEC, is observed. Typically, no more than 10%, preferably no more than 5% aggregation of the antibody is observed, ie, % high molecular weight species as determined, for example, by HP-SEC.

[0154] An antibody "maintains its physical stability" in a pharmaceutical formulation if it does not show a significant increase in aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and FTIR spectroscopy (which determines protein secondary structure).

[0155] An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of a protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or shearing (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping coupled to mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0156] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

[0157] The term "isotonic" means that the preparation of interest has substantially the same osmotic pressure as human blood. Isotonic preparations typically have an osmotic pressure of about 270-328 mOsm. Slightly hypotonic is 250-269, while slightly hypertonic is 328-350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or ice-type osmometer.

[0158] As used herein, "co-formulated" or "co-formulation" or "coformulation" or "coformulated" refers to at least two different antibodies or antigen-binding fragments thereof that are formulated together and stored as a combination product in a single vial or vessel (e.g., an injection device), rather than being formulated and stored individually and then mixed or administered separately prior to administration. In one embodiment, the co-formulation contains two different antibodies or antigen-binding fragments thereof.

[0159] Anti-ILT4 antibodies and antigen-binding fragments thereof

[0160] The formulations disclosed herein can be used with any antibody and antigen-binding fragment thereof (e.g., fully human antibody) that binds to ILT4. 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 an anti-ILT4 antibody or its antigen-binding fragment described herein (e.g., 1E1, 2A6, 3G7 or 2C1).

[0161] In one aspect, the formulations can be used with anti-ILT4 antibodies and antigen-binding fragments thereof as described herein having one or more of the following properties:

[0162] ● 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, e.g., as determined by hydrogen-deuterium exchange mass spectrometry;

[0163] ● Binds to human ILT4 at domain 1 (see Wilcox et al. BMC Structural Biology 2:6 (2002));

[0164] - Binds to the extracellular domain of human ILT4 or to the TM form of ILT4 expressed on the surface of cells, such as pre-B cells, Chinese hamster ovary cells, U937 cells or Jurkat JE6 cells.

[0165] Calculated pI ~ 7.29 (e.g., 7.29 or 7.30);

[0166] ●Experimentally determined pI ~7.2;

[0167] ● Characterized by a thermal map with Tm start > 60°C, Tm1 ~ 65.2°C, and Tm2 ~ 78.8°C;

[0168] ●About 1.7X 10 -8 M of K D Binding to human ILT4 (e.g., as determined by surface plasmon resonance, such as binding of anti-ILT4 to polyhistidine-tagged human ILT4);

[0169] Ka = 5.5 x 10 5 M -1 s -1 (e.g., binding of anti-ILT4 to polyhistidine-tagged human ILT4 as determined by surface plasmon resonance);

[0170] Kd = 9 x 10 -3 s -1 (e.g., binding of anti-ILT4 to polyhistidine-tagged human ILT4 as determined by surface plasmon resonance);

[0171] ● For example, as determined by surface plasmon resonance, for example, at about 0.25 micrograms / ml (+ 0.06 μg / ml) 50 Blocking 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);

[0172] ● blocking the binding of HLA-A, HLA-B (e.g., fluorescent dye-labeled dexamers of 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), for example, as determined by surface plasmon resonance;

[0173] ● blocking 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., a biotinylated ANGPTL protein), e.g., as determined by surface plasmon resonance;

[0174] ● does not bind to ILT2, ILT3, ILT5, LILRB5, LILRA1, LILRA2, ILT7, ILT8 and / or ILT11;

[0175] ● For example, 0.43 micrograms / ml ( + 0.14 μg / ml) 50 Reversal of ILT4-mediated IL2 suppression in ILT4-transfected 3A9 cells;

[0176] ● rescue of ILT4:HLA-G-induced inhibition of mast cell degranulation (e.g., in the presence of plate-bound HLA-G tetramers), e.g., wherein the mast cells express ILT4 and CD200RLa and are stimulated, e.g., with antibody-mediated cross-linking of CD200RLa;

[0177] ● Enhancement of lipopolysaccharide (LPS)-induced expression of pro-inflammatory myeloid cytokines such as GM-CSF and / or TNFα from peripheral blood mononuclear cells (PBMCs);

[0178] ● Enhancement of anti-CD3-induced expression of pro-inflammatory myeloid cytokines such as GM-CSF and / or TNFα from peripheral blood mononuclear cells (PBMCs);

[0179] Inhibiting tumor growth in a human or, e.g., other mammal, e.g., mouse (e.g., immunodeficient NSG mouse) reconstituted with, e.g., human hematopoietic stem cells with peripheral human CD45+ immune cells, e.g., wherein the tumor is, e.g., a human skin melanoma tumor from the cell line SKMEL5;

[0180] ● Alleviating MDSC-mediated tumor tolerance in a subject (e.g., a human subject) having a tumor;

[0181] ●Does not bind to cynomolgus monkey ILT4 and / or mouse pirB;

[0182] ●Staining for CD14+ human monocytes and / or CD11B+ human granulocytes; and / or

[0183] - Binds to one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10) of the human ILT4 haplotypes.

[0184] Antibody 1E1 (Q1E) Heavy Chain (IgG4)

[0185] Heavy chain

[0186]

[0187] VSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0188] (SEQ ID NO: 1; variable domain is underlined; CDR is double underlined)

[0189] Heavy chain variable domain

[0190] EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSS(SEQ ID NO:63)

[0191] Antibody 1E1 (Q1E, S54A) heavy chain (IgG4)

[0192] Heavy chain

[0193]

[0194]

[0195] (SEQ ID NO:2; variable domain is underlined; CDR is double underlined)

[0196] Heavy chain variable domain

[0197] EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHAGSTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSS(SEQ ID NO:57)

[0198] Antibody 1E1 heavy chain (IgG1)

[0199] Heavy chain

[0200]

[0201] (SEQ ID NO:44; variable domain is underlined; CDR is double underlined)

[0202] Heavy chain variable domain

[0203] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARLPTRWVTTRYFDLWGRGTLVTVSS(SEQ ID NO:69)

[0204] 1E1 Heavy Chain CDR

[0205] CDR-H1: GYYWS (SEQ ID NO: 16)

[0206] CDR-H2: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO: 17) (e.g., EINHSGSTNYNPSLKS (SEQ ID NO: 47) or EINHAGSTNYNPSLKS (SEQ ID NO: 48))

[0207] CDR-H3:LPTRWVTTRYFDL(SEQ ID NO:18)

[0208] Antibody 1E1 (Q1E) Light Chain (λ)

[0209] Light chain

[0210] (SEQ ID NO:3; variable domain is underlined; CDR is double underlined)

[0211] Light chain variable domain

[0212] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0213] (SEQ ID NO:70)

[0214] Antibody 1E1 (Q1E, S54A) light chain (λ)

[0215] Light chain

[0216] (SEQ ID NO:4; variable domain is underlined; CDR is double underlined)

[0217] Light chain variable domain

[0218] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNANRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0219] (SEQ ID NO:71)

[0220] Antibody 1E1 (Q1E, N53Q) light chain (λ)

[0221] Light chain

[0222] (SEQ ID NO:5; variable domain is underlined; CDR is double underlined)

[0223] Light chain variable domain

[0224] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGQSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0225] (SEQ ID NO:72)

[0226] Antibody 1E1 (Q1E, N53E) light chain (λ)

[0227] Light chain

[0228]

[0229] (SEQ ID NO:6; variable domain is underlined; CDR is double underlined)

[0230] Light chain variable domain

[0231] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGESNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0232] (SEQ ID NO:73)

[0233] Antibody 1E1 (Q1E, N53D) light chain (λ)

[0234] Light chain

[0235]

[0236] (SEQ ID NO:7; variable domain is underlined; CDR is double underlined)

[0237] Light chain variable domain

[0238] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0239] (SEQ ID NO:58)

[0240] Antibody 1E1 Light Chain (λ)

[0241] Light chain

[0242]

[0243]

[0244] (SEQ ID NO:45; variable domain underlined; CDRs double underlined)

[0245] Light chain variable domain

[0246] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYVFGGGTQLTVL

[0247] (SEQ ID NO:77)

[0248] 1E1 light chain CDR

[0249] CDR-L1:TGSSSNIGAGYDVH(SEQ ID NO:19)

[0250] CDR-L2: GX1X2NRPS; wherein X1 is N, Q, E or D, and X2 is S or A (SEQ ID NO:20) (e.g., 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))

[0251] CDR-L3: QSFDNSLSAYV(SEQ ID NO:21)

[0252] Includes 1E1 heavy and light chain CDRs or 1E1 V H and V L Antibodies and antigen-binding fragments thereof comprising the 1E1 or 1E1 heavy and light chains (or variants thereof, e.g., as described herein) may be referred to as "1E1".

[0253] Antibody 2A6(Q1E) Heavy Chain(IgG4)

[0254] Heavy chain

[0255] (SEQ ID NO:8; variable domain is underlined; CDR is double underlined)

[0256] Heavy chain variable domain

[0257] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDSSGWYKGGAFDIWGQGTMVTVSS

[0258] (SEQ ID NO:64)

[0259] Antibody 2A6 (Q1E, S102A, M119L) heavy chain (IgG4)

[0260] Heavy chain

[0261] (SEQ ID NO:9; variable domain is underlined; CDR is double underlined)

[0262] Heavy chain variable domain

[0263] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFDASGWYKGGAFDIWGQGTLVTVSS

[0264] (SEQ ID NO:65)

[0265] Antibody 2A6 (Q1E, D101S, M119L) heavy chain (IgG4)

[0266] Heavy chain

[0267] (SEQ ID NO: 10; variable domain is underlined; CDR is double underlined)

[0268] Heavy chain variable domain

[0269] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARYFSSSGWYKGGAFDIWGQGTLVTVSS

[0270] (SEQ ID NO:66)

[0271] The formulations disclosed herein can be used with antibodies and antigen-binding fragments thereof comprising 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 instead of E.

[0272] 2A6 Heavy Chain CDR

[0273] CDR-H1: SYAIS (SEQ ID NO: 22)

[0274] CDR-H2:GIIPIFGTANYAQKFQG(SEQ ID NO:23)

[0275] 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))

[0276] Antibody 2A6 Light Chain (λ)

[0277] Light chain

[0278] (SEQ ID NO: 11; variable domain is underlined; CDR is double underlined)

[0279] Light chain variable domain

[0280] QSVLTQPSSLSASPGASASLTCTLRSGINVDTYRIHWYQQKPGSPPQYLLRYKSDSDKHQGSGVPSRFSGSKDPSANAGILLISGLQSEDEADYYCAIWYSSTWVFGGGTQLTVL

[0281] (SEQ ID NO:74)

[0282] The formulations disclosed herein can be used with antibodies and antigen-binding fragments thereof comprising SEQ ID NO: 11 or 74, wherein residue 1 of SEQ ID NO: 11 or 74 is E instead of Q.

[0283] 2A6 light chain CDR

[0284] CDR-L1:TLRSGINVDTYRIH(SEQ ID NO:25)

[0285] CDR-L2:YKSDSDKHQGS(SEQ ID NO:26)

[0286] CDR-L3:AIWYSSTWV(SEQ ID NO:27)

[0287] including 2A6 heavy and light chain CDRs or 2A6 V H and V L Antibodies and antigen-binding fragments thereof that are derived from or contain the 2A6 heavy and light chains (or variants thereof, e.g., as described herein) may be referred to as "2A6."

[0288] Antibody 3G7(Q1E) Heavy Chain(IgG4)

[0289] Heavy chain

[0290]

[0291] (SEQ ID NO: 12; variable domain is underlined; CDRs are double underlined).

[0292] Heavy chain variable domain

[0293] EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGEWIQLWSPFDYWGQGTLVTVSS

[0294] (SEQ ID NO:67)

[0295] The formulations disclosed herein can be used 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 instead of E.

[0296] 3G7 heavy chain CDR

[0297] CDR-H1: SYAMH (SEQ ID NO: 28)

[0298] CDR-H2:VISYDGSNKYYADSVKG(SEQ ID NO:29)

[0299] CDR-H3:VGEWIQLWSPFDY(SEQ ID NO:30)

[0300] Antibody 3G7 Light Chain (κ)

[0301] Light chain

[0302]

[0303] (SEQ ID NO: 13; variable domain is underlined; CDR is double underlined)

[0304] Light chain variable domain

[0305] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKFLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK

[0306] (SEQ ID NO:75)

[0307] 3G7 light chain CDR

[0308] CDR-L1:RASQGISSWLA(SEQ ID NO:31)

[0309] CDR-L2:AASSLQS(SEQ ID NO:32)

[0310] CDR-L3: QQYNSYPPT(SEQ ID NO:33)

[0311] Includes 3G7 heavy and light chain CDRs or 3G7 V H and V L Antibodies and antigen-binding fragments thereof that are derived from or containing the 3G7 heavy and light chains (or variants thereof, e.g., as described herein) may be referred to as "3G7."

[0312] Antibody 2C1(Q1E) Heavy Chain(IgG4)

[0313] Heavy chain

[0314] (SEQ ID NO: 14; variable domain is underlined; CDR is double underlined)

[0315] Heavy chain variable domain

[0316] EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARAGPLYTIFGVVIIPDNWFDPWGQGTLVTVSS

[0317] (SEQ ID NO:68)

[0318] The formulations disclosed herein can be used 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.

[0319] 2C1 Heavy Chain CDR

[0320] CDR-H1: ELSMH (SEQ ID NO: 34)

[0321] CDR-H2:GFDPEDGETIYAQKFQG(SEQ ID NO:35)

[0322] CDR-H3:AGPLYTIFGVVIIPDNWFDP(SEQ ID NO:36)

[0323] Antibody 2C1 Light Chain (Q1E) (λ)

[0324] Light chain

[0325]

[0326] (SEQ ID NO: 15; variable domain is underlined; CDR is double underlined)

[0327] Light chain variable domain

[0328] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKTSASLAITGLQAEDEADYYCQSYDSSLSGSGVVFGGGTQLIIL

[0329] (SEQ ID NO:76)

[0330] The formulations disclosed herein can be used 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.

[0331] 2C1 light chain CDR

[0332] CDR-L1:TGSSSNIGAGYDVH(SEQ ID NO:37)

[0333] CDR-L2:GNSNRPS(SEQ ID NO:38)

[0334] CDR-L3: QSYDSSLSGSGVV(SEQ ID NO:39)

[0335] Includes 2C1 heavy and light chain CDRs or 2C1 V H and V L Antibodies and antigen-binding fragments thereof comprising or comprising the 2C1 heavy and light chains (or variants thereof, e.g., as described herein) may be referred to as "2C1."

[0336] In various embodiments of the antibody or antigen-binding fragment thereof, the C-terminal lysine of the heavy chain immunoglobulin is absent.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] 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:6.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 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:10 or 84 and a light chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:11.

[0347] 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.

[0348] 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:14 or 86 and a light chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO:15.

[0349] 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 shown 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 shown in SEQ ID NO:63, 57, 64, 65, 66, 67, 68 or 69.

[0350] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO:63 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:70.

[0351] 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:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:71.

[0352] 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:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:72.

[0353] 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:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:73.

[0354] 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:57 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:58.

[0355] In certain embodiments, the 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:70.

[0356] 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.

[0357] 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.

[0358] 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:66 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:74.

[0359] 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.

[0360] 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:68 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO:76.

[0361] In a further embodiment, the antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L L1, CDR-L2, and CDR-L3 of 1E1 (eg, SEQ ID NOs: 19-21). L ) domain; and an immunoglobulin heavy chain variable (V H ) domain.

[0362] In a further embodiment, the antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L L1, CDR-L2, and CDR-L3) comprising 2A6 (e.g., SEQ ID NOs: 25-27). L ) domain; and an immunoglobulin heavy chain variable (V H ) domain.

[0363] In a further embodiment, the antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L L1, CDR-L2, and CDR-L3 of 3G7 (e.g., SEQ ID NOs: 31-33). L) domain; and an immunoglobulin heavy chain variable (V H ) domain.

[0364] In a further embodiment, the antibody or antigen-binding fragment thereof that binds to ILT4 comprises an immunoglobulin light chain variable (V L L1, CDR-L2, and CDR-L3 of 2C1 (e.g., SEQ ID NOs: 37-39). L ) domain; and an immunoglobulin heavy chain variable (V H ) domain.

[0365] In one embodiment, the antibody or antigen-binding fragment comprises a V comprising: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS (SEQ ID NO: 49) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0366] In another embodiment, the antibody or antigen-binding fragment comprises a V comprising 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS (SEQ ID NO: 50) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0367] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0368] In another embodiment, the antibody or antigen-binding fragment thereof comprises 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS (SEQ ID NO: 52) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0369] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS (SEQ ID NO: 53) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0370] In another embodiment, the antibody or antigen-binding fragment thereof comprises V comprising 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 VL Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS (SEQ ID NO: 54) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0371] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0372] In another embodiment, the antibody or antigen-binding fragment thereof comprises 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS (SEQ ID NO: 56) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0373] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNSNRPS (SEQ ID NO: 49) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0374] In another embodiment, the antibody or antigen-binding fragment thereof comprises V comprising 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQSNRPS (SEQ ID NO: 50) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0375] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GESNRPS (SEQ ID NO: 51) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0376] In another embodiment, the antibody or antigen-binding fragment thereof comprises 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDSNRPS (SEQ ID NO: 52) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0377] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising: Hdomain: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GNANRPS (SEQ ID NO: 53) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0378] In another embodiment, the antibody or antigen-binding fragment thereof comprises V comprising 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GQANRPS (SEQ ID NO: 54) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0379] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises a V comprising: 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 L Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GEANRPS (SEQ ID NO: 55) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0380] In another embodiment, the antibody or antigen-binding fragment thereof comprises 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 VL Domains: CDR-L1: TGSSSNIGAGYDVH (SEQ ID NO: 19), CDR-L2: GDANRPS (SEQ ID NO: 56) and CDR-L3: QSFDNSLSAYV (SEQ ID NO: 21).

[0381] The formulations disclosed herein can be used together with an antibody or antigen-binding fragment thereof that binds to ILT4, wherein the antibody or antigen-binding fragment thereof comprises a V L domain (e.g., SEQ ID NO: 70, 71, 72, 73, 58 or 77) and / or V domain of antibody 1E1 H domain (e.g., SEQ ID NO: 63, 57 or 69).

[0382] The formulations disclosed herein can be used with an antibody or antigen-binding fragment thereof that binds to ILT4, wherein the antibody or antigen-binding fragment thereof comprises a V L domain (e.g., SEQ ID NO: 74) and / or V H domain (e.g., SEQ ID NO: 64, 65 or 66).

[0383] The formulations disclosed herein can be used with an antibody or antigen-binding fragment thereof that binds to ILT4, wherein the antibody or antigen-binding fragment thereof comprises a V L domain (e.g., SEQ ID NO: 75) and / or the V domain of antibody 3G7 H domain (e.g., SEQ ID NO:67).

[0384] The formulations disclosed herein can be used together with an antibody or antigen-binding fragment thereof that binds to ILT4, wherein the antibody or antigen-binding fragment thereof comprises a V L domain (e.g., SEQ ID NO: 76) and / or V domain of antibody 2C1 H domain (e.g., SEQ ID NO:68).

[0385] The formulations disclosed herein can be used with an antibody or antigen-binding fragment thereof that binds to ILT4, 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).

[0386] The formulations disclosed herein can be used with an antibody or antigen-binding fragment thereof that binds to ILT4, 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).

[0387] The formulations disclosed herein can be used with an antibody or antigen-binding fragment thereof that binds to ILT4, 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).

[0388] The formulations disclosed herein can be used with an antibody or antigen-binding fragment thereof that binds to ILT4, 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).

[0389] The formulations disclosed herein can be used with antibodies consisting of two heavy chains and two light chains, wherein each light chain comprises a V sequence of antibody 1E1, 2A6, 3G7 or 2C1. L or light chain immunoglobulins, and each heavy chain comprises a V H or heavy chain immunoglobulins.

[0390] 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.

[0391] 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.

[0392] 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.

[0393] 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, and the heavy chain further comprises the amino acid sequence shown in SEQ ID NO:89.

[0394] 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.

[0395] 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.

[0396] In one embodiment, the antibody or antigen-binding fragment thereof comprises V L (with or without a signal sequence), for example, V in any one of SEQ ID NOs: 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), for example, V in any one of SEQ ID NOs: 57 or 63-69. H , which have 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.

[0397] The formulations disclosed herein can be used with polypeptides comprising an amino acid sequence disclosed herein, e.g., SEQ ID NOs: 1-39, 44, 45, 47-58, 63-77, or 79-86, as well as polypeptides comprising such amino acid sequences having 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.

[0398] 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.

[0399] 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 to 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 to the amino acid sequence shown in SEQ ID NO:63, 57, 64, 65, 66, 67, 68 or 69.

[0400] 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, e.g., 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, e.g., comprising the amino acid sequence MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 42).

[0401] In one embodiment, the N-terminal glutamine (Q) of the immunoglobulin chain (e.g., heavy chain and / or light chain) described herein is replaced by pyroglutamic acid. In one embodiment, the N-terminal Q of the heavy chain immunoglobulin is replaced by pyroglutamic acid. In another embodiment, the N-terminal Q of the light chain immunoglobulin is replaced by 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 by pyroglutamic acid.

[0402] The formulations disclosed herein can also be used with antibodies or antigen-binding fragments that bind to the same ILT4 (e.g., human ILT4) epitope as any anti-ILT4 antibody or antigen-binding fragment 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 yet 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 comprising the heavy and light chain amino acid sequences shown below, respectively: SEQ ID NOs: 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 comprising the heavy chain variable domain and light chain variable domain amino acid sequences shown in the following, respectively: SEQ ID NOs: 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.

[0403] The formulations disclosed herein can be used with antibodies and antigen-binding fragments that cross-block any anti-ILT4 antibody or antigen-binding fragment thereof disclosed herein (e.g., 1E1, 2A6, 3G7 or 2C1) from binding to ILT4 (e.g., human ILT4), or compete with any anti-ILT4 antibody or antigen-binding fragment thereof disclosed herein (e.g., 1E1, 2A6, 3G7 or 2C1) for ILT4 (e.g., human ILT4). The cross-blocking antibodies and antigen-binding fragments thereof discussed herein can be identified based on their ability to block binding to ILT4 of any antibody or fragment specifically described herein in a binding assay (e.g., biomembrane interferometry (BLI; e.g., FORTEBIO OCTET binding assay; PallForteBio 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 acts as a biosensor, wherein the binding of an anti-ILT4 antibody or antigen binding fragment to ILT4 changes the interference pattern of white light reflected from the probe layer and the internal reference layer bound to ILT4. The offset indicates ILT4 / anti-ILT4 binding. In one embodiment of the invention, the tip coated with ILT4 is immersed in an analyte solution containing antibodies or antigen binding fragments, for example, 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 produce an orbital flow. In order to read the assay, white light is guided along the length of the fiber. As mentioned above, the interference between the light reflected from the reference layer and the fixed surface containing ILT4 at the tip produces a unique light pattern returning to the fiber. When a molecule binds to a fixed sensor surface, the pattern changes in proportion to the degree of binding. For example, such an assay can be used, wherein ILT4 (e.g., human ILT4) protein is fixed to a BLI probe or plate, a reference anti-ILT4 antibody or fragment is combined with ILT4 (e.g., at a saturating concentration), and a test anti-ILT4 antibody or fragment is added. Then determine the ability of the test antibody to compete with the reference antibody for ILT4 binding. In the BLI format, the light interference of the ILT4 complex is monitored to determine whether the test antibody effectively competes with the reference antibody, for example, monitoring the nanometers of the light wavelength shift over time, wherein the shift indicates the lack of additional binding and cross-blocking of 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 qualitatively considered to have occurred between antibodies. In one embodiment of the invention, as a control, confirm the cross-blocking of the reference antibody with itself; wherein if the reference antibody can cross-block itself and the binding of ILT4, the assay is determined to be correctly operated.The ability of the test antibody to inhibit the binding of the 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 therefore, 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 present invention also form part of the present invention. In one embodiment of the present invention, BLI is performed in a sandwich format, wherein a reference anti-ILT4 antibody or antigen-binding fragment is fixed to 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.

[0404] In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding to human ILT4 with an antibody or fragment comprising the heavy and light chain amino acid sequences shown in SEQ ID NOs: 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 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 below, respectively: SEQ ID NOs: 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.

[0405] The formulations disclosed herein can be used with anti-ILT4 antibodies and antigen-binding fragments thereof comprising N-linked glycans, which are typically added to immunoglobulins produced in Chinese hamster ovary cells (CHON-linked glycans) or modified yeast cells (modified yeast N-linked glycans) (such as, for example, Pichia pastoris). For example, in one embodiment, the anti-ILT4 antibodies and antigen-binding fragments thereof comprise one or more "modified 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 antibodies and antigen-binding fragments thereof comprise modified yeast N-linked glycans, i.e., G0 and / or G1 and / or G2, and optionally, further include Man5. In one embodiment, the anti-ILT4 antibody and its antigen-binding fragments comprise CHO N-linked glycans, i.e., G0-F, G1-F and G2-F, optionally, further comprising 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 N-linked glycans on the anti-ILT4 antibody and its antigen-binding fragments 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 modified yeast cell is GFI5.0 or YGLY8316 or a strain described in U.S. Pat. No. 7,795,002 or Zha et al. Methods Mol Biol. 988:31-43 (2013). See also International Patent Application Publication No. WO2013 / 066765.

[0406] Anti-ILT4 antibodies and antigen-binding fragments thereof that can be used with the various formulations disclosed herein (e.g., 1E1, 2A6, 3G7, and / or 2C1) can also be modified to include modifications within 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, antibodies and antigen-binding fragments thereof that can be used 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 antibody-drug conjugate) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody.

[0407] PD-1 antibodies and their antigen-binding fragments

[0408] In one aspect, the present invention provides a stable biologic comprising an anti-ILT4 antibody or antigen-binding fragment thereof co-formulated with an anti-human PD-1 antibody or antigen-binding fragment thereof that specifically binds to human PD-1 (e.g., a human or humanized anti-PD-1 antibody) as an active pharmaceutical ingredient (PD-1 API), and methods for using the formulations of the present invention. Any anti-PD-1 antibody or antigen-binding fragment thereof can be used in the co-formulations and methods of the present invention. In a particular embodiment, the PD-1 API is an anti-PD-1 antibody selected from pembrolizumab and nivolumab. In a specific embodiment, the anti-PD-1 antibody is pembrolizumab. In an alternative embodiment, the anti-PD-1 antibody is nivolumab. Table 3 provides amino acid sequences for exemplary anti-human PD-1 antibodies pembrolizumab and nivolumab. Alternative PD-1 antibodies and antigen-binding fragments that can be used in the co-formulations and methods of the present invention are shown in Table 4.

[0409] In some embodiments, the anti-human PD-1 antibody or antigen-binding fragment thereof used in the co-formulation of the present invention comprises the three light chain CDRs of CDRL1, CDRL2 and CDRL3 and / or the three heavy chain CDRs of CDRH1, CDRH2 and CDRH3.

[0410] In one embodiment of the invention, CDRL1 is SEQ ID NO:95 or a variant of SEQ ID NO:95, CDRL2 is SEQ ID NO:96 or a variant of SEQ ID NO:96, and CDRL3 is SEQ ID NO:97 or a variant of SEQ ID NO:97.

[0411] In one embodiment, CDRH1 is SEQ ID NO: 100 or a variant of SEQ ID NO: 100, CDRH2 is SEQ ID NO: 101 or a variant of SEQ ID NO: 101, and CDRH3 is SEQ ID NO: 102 or a variant of SEQ ID NO: 102.

[0412] In one embodiment, the three light chain CDRs are SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97, and the three heavy chain CDRs are SEQ ID NO:100, SEQ ID NO:101, and SEQ ID NO:102.

[0413] In an alternative embodiment of the invention, CDRL1 is SEQ ID NO: 105 or a variant of SEQ ID NO: 105, CDRL2 is SEQ ID NO: 106 or a variant of SEQ ID NO: 106, and CDRL3 is SEQ ID NO: 107 or a variant of SEQ ID NO: 107.

[0414] In one embodiment, CDRH1 is SEQ ID NO: 110 or a variant of SEQ ID NO: 110, CDRH2 is SEQ ID NO: 111 or a variant of SEQ ID NO: 111, and CDRH3 is SEQ ID NO: 11128 or a variant of SEQ ID NO:112.

[0415] In one embodiment, the three light chain CDRs are SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97, and the three heavy chain CDRs are SEQ ID NO:100, SEQ ID NO:101, and SEQ ID NO:102.

[0416] In an alternative embodiment, the three light chain CDRs are SEQ ID NO:105, SEQ ID NO:106, and SEQ ID NO:107, and the three heavy chain CDRs are SEQ ID NO:110, SEQ ID NO:111, and SEQ ID NO:112.

[0417] In a further embodiment of the invention, CDRL1 is SEQ ID NO: 115 or a variant of SEQ ID NO: 115, CDRL2 is SEQ ID NO: 116 or a variant of SEQ ID NO: 116, and CDRL3 is SEQ ID NO: 117 or a variant of SEQ ID NO: 117.

[0418] In yet another embodiment, CDRH1 is SEQ ID NO: 118 or a variant of SEQ ID NO: 118, CDRH2 is SEQ ID NO: 119 or a variant of SEQ ID NO: 119, and CDRH3 is SEQ ID NO: 120 or a variant of SEQ ID NO: 120.

[0419] In another embodiment, the three light chain CDRs are SEQ ID NO:115, SEQ ID NO:116, and SEQ ID NO:117, and the three heavy chain CDRs are SEQ ID NO:118, SEQ ID NO:119, and SEQ ID NO:120.

[0420] Some anti-human PD-1 antibodies and antigen-binding fragments of the present invention comprise a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region comprises SEQ ID NO: 98 or a variant of SEQ ID NO: 98, and the heavy chain variable region comprises SEQ ID NO: 103 or a variant of SEQ ID NO: 103. In further embodiments, the light chain variable region comprises SEQ ID NO: 108 or a variant of SEQ ID NO: 108, and the heavy chain variable region comprises SEQ ID NO: 113 or a variant of SEQ ID NO: 113. In further embodiments, the heavy chain variable region comprises SEQ ID NO: 121 or a variant of SEQ ID NO: 121, and the light chain variable region comprises SEQ ID NO: 122 or a variant of SEQ ID NO: 122, SEQ ID NO: 123 or a variant of SEQ ID NO: 123, or SEQ ID NO: 124 or a variant of SEQ ID NO: 124. In such embodiments, the variant light chain or heavy chain variable region sequence is identical to the reference sequence except having one, two, three, four or five amino acid substitutions. In some embodiments, the substitutions are located in the framework regions (i.e., outside the CDRs). In some embodiments, one, two, three, four or five amino acid substitutions are conservative substitutions.

[0421] In one embodiment of the co-formulation of the present invention, the anti-human PD-1 antibody or antigen-binding fragment comprises: a light chain variable region comprising or consisting of SEQ ID NO: 98 and a heavy chain variable region comprising or consisting of SEQ ID NO: 103. In a further embodiment, the anti-human PD-1 antibody or antigen-binding fragment comprises: a light chain variable region comprising or consisting of SEQ ID NO: 108 and a heavy chain variable region comprising or consisting of SEQ ID NO: 113. In one embodiment of the formulation of the present invention, the anti-human PD-1 antibody or antigen-binding fragment comprises: a light chain variable region comprising or consisting of SEQ ID NO: 122 and a heavy chain variable region comprising or consisting of SEQ ID NO: 121. In a further embodiment, the anti-human PD-1 antibody or antigen-binding fragment comprises: a light chain variable region comprising or consisting of SEQ ID NO: 123 and a heavy chain variable region comprising or consisting of SEQ ID NO: 121. In another embodiment, the antibody or antigen-binding fragment comprises: a light chain variable region comprising or consisting of SEQ ID NO: 124 and a heavy chain variable region comprising or consisting of SEQ ID NO: 121.

[0422] In another embodiment, the co-formulation of the present invention comprises an anti-human PD-1 antibody or antigen-binding protein having a VL domain and / or a VH domain having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 50% sequence homology with one of the above-mentioned VL domains or VH domains, and exhibits specific binding to PD-1. In another embodiment, the anti-human PD-1 antibody or antigen-binding protein of the co-formulation of the present invention comprises a VL and VH domain having up to 1, 2, 3, 4, or 5 or more amino acid substitutions, and exhibits specific binding to PD-1.

[0423] In any of the above embodiments, PD-1API can be a full-length anti-PD-1 antibody or an antigen-binding fragment thereof that specifically binds to human PD-1. In certain embodiments, PD-1API is a full-length anti-PD-1 antibody selected from any type of immunoglobulin, including IgM, IgG, IgD, IgA and IgE. Preferably, the antibody is an IgG antibody. Any IgG isotype can be used, including IgG1, IgG2, IgG3 and IgG4. Different constant domains can be attached to the VL and VH regions provided herein. For example, if the specific intended use of the antibody (or fragment) of the present invention requires a changed effector function, a heavy chain constant domain other than IgG1 can be used. Although IgG1 antibodies provide long half-life and effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, such activities may not be desired for all uses of antibodies. In such cases, for example, an IgG4 constant domain can be used.

[0424] In an embodiment of the present invention, PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 99, and the heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 104. In an alternative embodiment, PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 109, and the heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 114. In a further embodiment, PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 126, and the heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 31251. In another embodiment, the PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 127, and the heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 125. In yet another embodiment, the PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 128, and the heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 125. In some co-formulations of the present invention, the PD-1 API is pembrolizumab or a pembrolizumab biosimilar. In some co-formulations of the present invention, the PD-1 API is nivolumab or a nivolumab biosimilar.

[0425] Typically, the amino acid sequence variants of the anti-PD-1 antibodies and antigen-binding fragments and anti-ILT4 antibodies and antigen-binding fragments of the present invention will have an amino acid sequence with at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95, 98 or 99% amino acid sequence identity to the amino acid sequence of the reference antibody or antigen-binding fragment (e.g., heavy chain, light chain, VH, VL or humanized sequence). The identity or homology of the sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the anti-PD-1 residues after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity, and any conservative substitutions are not considered part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions to the antibody sequence should not be interpreted as affecting sequence identity or homology.

[0426] Sequence identity refers to the degree to which the amino acids in equivalent positions of two polypeptides are identical when the two sequences are optimally aligned. Sequence identity can be determined using the BLAST algorithm, wherein the parameters of the algorithm are selected to give the largest match between related sequences relative to the full length of the corresponding reference sequence. The following references relate to BLAST algorithms that are frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, SF, et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al., (1993) Nature Genet. 3: 266-272; Madden, TL, et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF, et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, JC, et al., (1993) Comput. Chem. 17: 149-163; Hancock, JM, et al., (1994) Comput. Appl. Biosci. 10: 67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, MO, et al., "A model of evolutionary change in proteins." in Atlas ofProtein Sequence and Structure, (1978) vol.5, suppl.3.MO Dayhoff (ed.), pp.345-352, Natl.Biomed.Res.Found., Washington, DC; Schwartz, RM, et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. MO Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, (1991) J. Mol. Biol. 219: 555-565; States, DJ, et al., (1991) Methods 3: 66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, SF, et al., (1993) J.Mol.Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.Natl.Acad.Sci.USA 87:2264-2268; Karlin, S., et al., (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, SF "Evaluating the statistical significance of multipledistinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York. .

[0427] Likewise, any type of light chain can be used in the compositions and methods herein. Specifically, kappa, lambda, or variants thereof can be used in the compositions and methods herein.

[0428] The formulation further comprises an anti-PD-1 antibody or antigen-binding fragment as exemplified below.

[0429] Table 3. Exemplary PD-1 antibody sequences

[0430]

[0431]

[0432]

[0433] Table 4. Additional anti-PD-1 antibodies and antigen-binding fragments useful in the formulations, methods and uses of the invention.

[0434]

[0435]

[0436] As used herein, "pembrolizumab variant" means a monoclonal antibody comprising heavy and light chain sequences that are substantially the same as those in pembrolizumab, except having three, two or one conservative amino acid substitutions at positions located outside the light chain CDRs and six, five, four, three, two or one conservative amino acid substitutions located outside the heavy chain CDRs, e.g., the variant positions are located in the FR region or the constant region, and optionally having a deletion of the C-terminal lysine residue of the heavy chain. In other words, pembrolizumab and pembrolizumab variants comprise the same CDR sequences, but differ from each other by having conservative amino acid substitutions at no more than three or six other positions in their full-length light and heavy chain sequences, respectively. Pembrolizumab variants are substantially the same as pembrolizumab in terms of the following properties: binding affinity to PD-1 and the ability to block the binding of PD-L1 and PD-L2 to PD-1, respectively.

[0437] In another aspect of the invention, the formulation comprises an anti-ILT4 antibody or antigen-binding fragment comprising 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); and an anti-PD-1 antibody or antigen-binding fragment comprising a light chain variable region sequence of SEQ ID NO: 98 and a heavy chain variable region sequence of SEQ ID NO: 103. In another embodiment, the formulation comprises an anti-ILT4 antibody, 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), and an anti-PD-1 antibody comprising a light chain sequence of SEQ ID NO: 5 and a heavy chain sequence of SEQ ID NO: 104. The present invention also provides preparations of anti-ILT4 antibodies or antigen-binding fragments thereof, whose heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 57, and whose light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 58; and anti-PD-1 antibodies, which comprise the light chain CDRL1 sequence of SEQ ID NO: 95, the CDRL2 sequence of SEQ ID NO: 96, and the CDRL3 sequence of SEQ ID NO: 97, and the heavy chain CDRH1 sequence of SEQ ID NO: 100, the CDRH2 sequence of SEQ ID NO: 101, and the CDRH3 sequence of SEQ ID NO: 102. In some preparations of the present invention, the anti-ILT4 antibody 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.In some embodiments, 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.

[0438] In a further aspect of the present invention, the preparation comprises: an anti-ILT4 antibody or an antigen-binding fragment thereof, which 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, and an anti-PD-1 antibody or antigen-binding fragment thereof, which comprises a light chain variable region sequence of SEQ ID NO:108 and a heavy chain variable region sequence of SEQ ID NO:113. The present invention also provides a preparation having an anti-ILT4 antibody or antigen-binding fragment and an anti-PD-1 antibody or antigen-binding fragment, wherein the anti-ILT4 antibody or antigen-binding fragment 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, and the anti-PD-1 antibody or antigen-binding fragment comprises a light chain variable region CDRL1 sequence of SEQ ID NO:105, a CDRL2 sequence of SEQ ID NO:106, and a CDRL3 sequence of SEQ ID NO:107, and a heavy chain variable region CDRH1 sequence of SEQ ID NO:110, a CDRH2 sequence of SEQ ID NO:111, and a CDRH3 sequence of SEQ ID NO:112.

[0439] The antibody or antigen-binding fragment of the preparation may include a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region includes SEQ ID NO: 98 or a variant of SEQ ID NO: 98, and the heavy chain variable region includes SEQ ID NO: 103 or a variant of SEQ ID NO: 103. In a further embodiment, the light chain variable region includes SEQ ID NO: 108 or a variant of SEQ ID NO: 108, and the heavy chain variable region includes SEQ ID NO: 113 or a variant of SEQ ID NO: 113. In a further embodiment, the heavy chain variable region includes SEQ ID NO: 121 or a variant of SEQ ID NO: 121, and the light chain variable region includes SEQ ID NO: 122 or a variant of SEQ ID NO: 1228, SEQ ID NO: 123 or a variant of SEQ ID NO: 123, or SEQ ID NO: 124 or a variant of SEQ ID NO: 124. In such embodiments, the variant light or heavy chain variable region sequence is identical to the reference sequence, except that it has one, two, three, four or five amino acid substitutions. In some embodiments, the substitutions are in the framework regions (ie, outside of the CDRs). In some embodiments, one, two, three, four, or five amino acid substitutions are conservative substitutions.

[0440] In another embodiment, the formulation of the present invention comprises an antibody or antigen-binding fragment having the same L Domain or V H One of the domains has at least 95%, 90%, 85%, 80%, 75% or 50% sequence homology to the V L Domain and / or V H domain, and exhibits specific binding to PD-1 or ILT4. In another embodiment, the antibody or antigen-binding fragment of the formulation of the present invention comprises a V having up to 1, 2, 3, 4 or 5 or more amino acid substitutions. L and V H domain, and exhibited specific binding to PD-1 or ILT4.

[0441] In an embodiment of the present invention, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 99, and a heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 104. In an alternative embodiment, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 109, and a heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 114. In a further embodiment, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 126, and a heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 125. In additional embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 127, and a heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 125. In yet further embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 128, and a heavy chain comprising or consisting of a sequence of amino acid residues as shown in SEQ ID NO: 125. In some formulations of the invention, the anti-PD-1 antibody is pembrolizumab or a pembrolizumab variant.

[0442] Typically, the amino acid sequence variants of the anti-PD-1 or anti-ILT4 antibodies and antigen-binding fragments of the invention will have amino acid sequences similar to those of the reference antibody or antigen-binding fragment (e.g., heavy chain, light chain, V H 、V L , framework or humanized sequence) has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95, 98 or 99% amino acid sequence identity. The identity or homology of the sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the anti-PD-1 or anti-ILT4 residues after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity, and any conservative substitutions are not considered part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions of the antibody sequence should not be interpreted as affecting sequence identity or homology.

[0443] In one embodiment, the ratio of anti-ILT4 antibody to anti-PD-1 antibody in the formulation is 1: 1, 1: 2 or 1: 3. In another embodiment, the molar ratio of anti-ILT4 antibody to anti-PD-1 antibody in the formulation is 1: 1, 2: 1, 3: 1, 3.5: 1, 4: 1, 5: 1 or 6: 1. In one embodiment, the molar ratio of anti-ILT4 antibody to anti-PD-1 antibody in the formulation is 4: 1. In another embodiment, the molar ratio of anti-ILT4 antibody to anti-PD-1 antibody in the formulation is 5: 1.

[0444] preparation

[0445] The co-formulations described herein can maintain the physical, chemical and / or biological stability of an anti-ILT4 antibody (e.g., 1E1, 2A6, 3G7 or 2C1) or an antigen-binding fragment thereof during storage (e.g., at least 6 months, 12 months, 24 months or 36 months at about 5°C) and / or under various stress conditions (e.g., agitation, freeze-thaw cycles or light exposure).

[0446] In one aspect, provided herein are various co-formulations of an anti-ILT4 antibody (e.g., 1E1, 2A6, 3G7, or 2C1) or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, comprising (i) an anti-ILT4 antibody or an antigen-binding fragment thereof (e.g., 1E1, 2A6, 3G7, or 2C1); (ii) an anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) a buffer (e.g., L-histidine buffer or acetate buffer); (iv) a non-reducing sugar (e.g., sucrose); (v) a non-ionic surfactant (e.g., PS-80); and (vi) an antioxidant (e.g., L-methionine).

[0447] In some aspects of the invention, the formulation of the present invention minimizes the formation of antibody aggregates (high molecular weight species) and microparticles, improves colloidal stability, minimizes fragmentation (low molecular weight species), or ensures that the antibody maintains its biological activity over time. In one aspect, the formulation comprises: about 3-300 mg / mL of anti-ILT4 antibody or its antigen-binding fragment and about 3-300 mg / mL of anti-PD-1 antibody or its antigen-binding fragment at a molar ratio of 4:1 to 5:1 (anti-ILT4 antibody to anti-PD-1 antibody or its antigen-binding fragment), a total excipient concentration of about 10-1000 mM selected from histidine, aspartic acid, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2 and FeCl2 One or more excipients, and a buffer having a pH of about 5-8. In one embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a molar ratio of 4:1 (anti-ILT4 antibody to anti-PD-1 antibody or its antigen-binding fragment). In another embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a molar ratio of 5:1 (anti-ILT4 antibody to anti-PD-1 antibody or its antigen-binding fragment). In another aspect, the formulation comprises: about 4-200 mg / mL of anti-ILT4 antibody or its antigen-binding fragment and about 4-200 mg / ml of anti-PD-1 antibody or its antigen-binding fragment. In one embodiment, one or more excipients selected from histidine, aspartic acid, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2 and FeCl2 are at a total excipient concentration of about 25-250 mM. In another embodiment, one or more excipients selected from histidine, aspartic acid, glutamine, glycine, proline, methionine, arginine or pharmaceutically acceptable salts thereof, NaCl, KCl, LiCl, CaCl2, MgCl2, ZnCl2 and FeCl2 are at a total excipient concentration of about 40-250 mM.

[0448] In one aspect, the excipient is arginine or its pharmaceutically acceptable salt at a concentration of about 15-250mM. In one aspect, the excipient is arginine or its pharmaceutically acceptable salt at a concentration of about 25-250mM. In another embodiment, the excipient is arginine or its pharmaceutically acceptable salt at a concentration of about 40-150mM. In another embodiment, the excipient is arginine or its pharmaceutically acceptable salt at a concentration of about 40-100mM. In another embodiment, the excipient is L-arginine or its pharmaceutically acceptable salt at a concentration of about 70mM. In another embodiment, the excipient is arginine or its pharmaceutically acceptable salt at a concentration of about 70-150mM. The example of a pharmaceutically acceptable salt of arginine (L or D form) includes but is not limited to L-arginine hydrochloride and L-arginine succinate. In other aspects of the aforementioned embodiments, the preparation further comprises a nonionic surfactant, a sugar or a polyol, or glutamine, glycine, proline or methionine.

[0449] In another aspect, the excipient is NaCl and arginine or a pharmaceutically acceptable salt thereof, with a total excipient concentration of about 25-250mM. In a further embodiment, the excipient is NaCl and arginine or a pharmaceutically acceptable salt thereof, with a total excipient concentration of about 70-100mM. In one embodiment, the concentration ratio of NaCl to arginine is 1:1. In another embodiment, the NaCl concentration is about 35mM, and the arginine concentration is about 35mM. In another embodiment, the NaCl concentration is about 50mM, and the arginine concentration is about 50mM.

[0450] In a further aspect, the excipient is NaCl, KCl or LiCl with about 40-150mM. In a further embodiment, the excipient is NaCl, KCl or LiCl with about 40-100mM. In a further embodiment, the excipient is NaCl, KCl or LiCl with about 70-130mM. In a further embodiment, the excipient is NaCl, KCl or LiCl with about 70-100mM. In a further embodiment, the excipient is NaCl with about 70mM. In other aspects of the aforementioned embodiments, the preparation further comprises a nonionic surfactant.

[0451] In a further aspect, the excipient is L-histidine at about 25-200 mM. In a further embodiment, the L-histidine is about 50-200 mM. In yet a further embodiment, the L-histidine is about 40-100 mM.

[0452] In a further aspect, the excipient is L-glutamine, L-glycine, L-proline or L-methionine or a combination thereof at about 25-200 mM. In a further embodiment, the excipient is about 50-200 mM. In yet a further embodiment, the excipient is about 40-100 mM. In yet a further embodiment, the excipient is about 70 mM.

[0453] In one embodiment, the excipient is about 25-200mM L-glutamine, L-glycine, L-aspartic acid or a combination thereof. In another embodiment, the excipient is about 20-50mM. In a further embodiment, the excipient is about 20mM. In another further embodiment, the excipient is about 40-100mM. In another further embodiment, the excipient is about 70mM. In another embodiment, the excipient is about 20mM L-aspartic acid and about 50mM L-glycine. In another embodiment, the excipient is about 20mM L-glutamine and about 50mM L-glycine.

[0454] In one embodiment, the co-formulated composition has a buffer having a pH of neutral or weakly acidic (pH 4.5-8), and arginine or a pharmaceutically acceptable salt thereof. In one embodiment, a buffer having a pH of about 5.5-6.5 is used in the composition. In one embodiment, a buffer having a pH of about 4.5-6.5 is used in the composition. In another embodiment, a buffer having a pH of about 5.5-6.0 is used in the composition. In a further embodiment, a buffer having a pH of about 5.0-6.0 is used in the composition. The buffer may have a concentration of about 5-1000mM. In another embodiment, the buffer may have a concentration of about 5-150mM. In a further embodiment, the buffer may have a concentration of about 5-300mM. In a further embodiment, the buffer may have a concentration of about 1-300mM. In another embodiment, the buffer may have a concentration of about 1-30mM. In another further embodiment, the buffer may have a concentration of about 5-30mM. In another further embodiment, the buffer may have a concentration of about 5-20mM. In yet a further embodiment, the buffer may have a concentration of about 8-12 mM. In one embodiment, the buffer is histidine, acetate or citrate. In some embodiments, the buffer contains about 10 mM histidine, acetate or citrate.

[0455] In one embodiment, the formulation comprises about 3-300 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof and 3-300 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof at a molar ratio of 4:1 to 5:1 (anti-ILT4 antibody to anti-PD-1 antibody or an antigen-binding fragment thereof), a sugar or a polyol; a non-ionic surfactant, a histidine buffer or an acetate buffer at a pH of about 4.5-8, about 10-1000 mM methionine (L or D form) or a pharmaceutically acceptable salt thereof, and optionally arginine, EDTA, DTPA, tryptophan (L or D form) or pyridoxine. In another embodiment, the formulation comprises about 4-250 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof and about 4-250 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof in a molar ratio of 4:1 to 5:1 (anti-ILT4 antibody to anti-PD-1 antibody or an antigen-binding fragment thereof), a sugar or a polyol; a non-ionic surfactant, about 50-500 mM histidine buffer at a pH of about 5-8, about 10-1000 mM monovalent cation salt selected from NaCl, KCl and LiCl or a multivalent cation salt selected from CaCl2, MgCl2, ZnCl2, FeCl2 and FeCl3, optionally about 10-1000 mM methionine (D or L form) or a pharmaceutically acceptable salt thereof, and optionally arginine, EDTA, DTPA, tryptophan and pyridoxine. In another aspect, the formulation comprises: about 4-200 mg / mL of an anti-ITL4 antibody or an antigen-binding fragment thereof and about 4-200 mg / ml of an anti-PD-1 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-ILT4 antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment have a molar ratio of 4:1 (anti-ILT4 antibody to anti-PD-1 antibody or its antigen-binding fragment). In another embodiment, the anti-ILT4 antibody or its antigen-binding fragment and the anti-PD-1 antibody or its antigen-binding fragment have a molar ratio of 5:1 (anti-ILT4 antibody to anti-PD-1 antibody or its antigen-binding fragment).

[0456] The preparation may include about 1-100uM, about 1-30uM, about 1-20uM, about 10uM-30uM DTPA or EDTA. The preparation may also include about 1-30mM L-methionine. In one embodiment, the preparation may also include about 1-20mM L-methionine. The preparation may also include about 5-15mM L-methionine. The preparation may also include about 5-15mM L-methionine. The preparation may also include about 5-20mM L-methionine. The preparation may also include about 10mM or at least about 10mM L-methionine. Sometimes during the production step and / or before the vial is closed, nitrogen overlay (covering, for example, only 5% or 10% residual O2 after nitrogen overlay) is used to stabilize the antibody for oxidation.

[0457] In another aspect of the present invention, the preparation further comprises sugar, polyol or nonionic surfactant or its combination. In one embodiment, sugar is selected from glucose, sucrose, trehalose and lactose or its combination. In one embodiment, sugar is disaccharide, such as sucrose, trehalose and maltose. In one embodiment, sugar is non-reducing sugar. In another embodiment, sugar is non-reducing disaccharide, such as sucrose or trehalose or its combination. In one embodiment, sugar is the concentration of about 10-200mg / ml. In another embodiment, sugar is the concentration of about 30-120mg / ml. In another embodiment, sugar is the concentration of about 30-80mg / ml. In a further embodiment, sugar is the concentration of about 50-90mg / ml.

[0458] In one embodiment, the polyol is selected from mannitol, sorbitol, glycerol and polyethylene glycol. In another embodiment, the polyol is a sugar alcohol. In one embodiment, sugar and polyol are selected from sucrose, trehalose, sorbitol, glycerol and polyethylene glycol. In a further embodiment, the polyol is a diol. In one embodiment, the diol is selected from ethylene glycol, propylene glycol and polyethylene glycol. In one embodiment, the polyol is a concentration of about 10-200mg / ml. In another embodiment, the polyol is a concentration of about 10-50mg / ml. In a further embodiment, the polyol is a concentration of about 5-30mg / ml.

[0459] In one embodiment, the preparation comprises about 10-250mg / ml of sucrose or trehalose. In another embodiment, the preparation comprises about 20-200mg / ml of sucrose or trehalose. In a further embodiment, the preparation comprises about 50-80mg / ml of sucrose or trehalose. In a further embodiment, the preparation comprises about 30-80mg / ml of sucrose or trehalose. In another embodiment, the preparation comprises about 50-90mg / ml of sucrose or trehalose. In another further embodiment, the preparation comprises about 70-80mg / ml of sucrose or trehalose. In another further embodiment, the preparation comprises at least about 50mg / ml of sucrose or trehalose. In another embodiment, the preparation comprises about 20-200mg / ml of sorbitol, PEG400 or glycerol. In a further embodiment, the preparation comprises about 20-50mg / ml of sorbitol, PEG400 or glycerol.

[0460] In one embodiment, the nonionic surfactant is selected from polysorbates and poloxamers. In yet another embodiment, the surfactant is selected from (Polysorbate 80), (Polysorbate 20), Pluoronic In one embodiment, the surfactant is polysorbate 20 or polysorbate 80, and the sugar is sucrose or trehalose. Polysorbate 80 or polysorbate 20 surfactants can be present in the preparation in an amount of about 0.005 to about 1 mg / ml. Polysorbate 80 or polysorbate 20 surfactants can be present in the preparation in an amount of about 0.02 to about 2 mg / ml. Polysorbate 80 or polysorbate 20 surfactants can be present in the preparation in an amount of about 0.05 to about 1 mg / ml. Polysorbate 80 or polysorbate 20 surfactants can be present in the preparation in an amount of about 0.1 to about 0.5 mg / ml. In another embodiment, polysorbate 80 or polysorbate 20 surfactants can be present in the preparation in an amount of about 0.005 mg / ml. Polysorbate 80 or polysorbate 20 surfactants can also be present in the preparation in an amount of about at least about 0.1 mg / ml. Polysorbate 80 surfactant may be present in the formulation in an amount of about 0.2 mg / ml.

[0461] In other aspects of the above formulations, the % high molecular weight (HMW) in the co-formulated anti-ILT4 antibody and anti-PD-1 antibody formulation is less than 5% after 10 days at 50°C as measured by size exclusion chromatography.

[0462] Liquid preparations

[0463] Liquid antibody preparations can be prepared by obtaining a bulk drug such as an aqueous pharmaceutical preparation and exchanging its buffer into a desired buffer as the last step of the purification process. The bulk drug in the final buffer is concentrated to the desired concentration. Excipients such as stabilizers and surfactants are added to the bulk drug, and it is diluted to the final protein concentration using an appropriate buffer. The final formulated bulk drug is filtered using a 0.22 μm filter and filled into a final container (e.g., a glass vial). The preparation can be stored in a vial and delivered by an injection device or vessel.

[0464] In another aspect of the invention, for a liquid co-formulated formulation comprising an anti-PD-1 antibody and an anti-ILT4 antibody (or an antigen-binding fragment thereof), the anti-ILT4 antibody or its antigen-binding fragment has a concentration of about 10-300 mg / ml. In another embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a concentration of about 15-250 mg / ml. In another embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a concentration of about 20-100 mg / ml. In a further embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a concentration of about 30-80 mg / ml. In a further embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a concentration of about 35-75 mg / ml. In a further embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a concentration of about 40-70 mg / mL. In a further embodiment, the anti-ILT4 antibody or its antigen-binding fragment has a concentration of about 45-65 mg / ml. In another embodiment, the anti-ILT4 antibody or its antigen-binding fragment is a concentration of about 45-60 mg / mL. In yet another further embodiment, the anti-ILT4 antibody or its antigen binding fragment is a concentration of about 45-65 or 55 mg / mL. In yet another further embodiment, the anti-ILT4 antibody or its antigen binding fragment is a concentration of about 40-60 mg / mL or 45-55 mg / mL. In yet another further embodiment, the anti-ILT4 antibody or its antigen binding fragment is a concentration of about 46-52 mg / mL. In yet another further embodiment, the anti-ILT4 antibody or its antigen binding fragment is a concentration of about 48-52 mg / mL. In yet another further embodiment, the anti-ILT4 antibody or its antigen binding fragment is a concentration of about 49 mg / mL. In yet another further embodiment, the anti-ILT4 antibody or its antigen binding fragment is a concentration of about 50 mg / mL. In yet another further embodiment, the anti-ILT4 antibody or its antigen binding fragment is a concentration of about 51 mg / mL.

[0465] In another aspect of the invention, the anti-PD-1 antibody or antigen-binding fragment thereof in the liquid formulation has a concentration of about 3-300 mg / ml. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 4-250 mg / ml. In another embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 40-100 mg / ml. In a further embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 10-60 mg / ml. In a further embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 20-30 mg / ml. In a further embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 10-30 mg / mL. In a further embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 15-50 mg / ml. In another embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 10-100 mg / mL. In another embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 20-30 or 25 mg / mL. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 21-29 mg / ml. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is at a concentration of about 22-28 mg / ml. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof has a concentration of about 24 mg / ml. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof has a concentration of about 25 mg / ml. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof has a concentration of about 26 mg / ml.

[0466] In one embodiment, the liquid preparation comprises a buffer having a pH of about 4.5-8, 5.0-6.5, 5.5-6.5, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2 and arginine or a pharmaceutically acceptable salt thereof. In one embodiment, the liquid preparation comprises a buffer having a pH of about 5-8. In one embodiment, the liquid preparation comprises a buffer having a pH of about 5.0-6.5. In one embodiment, the liquid preparation comprises a buffer having a pH of about 5.0-6.0. In other embodiments, the buffer is histidine. In another embodiment, the buffer is citrate or acetate. In a further embodiment, the liquid preparation comprises an acetate buffer having a pH of about 5-8, 5.0-6.5, 5.5-6.5, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2 and arginine or a pharmaceutically acceptable salt thereof.

[0467] The liquid antibody preparation of the present invention is suitable for parenteral administration, such as intravenous, intramuscular, intraperitoneal or subcutaneous injection; particularly suitable for subcutaneous injection.

[0468] Dosage and Administration

[0469] Toxicity is a consideration in selecting the appropriate dose of a therapeutic agent, such as a humanized anti-ILT4 or anti-PD-1 antibody (or its antigen-binding fragment). The toxicity and therapeutic effect of the antibody composition administered alone or in combination with an immunosuppressant can be determined by standard pharmaceutical procedures such as for determining LD50 (a dose lethal to 50% of the population) and ED50 (a dose effective for treatment in 50% of the population) in cell culture or experimental animals. The dose ratio between toxicity and efficacy is the therapeutic index, and it can be expressed as a ratio of LD50 to ED50. Antibodies that exhibit a high therapeutic index are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a circulating concentration range including ED50, with little or no toxicity. The dosage may vary within this range, depending on the dosage form used and the route of administration utilized.

[0470] Suitable routes of administration may, for example, include parenteral delivery, including intramuscular, intradermal, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal. The drug may be administered in various conventional ways, such as intraperitoneal, parenteral, intraarterial or intravenous injection. Wherein the mode of administration (e.g., subcutaneous administration) in which the volume of the solution must be limited requires that the lyophilized formulation be reconstituted at high concentrations.

[0471] Alternatively, the antibody can be administered in a local rather than systemic manner, for example, via injection of the antibody directly into a pathogen-induced lesion characterized by immunopathology, often in a depot or slow-release formulation. In addition, the antibody can be administered in a targeted drug delivery system, for example, in liposomes coated with tissue-specific antibodies that target, for example, pathogen-induced lesions characterized by immunopathology. The liposomes will be targeted to and selectively taken up by the diseased tissue.

[0472] The administration regimen selected for the therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the symptom level, the immunogenicity of the entity, and the accessibility of the target cells in the biomatrix. Preferably, the administration regimen maximizes the amount of the therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Therefore, the amount of the biologic delivered depends in part on the severity of the specific entity and the condition to be treated. Guidance in selecting the appropriate dose of antibodies, cytokines, and small molecules is available. 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 Edition); Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002).

[0473] The determination of the appropriate dosage is made by the clinician, for example, using parameters or factors known in the art or suspected to affect treatment or predicted to affect treatment. The appropriate dosage of the protein ("therapeutically effective amount") will depend on, for example, the condition to be treated, the severity and course of the condition, whether the protein is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the protein, the type of protein used, and the judgment of the attending physician. Typically, the dosage is started with an amount slightly less than the optimal dosage, and thereafter it is increased in small increments until the desired or optimal effect relative to any negative side effects is achieved. Important diagnostic measurements include symptoms, such as those measurements of inflammation or the levels of inflammatory cytokines produced. The protein is appropriately administered to the patient once or repeatedly. The protein can be administered alone or in combination with other drugs or therapies.

[0474] The antibody or antibody fragment can be provided by continuous infusion, or by providing doses at intervals of, for example, one day, 1-7 times per week, one week, two weeks, three weeks, monthly, every two months, etc. A preferred dosage regimen is one involving the maximum dose or dosage frequency that avoids significant undesirable side effects.

[0475] In certain embodiments, the pharmaceutical formulations of the invention will be administered by intravenous (IV) infusion or injection.

[0476] In other embodiments, the pharmaceutical preparations of the invention will be administered subcutaneously. Subcutaneous administration can be performed by using a syringe or other injection device (e.g., devices); injection pens; or needle-free devices (e.g., MediJector and ) injection to perform.

[0477] Subcutaneous administration can be performed by injection using a syringe, an autoinjector, an injection pen, or a needle-free injection device. Intravenous injection can be performed after diluting the formulation with a suitable commercial diluent, such as saline solution or 5% dextrose aqueous solution.

[0478] Although the high concentration solution formulations of the present invention are particularly advantageous for uses requiring high concentrations of antibodies, there is no reason why the formulations cannot be used at lower concentrations in situations where high concentrations are not required or desired. Lower concentrations of antibodies may be useful for low dose subcutaneous administration, or for other modes of administration (e.g., intravenous administration) where the volume that can be delivered is substantially greater than 1 ml. Such lower concentrations may include about 15, 10, 5, 2, 1 mg / ml or less.

[0479] use

[0480] The present invention provides a lyophilized or liquid preparation of an anti-ILT4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof for use in treating cancer and infection.

[0481] Those skilled in the art will recognize that the term "cancer" would be the name of a disease in which the cells of the body become abnormal and divide uncontrollably. Cancers that may be treated by the compounds, compositions and methods of the present invention include, but are not limited to: Heart: sarcomas (angiosarcomas, fibrosarcomas, rhabdomyosarcomas, liposarcoma), myxoma, rhabdomyomas, fibromas, lipomas and teratomas; Lung: bronchial protocarcinomas (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinomas), alveolar (bronchiolar) carcinomas, bronchial adenomas, sarcomas, lymphomas, chondromatous hamartomas, mesotheliomas; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcomas, lymphomas), stomach (carcinoma, lymphoma, leiomyosarcomas), pancreas (ductal adenocarcinomas, insulinomas, glucagonomas, gastrinomas, carcinoid tumors, vipoma), small intestine Colon: adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); Colorectum: adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma; Genitourinary tract: kidney (adenocarcinoma, Wilms' tumor [Nephroblastoma], lymphoma, leukemia); bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma); prostate (adenocarcinoma, sarcoma); testis (seminoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, vascular Sarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteochronfroma) (osteocartilaginous exostosis), benign enchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor; Nervous system: skull (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meninges (meningiomas, meningosarcomas, gliomatosis), brain (astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell Tumors [pinealoma], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibroma, meningioma, glioma, sarcoma); Gynecology: uterus (endometrial cancer), cervix (cervical cancer, preneoplastic cervical dysplasia), ovary (ovarian cancer [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], ovarian granulosa cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube (carcinoma), breast;Hematology: Blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevus dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and Adrenal: neuroblastoma. In one embodiment, the cancer is selected from colorectal cancer, gastric cancer, and head and neck cancer. ;

[0482] 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), etc. 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).

[0483] The buffers of the various formulations described herein have a pH in the range of about 4.5 to about 7.0, about 4.5 to about 6.8, about 5.0 to about 6.8, about 5.0 to about 6.5, 5.0 to about 6.0, about 5.5 to about 6.0. When obtaining exemplary formulations, the suitability of L-histidine and acetate buffers in the pH range of 5.0-6.8 was explored. When describing a range of pH values, such as "a pH of about pH 5.5 to about 6.0", the range is intended to include the values. For example, a range of 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 the case where the anti-ILT4 formulation described herein is a lyophilized formulation, the pH refers to the pH after the lyophilized formulation is reconstituted. The pH is typically measured at 25°C using a standard glass bulb pH meter. As used herein, a solution comprising "a histidine buffer at pH X" refers to a solution at pH X and comprising a histidine buffer, ie, the pH is intended to refer to the pH of the solution.

[0484] In some embodiments, the anti-ILT4 preparation 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 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 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 at about 6% (w / v) to about 8% (w / v). In one embodiment, sucrose is present at about 6% (w / v). In one embodiment, sucrose is present at about 7% (w / v). In one embodiment, sucrose is present at about 8% (w / v).

[0485] In certain embodiments, the anti-ILT4 formulation further comprises a surfactant. As used herein, a surfactant is a surface active agent that is amphipathic in nature. A surfactant can be added to the formulation herein to provide stability, reduce and / or prevent aggregation, or prevent and / or inhibit protein damage during processing conditions (e.g., purification, filtration, freeze drying, transportation, storage and delivery). In some embodiments, a surfactant can be used to provide additional stability for the active ingredient.

[0486] 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, marketed 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; Poloxamer (e.g., Poloxamer 188); X-100 (Union Carbide Corp., Houston, TX) and X-114; NP40; Span 20, Span 40, Span 60, Span 65, Span 80 and Span 85; copolymers of ethylene glycol and propylene glycol (e.g., A series of nonionic surfactants, such as F68, 10R5, F 108, F 127, 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.

[0487] The amount of nonionic 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 (i.e., anti-ILT4 antibody or antigen-binding fragment thereof (e.g., 1E1, 2A6, 3G7 or 2C1)) in the formulation. All percentages regarding nonionic surfactants are listed as % (w / v). Typically, the surfactant is present at a concentration of 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 about 0.01% to about 0.1%, about 0.01% to about 0.09%, about 0.01% to about 0.08%, about 0.01% to about 0.07%, about 0.01% to about 0.06%, about 0.01% to about 0.05%, about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.015% to about 0.04%, about 0.015% to about 0.03%, about 0.015% to about 0.02%, about 0.02% to about 0.04%, about 0.02% to about 0.035%, or 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%.

[0488] In a specific embodiment, the formulations described herein comprise about 0.01% to about 0.04% (w / v) PS-80. In one embodiment, the formulations described herein comprise 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%.

[0489] The formulations described herein also include 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 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.

[0490] Therefore, 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 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.

[0491] 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.

[0492] In some embodiments, the non-reducing sugar is sucrose.

[0493] In certain embodiments, the nonionic surfactant is PS-80 or PS-20. In one embodiment, the nonionic surfactant is PS-80. In another embodiment, the nonionic surfactant is PS-20.

[0494] In some embodiments, the antioxidant is L-methionine.

[0495] Therefore, in another aspect, provided herein is a pharmaceutical formulation for 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 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 L-methionine.

[0496] In some embodiments, the formulation comprises about 8 mM to about 12 mM L-histidine buffer.

[0497] In certain embodiments, the formulation comprises about 5 mM to about 10 mM L-methionine.

[0498] In other embodiments, the formulation comprises from about 0.01% to about 0.05% (w / v) PS-80.

[0499] In yet other embodiments, the formulation comprises an anti-ILT4 antibody or antigen-binding fragment thereof of about 10 mg / mL to about 150 mg / mL. In yet other embodiments, the concentration of the anti-ILT4 antibody or 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 antigen-binding fragment thereof is about 10 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 12.5 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 15 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 25 mg / mL. In one embodiment, the concentration of the anti-ILT4 antibody or antigen-binding fragment thereof is about 50 mg / mL. In another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 75 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 100 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 125 mg / mL. In yet another embodiment, the concentration of the anti-ILT4 antibody or its antigen-binding fragment is about 150 mg / mL.

[0500] Thus, in a specific embodiment, the formulation comprises about 25 mg / mL of an anti-ILT4 antibody or 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.

[0501] In another specific embodiment, the formulation comprises about 50 mg / mL of an anti-ILT4 antibody or 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.

[0502] In yet another specific embodiment, the formulation comprises about 75 mg / mL of an anti-ILT4 antibody or 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.

[0503] In another specific embodiment, the formulation comprises about 100 mg / mL of an anti-ILT4 antibody or 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.

[0504] In yet another specific embodiment, the formulation comprises about 125 mg / mL anti-ILT4 antibody or 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.

[0505] In certain embodiments of the various formulations provided herein, the formulation has a pH of about 5.0-about 6.8. In some embodiments, the formulation has a pH of about 5.5-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 yet another embodiment, the formulation has a pH of about 5.8. In yet another embodiment, the formulation has a pH of about 5.9. In yet another embodiment, the formulation has a pH of about 6.0.

[0506] Therefore, 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.

[0507] In certain embodiments of the various formulations disclosed herein, the anti-ILT4 antibody or its antigen-binding fragment is an anti-ILT4 antibody or its antigen-binding fragment described herein (e.g., 1E1, 2A6, 3G7, or 2C1). In some embodiments of the formulation, the anti-ILT4 antibody or its antigen-binding fragment is 1E1 or a variant thereof. In some embodiments of the formulation, the anti-ILT4 antibody or its antigen-binding fragment is 2A6 or a variant thereof. In some embodiments of the formulation, the anti-ILT4 antibody or its antigen-binding fragment is 3G7 or a variant thereof. In some embodiments of the formulation, the anti-ILT4 antibody or its antigen-binding fragment is 2C1 or a variant thereof.

[0508] In certain embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof includes 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).

[0509] In some embodiments of the various formulations provided herein, the anti-ILT4 antibody or antigen-binding fragment thereof includes 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).

[0510] 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.

[0511] 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 shown in SEQ ID NO:2 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0512] 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 shown in SEQ ID NO:80 and a light chain comprising the amino acid sequence shown in SEQ ID NO:7.

[0513] 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.

[0514] 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.

[0515] 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.

[0516] Thus, in some embodiments, provided herein is a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 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: 21). 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).

[0517] In certain embodiments, provided herein is a pharmaceutical formulation of an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 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: NO:19), CDR-L2: GDSNRPS (SEQ ID NO:52), and CDR-L3: QSFDNSLSAYV (SEQ ID NO:21).

[0518] In other embodiments, provided herein is a pharmaceutical formulation for an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 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.

[0519] In yet other embodiments, provided herein is a pharmaceutical formulation for an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 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.

[0520] In yet other embodiments, provided herein is a pharmaceutical formulation for an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 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.

[0521] In some embodiments, provided herein is a pharmaceutical formulation for an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 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.

[0522] In some embodiments, provided herein is a pharmaceutical formulation for an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 the 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.

[0523] In some embodiments, provided herein is a pharmaceutical formulation for an anti-ILT4 antibody or an antigen-binding fragment thereof, comprising: (i) 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 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.

[0524] 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 below. In yet another embodiment, the liquid formulation is a reconstituted solution from a lyophilized formulation.

[0525] 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 antigen-binding fragment thereof as determined by ultra-performance size exclusion chromatography is at least about 99%; (ii) the % monomer of the anti-ILT4 antibody or antigen-binding fragment thereof as determined by OD 350-500 The turbidity of the preparation is measured to be 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 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.

[0526] In some embodiments, the % monomer of the anti-ILT4 antibody or antigen-binding fragment thereof is at least about 99% as determined by ultra-performance size exclusion chromatography after storage of the formulation at about 3°C ​​to about 5°C for 6 months.

[0527] In some embodiments, after the formulation has been stored at about 3°C ​​to about 5°C for 6 months, the 350-500 The turbidity of the formulation was measured to be at most about 0.135.

[0528] 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 Variants 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 Variants 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 Variants of the anti-ILT4 antibody or its antigen-binding fragment is at most about 23%, and the Basic Variants of the anti-ILT4 antibody or its antigen-binding fragment is at most about 14% as determined by high performance ion exchange chromatography.

[0529] In some embodiments, after the formulation has been stored at about 3°C ​​to about 5°C for 6 months, the subvisible particle count for particles having a size of at least 2 μm as determined by microfluidic imaging is at most about 3500.

[0530] In some embodiments, 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 as determined by reduced peptide mapping analysis is less than about 4% after the formulation is stored at about 3° C. to about 5° C. for 6 months. In one embodiment, 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 as determined by reduced peptide mapping analysis is less than about 4% after the formulation is stored at about 3° C. to about 5° C. for 6 months. In one embodiment, 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 as determined by reduced peptide mapping analysis is less than about 4% after the formulation is stored at about 3° C. to about 5° C. for 6 months. In one embodiment, the % oxidation of three amino acid residues selected from W7, W102, M253, M359 and M429 in the heavy chain of an anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4% as determined by reduced peptide mapping analysis after the formulation is stored at about 3°C ​​to about 5°C for 6 months. In one embodiment, the % oxidation of four amino acid residues selected from W7, W102, M253, M359 and M429 in the heavy chain of an anti-ILT4 antibody as shown in SEQ ID NO: 2 or 80 is less than about 4% as determined by reduced peptide mapping analysis after the formulation is stored at about 3°C ​​to about 5°C for 6 months. In one embodiment, 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% as determined by reduced peptide mapping after the formulation is stored at about 3° C. to about 5° C. for 6 months.

[0531] The preparations described herein can be prepared as liquid preparations. Liquid antibody preparations can be prepared by obtaining a bulk drug (e.g., an anti-ILT4 monoclonal antibody) in a liquid form (e.g., an anti-ILT4 monoclonal antibody in an aqueous pharmaceutical preparation) and exchanging its buffer into a desired buffer as the last step of a purification process. There is no lyophilization step in this embodiment. The bulk drug in the final buffer is concentrated to the desired concentration. Excipients such as sucrose and PS80 are added to the bulk drug, and it is diluted to the final protein concentration using an appropriate buffer. The final prepared bulk drug is filtered using a 0.22 μm filter and filled into a final container (e.g., a glass vial).

[0532] The formulations described herein can also be reconstituted by lyophilized formulations. Lyophilized formulations of therapeutic proteins provide several advantages. Lyophilized formulations generally provide better chemical stability than solution formulations, and thus increased half-life. Lyophilized formulations can also be reconstituted with different concentrations depending on clinical factors such as route of administration or dosage. For example, lyophilized formulations can be reconstituted with high concentrations (i.e., with small volumes) for subcutaneous administration (if necessary), or reconstituted with lower concentrations if administered intravenously. If a high dose is required for a particular subject, particularly if subcutaneous administration (wherein the injection volume must be minimized), high concentrations may also be necessary. U.S. Patent No. 6,267,958 discloses a type of such lyophilized antibody formulation, which is incorporated herein by reference in its entirety. U.S. Patent No. 7,247,707 discloses another type of lyophilized formulation of therapeutic protein, which is incorporated herein by reference in its entirety.

[0533] Typically, lyophilized formulations are expected to be reconstituted with a high concentration of the drug product (DP, in an exemplary embodiment, an anti-ILT4 antibody or an antigen-binding fragment thereof), i.e., are expected to be prepared for reconstitution in a low volume of water. Subsequent dilution with water or an isotonic buffer can be easily used to dilute the DP to a lower concentration. Typically, excipients are included in the lyophilized formulations of the present invention at levels that will result in a roughly isotonic formulation when reconstituted at a high DP concentration, such as for subcutaneous administration. Reconstitution in a larger volume of water to give a lower DP concentration will inevitably reduce the osmotic pressure of the reconstituted solution, but such a reduction may not be significant in non-subcutaneous, such as 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.

[0534] The lyophilized preparation of the present invention is formed by freeze drying (freeze drying) of the pre-freeze drying solution. Freeze drying is achieved by freezing the preparation and then subliming water at a temperature suitable for primary drying. Under such conditions, the product temperature is lower than the eutectic point or collapse temperature of the preparation. Generally, the shelf temperature for primary drying ranges from about -30 to 25 ° C (provided that the product remains frozen during primary drying) at a suitable pressure of about 50 to 250 mTorr in the usual range. The size and type of the preparation, the container (e.g., glass vial) holding the sample, and the volume of the liquid will determine the time required for drying, which can range from a few hours to a few days (e.g., 40-60 hours). The secondary drying stage can be carried out at about 0-40 ° C, depending mainly on the type and size of the container and the type of protein used. The secondary drying time is determined by the residual moisture level 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 pressure can be the same as that used during the primary drying step. Freeze drying conditions can vary depending on the preparation and vial size.

[0535] In some cases, it may be desirable to lyophilize the protein formulation in the container in which the protein is to be reconstituted, so as to avoid a transfer step. In this case, the container may be, for example, a 3, 5, 10, 20, 50 or 100 cc vial.

[0536] The lyophilized formulation is reconstructed before administration. The protein can be reconstructed at a concentration of about 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg / mL, or a higher concentration such as 150 mg / mL, 200 mg / mL, 250 mg / mL or 300 mg / mL until about 500 mg / mL. In one embodiment, the protein concentration after reconstruction is about 10-300 mg / mL. In one embodiment, the protein concentration after reconstruction is about 20-250 mg / mL. In one embodiment, the protein concentration after reconstruction is about 150-250 mg / mL. In one embodiment, the protein concentration after reconstruction is about 180-220 mg / mL. In one embodiment, the protein concentration after reconstruction is about 50-150 mg / mL. In one embodiment, the protein concentration after reconstruction is about 150 mg / mL. In one embodiment, the protein concentration after reconstruction is about 125 mg / mL. In one embodiment, the protein concentration after reconstruction is about 100 mg / mL. In one embodiment, the protein concentration after reconstruction is about 75 mg / mL. In one embodiment, the protein concentration after reconstruction is about 50 mg / mL. In one embodiment, the protein concentration after reconstruction is about 25 mg / mL. When subcutaneous delivery of the reconstructed formulation is expected, high protein concentration is particularly useful. However, for other routes of administration, such as intravenous administration, lower protein concentrations (e.g., about 5-50 mg / mL) may be required.

[0537] Reconstitution is typically performed at a temperature of about 25°C to ensure complete hydration, although other temperatures may be used if desired. The time required for reconstitution will depend, for example, on the type of diluent, the amount of excipients and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0538] How to use

[0539] In another aspect, provided herein is a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of any formulation 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 for treating cancer in a human patient, comprising administering to the patient any formulation described herein.

[0540] In any of the methods of the invention, the cancer can be selected from the group consisting of melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, renal 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 prostate adenocarcinoma), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma, and other neoplastic malignancies.

[0541] In some embodiments, the lung cancer is non-small cell lung cancer.

[0542] In alternative embodiments, the lung cancer is small cell lung cancer.

[0543] In some embodiments, the lymphoma is Hodgkin's lymphoma.

[0544] In other embodiments, the lymphoma is non-Hodgkin's lymphoma.In specific embodiments, the lymphoma is mediastinal large B-cell lymphoma.

[0545] In some embodiments, the breast cancer is triple-negative breast cancer.

[0546] In a further embodiment, the breast cancer is ER+ / HER2- breast cancer.

[0547] In some embodiments, the bladder cancer is urothelial carcinoma.

[0548] 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.

[0549] In some embodiments, the cancer is a solid tumor with high levels of microsatellite instability (MSI-H).

[0550] In some embodiments, the cancer is a solid tumor with a high mutational burden.

[0551] In some embodiments, the cancer is metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

[0552] 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.

[0553] In other embodiments of the above methods of treatment, 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's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL) or small lymphocytic lymphoma (SLL).

[0554] Malignancies for which improved disease-free and overall survival associated with the presence of tumor-infiltrating lymphocytes in biopsy or surgical material are demonstrated to be included in the methods and treatments described herein, 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. In addition, refractory or recurrent malignancies are also included, the growth of which can be inhibited using the antibodies described herein.

[0555] Additional cancers that may benefit from treatment with the formulations described herein include those associated with persistent infection by viruses, such as human immunodeficiency virus, hepatitis A, B, and C viruses, Epstein-Barr virus, human papillomavirus known to be causally linked to, for example, Kaposi's sarcoma, liver cancer, nasopharyngeal carcinoma, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer.

[0556] The preparation can also be used to prevent or treat infection and infectious diseases. Therefore, the invention provides a method for treating a chronic infection in a mammalian subject, comprising administering an effective amount of the preparation of the present invention to the subject. In some specific embodiments of the method, the preparation is administered to the subject via intravenous administration. In other embodiments, the preparation is administered to the subject by subcutaneous administration.

[0557] These agents can be used alone or in combination with vaccines to stimulate immune responses against pathogens, toxins and self-antigens. Antibodies or antigen-binding fragments thereof can be used to stimulate immune responses 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 immune responses against infections by bacteria or fungal parasites and other pathogens. Viral infections by hepatitis B virus, hepatitis C virus and HIV are among those considered chronic viral infections.

[0558] The formulation of the present invention can be administered to a patient in combination with one or more "additional therapeutic agents". Additional therapeutic agents can be biological therapeutic agents (including but not limited to antibodies to VEGF, EGFR, Her2 / neu, VEGF receptors, 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 genes encoding immunostimulatory cytokines such as but not limited to GM-CSF).

[0559] As noted above, in some embodiments of the methods of the present invention, the method further comprises administering an additional therapeutic agent. In specific 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-ILT4 antibody or an antigen-binding fragment thereof, an anti-ILT4 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 further embodiments, the additional therapeutic agent is a STING agonist.

[0560] Suitable routes of administration may, for example, include parenteral delivery, including intramuscular, subcutaneous, and intrathecal, direct intraventricular, intravenous, intraperitoneal. The drug may be administered in various conventional ways, such as intraperitoneal, parenteral, intraarterial, or intravenous injection. Wherein the mode of administration (e.g., subcutaneous administration) in which the volume of the solution must be limited requires that the lyophilized formulation be reconstituted at high concentrations.

[0561] The dosage of the additional therapeutic agent is selected depending 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. Therefore, the dosage and frequency of administration of each additional therapeutic agent (e.g., a biotherapeutic or chemotherapeutic agent) will depend in part on the specific therapeutic agent, the severity of the cancer to be treated, and the patient's characteristics. Guidance in selecting appropriate dosages of antibodies, cytokines, and small molecules is available. 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. 345:783-792. 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 Edition); Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002). Determination of an appropriate dosage regimen can be made by a clinician, for example, using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend on, for example, the patient's clinical history (e.g., previous treatments), the type and stage of cancer to be treated, and biomarkers of response to one or more therapeutic agents in the combination therapy.

[0562] Various literature references may be used to facilitate the selection of pharmaceutically acceptable carriers or excipients for additional therapeutic agents. See, for example, Remington's Pharmaceutical Sciences and US 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: Parental 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.

[0563] Drug antibody preparations can be administered by continuous infusion, or by being administered at intervals such as one day, 1-7 times per week, one week, two weeks, three weeks, monthly, every two months. A preferred dosage regimen is a dosage regimen involving the maximum dose or dosage frequency that avoids significant undesirable side effects. The total weekly dose is generally 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 dosage of small molecule therapeutics such as peptide mimetics, natural products or organic chemicals is about the same as that of antibodies or polypeptides on a mole / kg basis.

[0564] Embodiments of the invention also include one or more biological agents described herein, which are (i) for use in the following, (ii) as a medicament or composition for the following, or (iii) for the preparation of a medicament for the following: (a) (e.g., human) treatment; (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 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 similar untreated patients; (m) inducing complete or partial remission of a cancerous condition or other PD-1-related disease or an ILT4-related disease; (n) treating cancer; or (o) treating a chronic infection.

[0565] General approach

[0566] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2nd edition, 2001 3rd edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd edition, 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 describes cloning and DNA mutagenesis in bacterial cells (Volume 1), cloning in mammalian cells and yeast (Volume 2), glycoconjugates and protein expression (Volume 3), and bioinformatics (Volume 4).

[0567] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modifications, post-translational modifications, production of fusion proteins, 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) Bio Directory, Piscataway, NJ, pp. 384-391). The 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, supra). 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).

[0568] 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). 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. Pat. No. 6,329,511).

[0569] An alternative approach to humanization is to 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).

[0570] Purification of the antigen is not necessary for the production of antibodies. Animals can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animal and fused with a myeloma cell line to produce hybridomas (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

[0571] The antibody can be conjugated to, for example, a small drug molecule, an enzyme, a liposome, polyethylene glycol (PEG). The antibody can be used for treatment, diagnosis, kits or other purposes, and includes antibodies coupled to, for example, dyes, radioisotopes, enzymes or metals such as colloidal gold (see, e.g., 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).

[0572] 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 for use as, e.g., diagnostic reagents are available (Molecular Probesy (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0573] 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, Philadelphia, PA; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0574] Software packages and databases are available for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (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).

[0575] Analytical methods

[0576] Analytical methods suitable for assessing product stability include size exclusion chromatography (SEC), dynamic light scattering assay (DLS), differential scanning calorimetry (DSC), isoaspartate quantification, potency, UV at 340 nm, UV spectroscopy, and FTIR. SEC (J. Pharm. Scien., 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 percent 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 protein denaturation temperature and 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 protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45:231 (1998); Pharm. Res., 12:1250 (1995); J. Pharm. Scien., 85:1290 (1996); J. Pharm. Scien., 87:1069 (1998)) measures the IR spectrum in the amide region and gives information on the secondary structure of the protein.

[0577] The isoaspartate content in the sample was measured using the Isoquant Isoaspartate Detection System (Promega). The kit uses the enzyme protein isoaspartate 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 isoaspartate at the α-carboxyl position, generating S-adenosyl-L-homocysteine ​​(SAH) in the process. This is a relatively small molecule and can usually be separated and quantified by reverse phase HPLC using the SAH HPLC standard provided in the kit.

[0578] The efficacy or bioidentity of an antibody can be measured by its ability to bind to its antigen. The specific binding of an antibody to its antigen can be quantified by any method known to those skilled in the art, such as an immunoassay, such as ELISA (enzyme-linked immunosorbent assay).

[0579] All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methodologies and materials that might be used in connection with the present invention.

[0580] Various embodiments of the present invention have been described herein with reference to the accompanying drawings, but it should be understood that the invention is not limited to those precise embodiments and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0581] Embodiment 1 provides a formulation comprising: (i) about 10 mg / mL to about 200 mg / mL of an anti-human immunoglobulin-like transcript 4 ("anti-ILT4") antibody or an antigen-binding fragment thereof; (ii) about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) about 5 mM to about 20 mM buffer; (iv) about 6% to about 8% weight / volume (w / v) of a non-reducing sugar; (v) about 0.01% to about 0.10% (w / v) of a non-ionic surfactant; and (vi) about 1 mM to about 20 mM of an antioxidant, 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: 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 the following: 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 anti-PD-1 antibody or its antigen-binding fragment comprises a variable light chain region comprising the following: CDRL1 of SEQ ID NO: 95, CDRL2 of SEQ ID NO: 96, and CDRL3 of SEQ ID NO: 97, and a variable heavy chain region comprising the following: CDRH1 of SEQ ID NO: 100, CDRH2 of SEQ ID NO: 101, and CDRH3 of SEQ ID NO: 102. The CDRH3 of NO:102, wherein the buffer is L-histidine buffer, acetate buffer or 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.

[0582] Embodiment 2 provides a formulation of embodiment 1, comprising 40-60 mg / mL of an anti-ILT4 antibody; 15-35 mg / mL of an anti-PD-1 antibody; about 7% sucrose or trehalose; about 0.025% polysorbate 80 or polysorbate 20; about 3-30 mM L-histidine buffer at a pH of about 5.0-6.5; and about 5-20 mM methionine or a pharmaceutically acceptable salt thereof.

[0583] Embodiment 3 provides a formulation of embodiment 1, comprising 45-55 mg / mL of an anti-ILT4 antibody; 20-30 mg / mL of an anti-PD-1 antibody; about 7% sucrose or trehalose; about 0.025% polysorbate 80 or polysorbate 20; about 3-30 mM L-histidine buffer at a pH of about 5.0-6.5; and about 10-20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0584] Embodiment 4 provides the formulation of embodiment 1, wherein the buffer is an L-histidine buffer.

[0585] Embodiment 5 provides the formulation of any one of embodiments 1-4, wherein the non-reducing sugar is sucrose.

[0586] Embodiment 6 provides the formulation of any one of embodiments 1-5, wherein the nonionic surfactant is polysorbate 80.

[0587] Embodiment 7 provides the formulation of any one of embodiments 1-6, wherein the antioxidant is L-methionine.

[0588] Embodiment 8 provides a formulation comprising: (i) about 10 mg / mL to about 200 mg / mL of an anti-ILT4 antibody ("anti-ILT4") or an antigen-binding fragment thereof; (ii) about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) about 5 mM to about 20 mM L-histidine buffer; (iv) about 6% to about 8% weight / volume (w / v) sucrose; (v) about 0.01% to about 0.10% (w / v) polysorbate 80; and (vi) about 1 mM to about 20 mM L-methionine, 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: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO: 17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO: 18). NO:18); and a light chain variable domain comprising the following: 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); and wherein the anti-PD-1 antibody or its antigen-binding fragment comprises a variable light chain region comprising the following: CDRL1 of SEQ ID NO:95, CDRL2 of SEQ ID NO:96, and CDRL3 of SEQ ID NO:97, and a variable heavy chain region comprising the following: CDRH1 of SEQ ID NO:100, CDRH2 of SEQ ID NO:101, and CDRH3 of SEQ ID NO:102, or a pharmaceutically acceptable salt thereof.

[0589] Embodiment 9 provides the formulation of embodiment 8, comprising about 8 mM to about 12 mM L-histidine buffer.

[0590] Embodiment 10 provides the formulation of any of embodiments 8 or 9 comprising about 5 mM to about 10 mM L-methionine.

[0591] Embodiment 11 provides the formulation of any one of embodiments 8-10, comprising about 0.01% to about 0.05% (w / v) polysorbate 80.

[0592] Embodiment 12 provides the formulation of any one of embodiments 1 or 4-11, comprising about 10 mg / mL to about 150 mg / mL of the anti-ILT4 antibody or antigen-binding fragment thereof.

[0593] Embodiment 13 provides the formulation of any one of embodiments 1 or 4-11, comprising about 10 mg / mL to about 150 mg / mL of the anti-PD-1 antibody or antigen-binding fragment thereof.

[0594] Embodiment 14 provides a formulation of any one of Embodiment 12, wherein the concentration of the anti-ILT4 antibody or 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.

[0595] Embodiment 15 provides the formulation of any one of Embodiment 12, wherein the concentration of the anti-PD-1 antibody or 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.

[0596] Embodiment 16 provides a formulation of any one of embodiments 1-13, comprising about 25 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mg / mL of an anti-PD-1 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.

[0597] Embodiment 17 provides a formulation of any one of embodiments 1-13, comprising about 50 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 25 mg / mL of an anti-PD-1 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.

[0598] Embodiment 18 provides a formulation of any one of embodiments 1-13, comprising about 75 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 50 mg / mL of an anti-PD-1 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.

[0599] Embodiment 19 provides a formulation of any one of embodiments 1-13, comprising about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 75 mg / mL of an anti-PD-1 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.

[0600] Embodiment 20 provides a formulation of any one of embodiments 1-13, comprising about 125 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 100 mg / mL of an anti-PD-1 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.

[0601] Embodiment 21 provides the formulation of any one of embodiments 1-20, wherein the formulation has a pH of about 5.0 to about 6.8.

[0602] Embodiment 22 provides the formulation of any one of embodiments 1-21, wherein the formulation has a pH of about 5.5 to about 6.0.

[0603] Embodiment 23 provides the formulation of any one of embodiments 1-22, wherein the formulation has a pH of about 5.5.

[0604] Embodiment 24 provides a formulation of an anti-human immunoglobulin-like transcript 4 ("anti-ILT4") antibody or an antigen-binding fragment thereof, comprising: (i) about 50 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or an antigen-binding fragment thereof; (iii) about 10 mM L-histidine buffer, pH about 5.5; (iv) about 7% weight / volume (w / v) sucrose; (v) about 0.025% (w / v) polysorbate 80; and (vi) about 10 mM L-methionine, 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: EINHXGSTNYNPSLKS, wherein X is S or A (SEQ ID NO: 17). NO:17), and CDR-H3: LPTRWVTTRYFDL (SEQ ID NO:18); and a light chain variable domain comprising the following: 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), and wherein the anti-PD-1 antibody or antigen-binding fragment thereof includes a variable light chain region comprising the following: CDRL1 of SEQ ID NO:95, CDRL2 of SEQ ID NO:96, and CDRL3 of SEQ ID NO:97, and a variable heavy chain region comprising the following: CDRH1 of SEQ ID NO:100, CDRH2 of SEQ ID NO:101, and CDRH3 of SEQ ID NO:102.

[0605] Embodiment 25 provides a formulation of any one of embodiments 1-24, 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: 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, 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).

[0606] Embodiment 26 provides a formulation of any one of embodiments 1-24, 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).

[0607] Embodiment 27 provides the formulation of any one of embodiments 1-24, 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.

[0608] Embodiment 28 provides the formulation of any one of embodiments 1-24, 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.

[0609] Embodiment 29 provides the formulation of any one of embodiments 1-24, 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.

[0610] Embodiment 30 provides the formulation of any one of embodiments 1-24, 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.

[0611] Embodiment 31 provides the formulation of any one of embodiments 1-24, 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.

[0612] Embodiment 32 provides the formulation of any one of embodiments 1-24, 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.

[0613] Embodiment 33 provides the formulation of any one of embodiments 1-24, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: a variable light region comprising the amino acid sequence shown in SEQ ID NO:98 and a variable heavy region comprising the amino acid sequence shown in SEQ ID NO:103.

[0614] Embodiment 34 provides the formulation of any one of embodiments 1-24, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: a variable light region comprising the amino acid sequence shown in SEQ ID NO:99 and a variable heavy region comprising the amino acid sequence shown in SEQ ID NO:104.

[0615] Embodiment 35 provides the formulation of any one of embodiments 1-24, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof consists of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence shown in SEQ ID NO: 99, and the two heavy chains consist of the amino acid sequence shown in any one of SEQ ID NO: 104-109.

[0616] Embodiment 37 provides the formulation of any one of embodiments 1-35, wherein the anti-human PD-1 antibody is pembrolizumab.

[0617] Embodiment 37 provides the formulation of any one of embodiments 1-36, wherein the ratio of the anti-ILT4 antibody to the anti-PD-1 antibody is 1:1 or 1:2.

[0618] Embodiment 38 provides a formulation of any one of embodiments 1-37, wherein after 9 months at 5°C: (i) the % monomer of the anti-ILT4 antibody as determined by size exclusion chromatography is ≥95%; (ii) the % heavy and light chains of the anti-ILT4 antibody as measured by reducing CE-SDS are ≥90%; (iii) the % heavy and light chains of the anti-ILT4 antibody as measured by reducing CE-SDS are ≥95%; (iv) the % intact IgG of the anti-ILT4 antibody as measured by non-reducing CE-SDS is ≥90%; and / or (v) the % intact IgG of the anti-ILT4 antibody as measured by non-reducing CE-SDS is ≥95%.

[0619] Embodiment 39 provides the formulation of any one of embodiments 1-38, wherein the formulation is a liquid formulation stored at about 3°C ​​to about 5°C, or is a reconstituted solution from a lyophilized formulation.

[0620] Embodiment 40 provides the formulation of any one of embodiments 1-24, wherein after storage for up to 9 months at about 3°C ​​to about 5°C: (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 of at least 2 μm in size is at most about 3500 as determined by microfluidic imaging; and / or (v) as determined by reduced peptide mapping analysis, the % main peak of the anti-ILT4 antibody or antigen-binding fragment thereof is at most 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% 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 set forth in NO: 2 or 80 is less than about 4%.

[0621] Embodiment 41 provides the formulation of any one of embodiments 1-34 or 37-41, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0622] Embodiment 42 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 1-40.

[0623] Embodiment 43 provides the method of embodiment 42, 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.

[0624] Embodiment 44 provides the use of any one of the formulations of embodiments 1-41 for the preparation of a medicament for treating cancer.

[0625] Embodiment 45 provides the use of any one of the formulations of embodiments 1-42 for treating cancer in a human patient.

[0626] Embodiment 46 provides the use of embodiment 45, 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.

[0627] Example

[0628] Example 1: Preparation of anti-ILT4 / anti-PD-1 co-formulation

[0629] Anti-ILT4 monoclonal antibody and anti-PD-1 antibody ("anti-ILT4 / anti-PD-1 co-formulation") injection is prepared as a sterile, preservative-free solution that requires dilution for intravenous infusion. The anti-ILT4 / anti-PD-1 co-formulation is a fixed-dose combination of anti-ILT4 and pembrolizumab antibodies. The co-formulation composition is manufactured by combining a fully formulated anti-ILT4 monoclonal antibody process 2 drug substance and the pembrolizumab drug substance. The anti-ILT4 drug substance contains 50.0 mg / mL anti-ILT4 in 0.29 mg / mL L-histidine, 1.71 mg / mL L-histidine monohydrochloride monohydrate, 1.49 mg / mL L-methionine, 70.0 mg / mL sucrose and 0.25 mg / mL polysorbate 80, pH 5.5. The pembrolizumab drug substance contained 25.0 mg / mL pembrolizumab in 0.29 mg / mL L-histidine, 1.71 mg / mL L-histidine monohydrochloride monohydrate, 70 mg / mL sucrose, and 0.20 mg / mL polysorbate 80, pH 5.5. The following formulation attributes were considered during formulation optimization: protein concentration, buffer, pH, charge species, antioxidants, and surfactants.

[0630] Example 2: Analysis of pre-anti-ILT4 / pembrolizumab co-formulation

[0631] Table 6 summarizes the pre-coformulation development plan. This study was conducted to evaluate the sensitivity of the anti-ILT4 drug product (DP), single entity (SE), and co-formulation (CF) with pembrolizumab to different formulation and process parameters such as light exposure, oxygen, and residual levels of peroxides and metals, which the product experiences during the manufacturing process and are previously known to affect oxidation. Filtered bulk DP of anti-ILT4 (30 mg / mL), pembrolizumab (7.5 mg / mL), and anti-ILT4+pembrolizumab (30 mg / mL+7.5 mg / mL) were spiked with residual levels of peroxides (0, 20, and 200 ppb) and metals (0, 25, and 250 ppb) and subjected to light stress (0, 0.2X, and 0.4X ICH combined CWL+UV, where 1X ICH exposure level corresponds to 1.2 million lux hours of white light and 200 watt hours / m 2UV energy), followed by staging for stability up to 12 weeks at 5°C and 4 weeks at 40°C to evaluate the potential oxidative susceptibility of anti-ILT4. The range of test stress levels for metal, peroxide and light exposure was selected to cover the target exposure during DP manufacturing at a commercial manufacturing site, and then appropriately bracketed for control and worse cases. In addition, this study also acted to measure free L-methionine concentration (7, 10 and 15mM) as an excipient for controlling protein oxidation. Samples were analyzed by HIC, IEX, SEC, L-methionine content, PS-80 content, sub-visible particles and reduced peptide mapping assays.

[0632] The composition of the anti-ILT4 / anti-PD-1 co-formulations tested in this study are presented in Table 6. L-methionine concentrations (7, 10, and 15 mM) were measured as an excipient for controlling protein oxidation

[0633] Table 6. Composition of anti-ILT4 / anti-PD-1 CF, anti-ILT4 SE, and pembrolizumab SE qs = sufficient quantity

[0634] L-methionine concentrations were measured as part of the DOE study design

[0635] Example 3: Evaluation of Oxidative Sensitivity

[0636] The five key parameters studied in the study were L-Met, peroxide, metal, fill volume, and light stress. A D optimal split-plot design was approved for the program, with 36 formulations each for anti-ILT4 / anti-PD-1 co-formulated products, anti-ILT4 single entity (SE), and pembrolizumab SE, for a total of 108 formulations. Since light stress is a factor that is difficult to change, the 36 formulations (data not shown) have 6 whole plots with constant light stress levels for each whole plot. Each whole plot is further divided by 2 sub-plots to keep the L-Met level the same within the whole plot. The study ranges for the five factors are given in Table 7.

[0637] Table 7. Studies evaluating the oxidative sensitivity of anti-ILT4 / anti-PD-1 co-formulations

[0638]

[0639] N / A = Not Applicable

[0640] 1 Metals will be tested by spiking equal amounts of FeCl2 and CuCl2. That is, 100 ppb metal corresponds to a solution containing 50 ppb FeCl2 and 50 ppb CuCl2

[0641] 2 Peroxides will be tested by spiking equal amounts of t-BHP and AAPH. That is, 100 ppb peroxide corresponds to a solution containing 50 ppb t-BHP and 50 ppb AAPH.

[0642] 3 The 1XICHQ1B exposure level corresponds to 1.2 million lux hours of white light and 200 watt hours / m 2 UV energy 4 Fill volume acts as a surrogate for controlling O2 levels

[0643] 5 All samples will be staged for stability (0, 4 and 12 weeks at 5°C; 0, 2 and 4 weeks at 40°C)

[0644] Example 4: Preparation of Metal and Peroxide Spiked Vials

[0645] A diluent (consisting of a protein-free solution) having a similar composition to the anti-ILT4 / anti-PD-1 co-formulation, anti-ILT4 SE, and pembrolizumab SE stock solutions was used for sample preparation. The metal stock solution included equal amounts of FeCl2 and CuCl2. Specifically, the 100 ppb metal stock solution included 50 ppb FeCl2 and 50 ppb CuCl2. The peroxide stock solution included equal parts of t-BHP and AAPH. Specifically, the 100 ppb peroxide stock solution included 50 ppb t-BHP and 50 ppb AAPH. Using the diluent, peroxide stock solution, and metal stock solution, appropriate amounts of these solutions were added to the filtered anti-ILT4 / anti-PD-1 co-formulation, anti-ILT4 SE, and pembrolizumab SE stock formulations in a ratio of 1 μL of diluent, peroxide stock solution, or metal stock solution for each 1 mL of stock formulation used. Thus, multiple combinations of peroxide and metal concentrations were targeted. Specifically, the peroxide concentrations evaluated were 0, 20 and 200 ppb, while the metal concentrations were 0, 25 and 250 ppb. When taking into account different L-methionine concentrations (7, 10 and 15 mM), fill volumes (1.2, 2.2 and 3.2 mL) and light exposure levels (0, 0.2X and 0.4XICH CWL+UV), a total of 108 independent formulations of anti-ILT4 / anti-PD-1 co-formulations, anti-ILT4SE and pembrolizumab SE were prepared. Table 7 summarizes the oxidation-stress conditions for each of the 108 formulations prepared, as well as the volume of the stock solution used to prepare them. For the study, PS-80 levels remained unchanged in all formulations, and degradation was monitored within the study parameters and duration.

[0646] In a laminar flow hood, filtered anti-ILT4 / anti-PD-1 co-formulations, anti-ILT4 SE, and anti-ILT4 SE formulations were filled into 2R vials with variable fill volumes (1.2, 2.2, and 3.2 mL). The different fill volumes used in this study were selected to control the amount of oxygen available in the vials. 2R vials were used instead of the expected commercial images (20 mL and 30 mL vials were used for single entity and co-formulation images, respectively) as a scaled-down model and to minimize material use.

[0647] The same 36 formulations were prepared for anti-ILT4 / anti-PD-1 CF, anti-ILT4 SE and pembrolizumab SE (data not shown). The stock formulations corresponded to the formulation stocks of the corresponding co-formulations or single entities and depended on the L-methionine concentration (7, 10 or 15 mM).

[0648] Example 5: Light exposure and stability studies

[0649] All vials from the same formulation pool (i.e., anti-ILT4 / anti-PD-1 co-formulation, anti-ILT4 SE, or pembrolizumab SE) were placed in a photostability chamber at 25°C for light exposure (0, 0.2X, and 0.4X ICH CWL+UV). Samples intended for 0X ICH light exposure should be protected from light; nonetheless, these samples were also left in the chamber for the duration of the experiment. Samples intended for 0.2X and 0.4X ICH were placed in the chamber in a horizontal position to maximize exposure.

[0650] After all vials were exposed to light, samples were staged for stability up to 12 weeks at 5°C and up to 4 weeks at 40°C according to the scheme in Table 2. At each stability time point, samples were removed and frozen at -80°C and then submitted for analytical testing (with appropriate controls).

[0651] Example 6: Qualitative analysis

[0652] Visual analysis of the analytical data suggested that there was no effect from increasing residual peroxide levels (from 0-200 ppb) or changes in fill volume (from 1.2 to 3.2 mL). On the other hand, changes in light exposure (from 0 to 0.4X ICH) and residual metal content (0–250 ppb) resulted in increased drug product degradation, as evidenced by changes in high molecular weight species from UP-SEC ( Figure 1 ), degradation of PS80 ( Figure 2 ) and from reduced peptide mapping analysis ( Figure 3 , Figure 4 and Figure 5 ) and HIC( Figure 6) was observed for oxidation of methionine and tryptophan residues in the samples stored at 40°C. Note that an increase in observed physical and chemical degradation was detected for samples stored for up to 4 weeks at 40°C, while no changes were observed for those samples stored for up to 3 months at 5°C. As captured in the data summary, results from other attributes such as charged variants (by IEX) and subvisible particles (by MFI) showed minimal to no changes with changing peroxide, metal, light, or fill volume levels.

[0653] An overall inspection of the data from the study showed that exposure to light and metal stress were the most important factors affecting oxidation of anti-ILT4 / anti-PD-1 co-formulations. Nevertheless, the effects from these stressors were reduced by increasing the concentration of L-methionine. Therefore, the data suggest that a concentration of 15mM L-methionine provides the lowest level of aggregation and chemical degradation. It is worth noting that such L-methionine concentrations are only effective for anti-ILT4 / anti-PD-1 co-formulations, as no improvements in physical and chemical properties were observed for anti-ILT4 monoclonal antibody SE and pembrolizumab SE at L-methionine concentrations greater than 10mM.

[0654] Example 7: UP-SEC (Ultra-High Performance Size Exclusion Chromatography)

[0655] The increase of high molecular weight species (HMW) and low molecular weight species (LMW) indicates protein degradation via aggregation and fragmentation. The figure (data not shown) indicates that for samples at 40C, higher light exposure levels provide higher %HMW and %LMW and increase over time, while it gives lower monomers and further decreases over time.

[0656] Statistical analysis of the data was performed to assess the effects from different formulation and process factors (ie, L-methionine concentration, peroxide content, metal content, light exposure and fill volume). Based on this analysis, contour plots are presented only for the % HMW for all three formulations.

[0657] Figure 7 Depicted are contour plots for HMW co-formulations, where predicted values ​​at 40°C are derived from model fits for different settings of three key parameters (L-methionine, light exposure, metal content) over time. The contour plots show that predicted HMW levels increase with increasing light stress and lower levels of L-Met.

[0658] Similar to co-formulated preparations, e.g. Figure 8 As shown in , the contour plot for anti-ILT4 monoclonal antibody SE shows that with increasing light exposure and metal content, the level of HMW also increases when the L-methionine concentration is below 10 mM.

[0659] Contour plots for pembrolizumab SE are shown in Fig. 9 Similar to anti-ILT4 SE, statistical analysis showed that the levels of HMW increased with light exposure and metal content when the L-methionine concentration was below 10 mM.

[0660] Changes in charge variants can be the result of oxidation, deamidation, and isomerization. Variability plots (data not shown) indicate that, under all conditions, higher light stress levels increase % acidic variants and % basic variants and decrease % primary. The same conclusions can also be seen in the charge variants of pembrolizumab (for both co-formulations and pembrolizumab SE).

[0661] Statistical analysis of the data was performed to evaluate the effects from different formulation and process factors (i.e., L-methionine concentration, peroxide content, metal content, light exposure, and fill volume). Based on this analysis, Figure 10-18 Contour plots of % acidic variant, % major and % basic variant for all three formulations are given in .

[0662] Fig.10 The contour plots show the predicted values ​​for the % acidic variants of anti-ILT4 in co-formulations at different factor levels at 40 °C for 4 weeks.

[0663] Fig.11 The contour plot of shows the % major for anti-ILT4 in the co-formulations according to different factors (L-methionine concentration, light exposure and metal content). The figure illustrates that the increase of light stress reduces the % major for anti-ILT4.

[0664] Fig.12 The contour plot of shows the % basic variants of anti-ILT4 in the co-formulations according to different factors (L-methionine concentration, light exposure and metal content). The figure illustrates that the increase of light stress increases the % basic variants of anti-ILT4.

[0665] Fig.13 The contour plot of shows the predicted values ​​from the model fit at different settings of the three parameters (L-methionine concentration, light exposure, metal content) over time. The contour plot shows that as light stress increases, the predicted levels of % acidic variants of pembrolizumab in the co-formulation remain constant.

[0666] Fig.14 The contour plot of shows the % α for pembrolizumab in the co-formulation according to different factors (L-methionine concentration, light exposure and metal content). The figure illustrates that light stress and increase in metal content reduce the % α for pembrolizumab. In contrast, increasing the L-methionine concentration above 10 mM reduces the rate of α reduction after light stress.

[0667] Fig.15 The contour plot of shows the % basic variants for pembrolizumab in the co-formulations according to different factors (L-methionine concentration, light exposure and metal content). The figure illustrates that the increase of light stress reduces the % basic variants for pembrolizumab, while increasing the L-methionine concentration above 10 mM reduces the rate of the major reduction after light stress.

[0668] Fig.16 The contour plot of shows the predicted values ​​from the model fit at different settings of three parameters (L-methionine concentration, light exposure, metal content) over time. The contour plot shows that as light stress increases, the predicted levels of % acidic variants of anti-ILT4 in the anti-ILT4 SE preparations increase.

[0669] Fig.17 The contour plot of shows the % major for anti-ILT4 in anti-ILT4 SE formulations according to different factors (L-methionine concentration, light exposure and metal content). The figure illustrates that light stress and increase in metal content reduce the % major for anti-ILT4.

[0670] Fig.18 The contour plot of shows the % basic variants of anti-ILT4 in anti-ILT4 SE formulations according to different factors (L-methionine concentration, light exposure and metal content). The figure illustrates that an increase in light stress increases the % basic variants of anti-ILT4.

[0671] Overall, the data provide key information about the five key parameters for studying the oxidation process. In some responses, some values ​​that deviate from the trend introduce noise in the data and make statistical interpretation difficult. However, an overall review of the data based on statistical models, predictive analyzers and contour maps was completed to propose the final formulations for co-formulations and anti-ILT4 SE formulations. Based on the responses of the properties of measuring oxidation, high levels of light stress always have a significant effect on all responses. L-methionine concentration and peroxide and metal content are also significant for some responses. Metal content has a significant effect for pembrolizumab SE formulations in some cases. Fill weight does not have any significant effect in most responses, and the factor range can be maintained at the proposed 1.2mL to 3.2mL level. For prediction purposes, the fill volume is set to a target level of 2.2mL.

[0672] Based on statistical predictions, data summaries, graphs and scientific input for key responses (UPSEC, IEX, PS-80, L-methionine, turbidity), the final formulations selected for SE and co-formulated products are given in Table 8.

[0673] Table 8: Final formulation composition of co-formulations and anti-ILT4 SE formulations

[0674]

[0675] The long-term stability of two lead formulation prototypes was evaluated in this study. The final two compositions of the anti-ILT4 / anti-PD-1 co-formulations (with 10mM and 15mM L-methionine, respectively) were staged for stability at 5°C for 12 months and at 25°C and 40°C for 6 months. The formulation compositions of co-formulation 1 (CF 1, 15mM L-methionine) and co-formulation 2 (CF2, 10mM L-methionine) are captured in Table 9. All data are captured in Tables 10–12. Excess 5mM L-methionine was spiked as a powder and gently mixed for dissolution. Samples were analyzed by HIC, IEX, SEC, L-methionine content, PS-80 content, sub-visible particles, and reduced peptide mapping assays.

[0676] like Fig.19 The L-methionine concentrations at all stability time points for all three formulations are shown in Figure 1. The results show that the effects from all different factors on the stability of L-methionine are negligible.

[0677] Table 9. Composition of anti-ILT4 / anti-PD-1 co-formulations used for pre-probing stability

[0678]

[0679]

[0680]

[0681]

[0682]

[0683] Example 8: Pre-detection stability study of anti-ILT4 / anti-PD-1 co-formulation

[0684] like Fig. 20 The turbidity values ​​for all three formulations at 5°C and 40°C are shown in Figure . Fig. 20 Statistical analysis of the data in to evaluate the effects from different formulations and process factors (i.e., L-methionine concentration, peroxide content, metal content, light exposure, and fill volume). Based on this analysis, a predictive analyzer for turbidity for all three formulations was given. An increase in solution turbidity indicates increased aggregation in the protein formulation.

[0685] Figure 21-25Peptide mapping (PTM) for single entities and co-formulations at initial and 40°C conditions are depicted. For a subset of conditions, key response data on PTMs were collected at 40°C for co-formulations and anti-ILT4 SE formulations at initial and 4 weeks. A statistical model was fitted for all PTM responses, with main effects and two-way interactions with 2 stability time points at 40°C. Reduced peptide mapping was used to monitor M105 oxidation for anti-PD-1 and multiple oxidation hotspots for anti-ILT4 monoclonal antibodies within anti-ILT4 / anti-PD-1 co-formulations. Data on PTMs supported the final formulations selected for co-formulations and anti-ILT4 SE formulations. Specifically, Fig.21 Depicts M253 (shared), Fig. 22 Depicts M429 (shared), Fig.23 W7 (anti-ILT4) is depicted, Fig.24 W102 (anti-ILT4) is depicted, while Fig.25 M105 (pembrolizumab) is depicted.

[0686] M105 oxidation on the anti-PD-1 mAb component is a known adverse factor from experience with other pembrolizumab (anti-PD-1 mAb) co-formulated products, and the effect of adding L-methionine (15 mM for CF 1) on stability was evaluated by monitoring changes in the M105 residue. Fig.25 An increasing trend for stability at 6 months at 40°C is depicted for all residue pairs for CF 1 and CF 2. CF 1 (15 mM L-methionine) showed a marginal decrease in M105 oxidation at 3 and 6 months at 40°C compared to CF 2 (10 mM L-methionine). A slight increase in the M105 residue was observed after 6 months at 25°C and 12 months at 5°C, however, the slope of the increase was low, indicating a minimal impact on stability at the recommended 5°C storage conditions. Similarly, a stability trend was observed for oxidation of the anti-ILT4 monoclonal antibody W102 and W7 residues in CF 1 and CF 2, where oxidation increased after 6 months at 40°C and there was no measurable change after 12 months at the recommended 5°C storage conditions.

[0687] Soluble aggregates (UP-SEC)

[0688] like Fig.26As shown in the graph, high molecular weight (HMW) and low molecular weight (LMW) species as determined by UP-SEC in co-formulation 1 (CF1) and co-formulation 2 (CF2) when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles), and 40°C (downward triangles). The lines correspond to third-order polynomial fits, and they are used only for guidance in the figures. Both formulations show an increasing trend in HMW with respect to stability over 6 months of storage at 40°C. There is a slight increase in HMW over 6 months at 25°C. The aggregate content did not change for up to 12 months at 5°C, indicating adequate physical stability under the recommended storage conditions. No measurable differences were observed in soluble aggregates between co-formulation prototypes with varying L-methionine content from 10 mM to 15 mM.

[0689] Charge variants (HP-IEX)

[0690] like Fig. 27 As shown in the graphs, anti-ILT4 and pembrolizumab charge variants as determined by IEX in CF 1 and CF 2 when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles), and 40°C (downward triangles). The lines correspond to third-order polynomial fits and are used only for guidance in the figures.

[0691] CF 1 and CF 2 were evaluated for chemical degradation by IEX combined method, where two single entities (anti-ILT4 mAb and pembrolizumab) were separated for main peak and acidic / basic charged variants. Both formulations showed a decrease in the main peak based on stability and an increase in acidic and basic variants at 40°C. A slight decrease in the main peak and an increase in charge variants were observed for 6 months at 25°C, while minimal changes in charge species were observed for 12 months at 5°C. No measurable differences in chemical degradation were observed in the co-formulation prototypes as the L-methionine content increased to 15 mM.

[0692] like Fig.28 Shown in FIG. 1 are the total L-methionine contents in CF 1 and CF 2 when stored for 12 months at 5° C. (circles), 6 months at 25° C. (downward triangles) and 40° C. (upward triangles).

[0693] like Fig.29As shown in the graph, the total PS-80 content in CF1 and CF2 when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles) and 40°C (downward triangles). The lines correspond to third-order polynomial fits, and they are used only for guidance in the figures. After 6 months storage at 25°C and 40°C, a temperature-dependent decrease in PS-80 was observed. Within the analytical assay variability, no changes were observed for the PS-80 content in CF1 and CF2 after 12 months storage at 5°C.

[0694] Fig.30 Subvisible particle counts in CF1 and CF2 are shown when stored for 12 months at 5°C (light grey), 6 months at 25°C (dark grey), and 40°C (black). Regarding stability, a stability temperature-dependent increase in particle count was observed for both CF1 and CF2 formulations. Overall, particle counts for both smaller particles (≥2 μm and ≥5 μm) and larger particles (≥10 μm and ≥25 μm) remained low with respect to stability.

[0695] Fig.31 Shown is the oxidation of M105 (anti-PD-1), M429 (shared residues) and M253 (shared residues) when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles) and 40°C (downward triangles), as determined by reduced peptide mapping of CF 1 and CF 2. The line corresponds to a third order polynomial fit and it is used only for guidance in the figures.

[0696] Fig.32 Shown is the oxidation of W102 (anti-ILT4 monoclonal antibody) and W7 (anti-ILT4 monoclonal antibody) when stored for 12 months at 5°C (circles), 6 months at 25°C (upward triangles) and 40°C (downward triangles), as determined by reduced peptide mapping of CF 1 and CF 2. The line corresponds to a third order polynomial fit and it is used only for guidance in the figures.

[0697] Formulation studies with fixed dose ratios of anti-ILT4 mAb and anti-PD-1 mAb indicated the feasibility of developing an anti-ILT4 / anti-PD-1 co-formulation to accommodate a fixed dose of 800 mg of anti-ILT4 mAb and 200 mg of anti-PD-1 mAb.

[0698] Based on the experimental design and preliminary exploratory stability studies on co-formulations, the following conclusions were drawn:

[0699] • The anti-ILT4 / anti-PD-1 co-formulation had a pH of 5.5, which was identified as optimal for stability.

[0700] L-histidine buffer (10 mM) was confirmed as an acceptable buffer system for anti-ILT4 / anti-PD-1 co-formulation.

[0701] • 10 mM L-histidine pH 5.5 matrix supplemented with sucrose at a concentration of 70 mg / mL as a heat stabilizer.

[0702] • PS80 at 0.25 mg / mL was chosen as the surfactant of choice based on its ability to improve stability against agitation-induced stress.

[0703] L-methionine was added as an antioxidant at 2.24 mg / mL (15 mM) to minimize oxidation under light stress.

[0704] The formulation feasibility (experimental design) study evaluated the thermal stability of the anti-ILT4 / anti-PD-1 co-formulation at 5°C for 12 weeks and at 40°C for 4 weeks after subjecting the samples to different stress conditions (process and formulation factors).

[0705] Example 9: Manufacturing and DP Image Considerations

[0706] Diluent selection:

[0707] For the anti-ILT4 / anti-PD-1 co-formulation, process diluents will be added during the drug product manufacturing process in order to maintain the recommended levels of L-methionine (15mM or 2.24mg / mL) and PS-80 (0.25mg / mL) in the final composition. Based on the scope of previous co-formulation manufacturing experience at the DP manufacturing site (Carlow), an aqueous solution for injection containing 0.29mg / mL L-histidine, 1.71mg / mL L-histidine monohydrochloride monohydrate, 17.46mg / mL L-methionine, 70mg / mL sucrose and 0.45mg / mL polysorbate 80 was selected as the process diluent of the optimal composition. In order to maintain the drug product ratio and accommodate the diluent in an appropriate fill volume, the final ratio was optimized to 8:1:4 (v:v:v) of anti-ILT4 monoclonal antibody: diluent: anti-PD-1 monoclonal antibody.

[0708] Fill volume:

[0709] The final fill volume is optimized based on the following considerations:

[0710] 1. Dilution to ensure that the ratio of 8:1:4 of anti-ILT4:diluent:anti-PD-1 is maintained in the final DP image

[0711] 2. Round the recoverable fill volume to the appropriate decimal place so that the healthcare professional can use the syringe's calibration to accurately measure the DP volume (e.g., 26.00 ml is the preferred volume to draw 26.7 mL using a 30 mL syringe)

[0712] The final composition of 30.8 mg / mL anti-ILT4 monoclonal antibody, 7.7 mg / mL anti-PD-1 monoclonal antibody, 0.29 mg / mL L-histidine, 1.71 mg / mL L-histidine monohydrochloride monohydrate, 2.24 mg / mL L-methionine, 70 mg / mL sucrose, 0.25 mg / mL polysorbate 80 at pH 5.5 was found to be stable under refrigerated conditions (Table 12).

[0713] The stability of the final drug product was evaluated under storage conditions of 5°C ± 3°C, accelerated storage conditions of 25°C / 60% RH ending after six months, and stress conditions of 40°C / 75% RH ending after six months.

[0714] Tables 13-15 below summarize the stability data for the co-formulations. Under the recommended storage condition of 5°C, there were no significant changes in stability observed for the final drug product from any assay. Test results for visible particles under all conditions evaluated so far are reported as "liquids are substantially free of visible particles", which meets the acceptance criteria. Observations do not change due to storage conditions or time. Tables 13-15 capture the potency stability data under storage conditions of 5°C, 25°C, and 40°C. Across the accelerated condition of 40°C, there is a decrease within the first 6 months. Under the stress condition of 25°C and the recommended condition of 5°C, very small changes in potency are visible. For the anti-PD-1 monoclonal antibody component, a small increase in concentration is visible under the stress condition of 6M. Across all time points and conditions, no other changes were observed in protein concentration.

[0715] Table 13. Composition of anti-ILT4 / anti-PD-1 co-formulated drug product

[0716]

[0717]

[0718] a Provides 1.0 mL overfill to ensure label-required recovery of 800 mg / vial of anti-ILT4 mAb and 200 mg / vial of anti-PD-1 mAb b qs = sufficient quantity; pH ​​of the preparation is 5.5

[0719] Studies were conducted to demonstrate that the amount of PS-80 in the final composition protects against freeze-thaw (F / T) and agitation-induced particle formation and aggregation. Freeze-thaw (F / T) and agitation stress studies were performed on selected formulation compositions listed in Table 14. As shown below, two F / T excursion conditions were studied; a. a temperature excursion study consisting of up to five temperature exposure cycles alternating between -40°C and 25°C; and b. a temperature excursion study consisting of up to five temperature exposure cycles alternating between -80°C and 25°C.

[0720]

[0721] Agitation stress studies were conducted at ambient conditions with drug product vials agitated at 300 rpm for 3 and 7 days as shown in Table 15. Drug product vials also at ambient conditions but not subjected to agitation served as controls.

[0722]

[0723] After freeze-thaw exposure for up to five freeze-thaw cycles (5XF / T) between -40°C and 25°C, there was no change in turbidity, high molecular weight species, charge variants, and subvisible particle counts by MFI. A decrease in PS-80 and L-methionine content was observed for 5X F / T at -40°C to 25°C and -80°C to 25°C. The results are captured in Table 20.

[0724] After agitation at 300 rpm for up to 7 days under ambient temperature conditions, there were no changes in turbidity, high molecular weight species, charge variants, L-methionine content, and PS-80 content. For particles ≥2 μm and ≥5 μm, a small increase in subvisible particle counts was observed in the MFI results for the drug product for 7-day agitation compared to the 7-day control. The results are captured in Table 21.

[0725] Data from freeze-thaw stress and agitation stress studies did not show any significant property changes, indicating that the selected S-80 level (0.25 mg / mL) in the final co-formulation composition was sufficient to protect against freeze-thaw and agitation-induced particle formation and aggregation.

[0726] Example 10: Transparency and opalescence of pharmaceutical products

[0727] The test results for clarity and opalescence of the drug product are captured in Tables 16-18. The results from the color analysis of the drug product at all conditions evaluated to date meet the acceptance criteria of "not more intense than reference Y5". No significant changes in PS-80 content were observed in the 5°C condition until nine months. The 25°C and 40°C conditions trended downward until six months, but were still within specifications.

[0728] No changes in L-methionine were observed across conditions and time points throughout the study.

[0729] The nine-month HP-IEX data showed that there was no significant change in acidic variants after storage under the proposed 5°C storage conditions. At 25°C, there was a slight increase in % acidic variants, with a significant increase at 40°C until six months. For anti-PD-1 monoclonal antibodies, no changes were observed in % basic variants across all conditions studied, with a reduction visible at 40°C for anti-ILT4 / anti-PD-1 co-formulations until six months. For anti-ILT4 / anti-PD-1 co-formulations, a significant reduction in % major was seen at 40°C, with a smaller reduction observed for anti-PD-1 monoclonal antibodies until 6 months. For anti-ILT4 / anti-PD-1 co-formulations, a slight reduction was also observed at 25°C. For the remaining conditions until nine months, no changes were observed for any component. For the % combined variants of the tested formulations, no changes were observed across conditions and time points. Anti-PD-1 monoclonal antibody % was slightly reduced until six months, with no changes for the remaining conditions explored in the study. For the tested formulations, no changes were observed for % mixed basic variants across conditions until nine months.

[0730] For the formulations tested through 9 months, no changes were observed in % monomer % or high molecular weight species at 5C and 25C. At 40°C, a gradual decrease was seen until six months, with the results for % monomer being below specification at six months. After three months, the results for % high molecular weight species at 40°C exceeded the upper specification limit.

[0731] An increase in % low molecular weight species was observed for the 40°C condition, which was below the specification limit after two months, where no changes were observed for the formulations tested at the 5C and 25°C conditions.

[0732] For the formulations tested, no appreciable changes were observed in the % intact IgG at 5C and 25C conditions. Results from the 40C condition showed a decline near the six month time point, with the data falling below specification after three months.

[0733] The formulations tested showed no observed changes for % heavy chain + light chain under the recommended storage condition of 5C and stress condition of 25C. At 40C, the % heavy chain + light chain results decreased and were below specification after three months. Across the conditions and time points for the formulations tested, no changes were observed for the duration of the study.

[0734] Throughout the study and across various conditions, particle counts were within specifications for the formulations tested. Results from the full analytical test panel were within acceptance criteria for up to 9 months under recommended storage conditions.

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741] Example 11: Ion Exchange (IEX) Method for Measuring Acidic Species of Anti-PD-1 Antibodies

[0742] For the IEX method, a Waters Alliance LC system (Milford, MA, USA) was used, and ThermoScientific's ProPac WCX-10 (p / n: 054993, particle size 10 um, diameter 4 mm, length 250 mm) was selected with a loading of 80 μg of sample. Mobile phase (A) 24 mM MES pH 6.1 with 4% acetonitrile, and mobile phase (B) 20 mM sodium phosphate, 95 mM NaCl pH 8.0 with 4% acetonitrile were used as nonlinear sigmoidal pH gradients, and the separation was monitored over 34 minutes with a flow rate of 0.5 mL min-1, with a column temperature of 35°C. The gradient used was: 22%–22% B for 0–0.6 min; 22%–29% B for 0.6–15.0 min; 29%–70% B for 15.0–30.0 min; 70%–100% B for 30.0–30.5 min; and 100%–100% B for 30.5–33.0 min. 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 min, followed by re-equilibration with 22% B at 1.0 mL min-1 from 34.5–44.5 min. From 44.5–45 min, the flow rate was reduced to 0.5 mL min-1. Elution was monitored at 280 nm for peak detection. Assay variability was determined to be within 1%.

[0743] U.S. Provisional Application No. 63 / 143,461 is incorporated by reference in its entirety. All references cited herein are incorporated by reference to the extent that each individual publication, database entry (e.g., GenBank sequence or GeneID entry), patent application or patent specifically and individually indicates incorporation by reference. Each and every individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application or patent is clearly identified in accordance with 37 C.FR § 1.57 (b) (2) individually, even if such citations are not immediately adjacent to the special statement incorporated by reference. The inclusion (if any) of the special statement incorporated by reference in the specification does not in any way weaken the general statement incorporated by reference. The citation of this paper reference does not mean that the reference is recognized as a related prior art, nor does it constitute any recognition of the content or date of these publications or files. The definition provided in this specification should be used to explain the present invention insofar as the reference provides a definition of the claimed term that conflicts with the definition provided in this specification.

Claims

1. A preparation comprising: (i) about 10 mg / mL to about 200 mg / mL of an anti-human immunoglobulin-like transcript 4 ("anti-ILT4") antibody or an antigen-binding fragment thereof; (ii) about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof; (iii) a buffer of about 5 mM to about 20 mM; (iv) about 6% to about 8% weight / volume (w / v) non-reducing sugars; (v) from about 0.01% to about 0.10% (w / v) of a nonionic surfactant; and (vi) about 1 mM to about 20 mM of an antioxidant, 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, 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), The anti-PD-1 antibody or antigen-binding fragment thereof comprises: a variable light chain region comprising: CDRL1 of SEQ ID NO:95, CDRL2 of SEQ ID NO:96, and CDRL3 of SEQ ID NO:97, and a variable heavy chain region comprising: CDRH1 of SEQ ID NO: 100, CDRH2 of SEQ ID NO: 101, and CDRH3 of SEQ ID NO: 102, wherein the buffer is L-histidine buffer, acetate buffer or citrate buffer, wherein the non-reducing sugar is a disaccharide, wherein the nonionic surfactant is polysorbate 20 or polysorbate 80, and The antioxidant is methionine or a pharmaceutically acceptable salt thereof.

2. The formulation of claim 1, comprising 40-60 mg / mL of an anti-ILT4 antibody; 15-35 mg / mL of an anti-PD-1 antibody; about 7% sucrose or trehalose; about 0.025% polysorbate 80 or polysorbate 20; about 3-30 mM L-histidine buffer at a pH of about 5.0-6.5; and about 5-20 mM methionine or a pharmaceutically acceptable salt thereof.

3. The formulation of claim 1, comprising 45-55 mg / mL of an anti-ILT4 antibody; 20-30 mg / mL of an anti-PD-1 antibody; about 7% sucrose or trehalose; about 0.025% polysorbate 80 or polysorbate 20; about 3-30 mM L-histidine buffer at a pH of about 5.0-6.5; and about 10-20 mM L-methionine or a pharmaceutically acceptable salt thereof. The formulation according to claim 1 , wherein the buffer is an L-histidine buffer.

5. The formulation of any one of claims 1-4, wherein the non-reducing sugar is sucrose.

6. The formulation of any one of claims 1-5, wherein the non-ionic surfactant is polysorbate 80.

7. The formulation of any one of claims 1-6, wherein the antioxidant is L-methionine.

8. A preparation comprising: (i) about 10 mg / mL to about 200 mg / mL of an anti-ILT4 antibody ("anti-ILT4") or an antigen-binding fragment thereof; (ii) about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof; (iii) about 5 mM to about 20 mM L-histidine buffer; (iv) about 6% to about 8% weight / volume (w / v) sucrose; (v) about 0.01% to about 0.10% (w / v) polysorbate 80; and (vi) about 1 mM to about 20 mM L-methionine, 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, 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); and The anti-PD-1 antibody or antigen-binding fragment thereof comprises: a variable light chain region comprising: CDRL1 of SEQ ID NO:95, CDRL2 of SEQ ID NO:96, and CDRL3 of SEQ ID NO:97, and A variable heavy chain region comprising: CDRH1 of SEQ ID NO: 100, CDRH2 of SEQ ID NO: 101, and CDRH3 of SEQ ID NO: 102, or a pharmaceutically acceptable salt thereof.

9. The formulation of claim 8 comprising about 8 mM to about 12 mM L-histidine buffer.

10. The formulation of claim 8 or 9 comprising about 5 mM to about 10 mM L-methionine.

11. The formulation of any one of claims 8-10, comprising about 0.01% to about 0.05% (w / v) polysorbate 80.

12. The formulation of any one of claims 1 or 4-11, comprising about 10 mg / mL to about 150 mg / mL of an anti-ILT4 antibody or antigen-binding fragment thereof.

13. The formulation of any one of claims 1 or 4-11, comprising about 10 mg / mL to about 150 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof.

14. The formulation of claim 12, wherein the concentration of the anti-ILT4 antibody or 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.

15. The formulation of claim 12, wherein the concentration of the anti-PD-1 antibody or 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.

16. The formulation of any one of claims 1-13, comprising about 25 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 10 mg / mL of an anti-PD-1 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.

17. The formulation of any one of claims 1-13, comprising about 50 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 25 mg / mL of an anti-PD-1 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.

18. The formulation of any one of claims 1-13, comprising about 75 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 50 mg / mL of an anti-PD-1 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.

19. The formulation of any one of claims 1-13, comprising about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 75 mg / mL of an anti-PD-1 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.

20. The formulation of any one of claims 1-13, comprising about 125 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof, about 100 mg / mL of an anti-PD-1 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.

21. The formulation of any one of claims 1-20, wherein the formulation has a pH of about 5.0 to about 6.

8.

22. The formulation of any one of claims 1-21, wherein the formulation has a pH of about 5.5 to about 6.

0.

23. The formulation of any one of claims 1-22, wherein the formulation has a pH of about 5.

5.

24. A preparation of an anti-human immunoglobulin-like transcript 4 ("anti-ILT4") antibody or an antigen-binding fragment thereof, comprising: (i) about 50 mg / mL to about 100 mg / mL of an anti-ILT4 antibody or an antigen-binding fragment thereof; (ii) about 10 mg / mL to about 200 mg / mL of an anti-PD-1 antibody or antigen-binding fragment thereof; (iii) about 10 mM L-histidine buffer, pH about 5.5; (iv) about 7% weight / volume (w / v) sucrose; (v) about 0.025% (w / v) polysorbate 80; and (vi) about 10 mM L-methionine, 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, 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), and The anti-PD-1 antibody or antigen-binding fragment thereof comprises: a variable light chain region comprising: CDRL1 of SEQ ID NO:95, CDRL2 of SEQ ID NO:96, and CDRL3 of SEQ ID NO:97, and A variable heavy chain region comprising: CDRH1 of SEQ ID NO:100, CDRH2 of SEQ ID NO:101, and CDRH3 of SEQ ID NO:

102.

25. The formulation of any one of claims 1-24, 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: 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, 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).

26. The formulation of any one of claims 1-24, 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).

27. The formulation of any one of claims 1-24, 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.

28. The formulation of any one of claims 1-24, 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.

29. The formulation of any one of claims 1-24, 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.

30. The preparation according to any one of claims 1-24, 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.

31. The preparation according to any one of claims 1-24, 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.

32. The preparation according to any one of claims 1-24, 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.

33. The formulation of any one of claims 1-24, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: a variable light region comprising the amino acid sequence shown in SEQ ID NO: 98 and a variable heavy region comprising the amino acid sequence shown in SEQ ID NO:

103.

34. The formulation of any one of claims 1-24, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises: a variable light region comprising the amino acid sequence shown in SEQ ID NO: 99 and a variable heavy region comprising the amino acid sequence shown in SEQ ID NO:

104.

35. The formulation according to any one of claims 1-24, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof consists of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence shown in SEQ ID NO: 99, and the two heavy chains consist of the amino acid sequence shown in any one of SEQ ID NOs: 104-109.

36. The formulation of any one of claims 1-35, wherein the anti-human PD-1 antibody is pembrolizumab.

37. The formulation of any one of claims 1-36, wherein the ratio of anti-ILT4 antibody to anti-PD-1 antibody is 1:1 or 1:

2.

38. The formulation of any one of claims 1-37, wherein after 9 months at 5°C: (i) the % monomer of the anti-ILT4 antibody is ≥ 95% as determined by size exclusion chromatography; (ii) the % heavy chain and light chain of the anti-ILT4 antibody is ≥ 90% as measured by reducing CE-SDS; (iii) the % heavy chain and light chain of the anti-ILT4 antibody is ≥ 95% as measured by reducing CE-SDS; (iv) the % intact IgG of the anti-ILT4 antibody is ≥ 90% as measured by non-reducing CE-SDS; and / or (v) the % intact IgG of the anti-ILT4 antibody is ≥ 95% as measured by non-reducing CE-SDS; 39. The formulation of any one of claims 1-38, wherein the formulation is a liquid formulation stored at about 3°C ​​to about 5°C, or is a reconstituted solution from a lyophilized formulation.

40. The formulation of any one of claims 1-24, wherein after storage for up to 9 months at about 3°C ​​to about 5°C: (i) the % monomer of the anti-ILT4 antibody or antigen-binding fragment thereof is at least about 99% as determined by ultra-performance size exclusion chromatography; (ii) If through OD 350-500 The turbidity of the formulation is measured to be at most about 0.135; (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) a subvisible particle count of at most about 3500 particles having a size of at least 2 μm as determined by microfluidic 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 set forth in SEQ ID NO: 2 or 80 is less than about 4% as determined by reduced peptide mapping analysis.

41. The formulation of any one of claims 1-34 or 37-41, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is a monoclonal antibody.

42. A method of treating cancer in a human patient in need thereof, the method comprising administering an effective amount of the formulation of any one of claims 1-41.

43. The method of claim 42, wherein the cancer is selected from the group consisting of colorectal cancer, esophageal cancer, melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, and small cell lung cancer.

44. Use of a formulation according to any one of claims 1 to 41 for the preparation of a medicament for the treatment of cancer.

45. Use of a formulation according to any one of claims 1-42 for treating cancer in a human patient.

46. ​​The use according to claim 45, 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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