Anti-TREM-1 antibody and its use

Abstract

Provided herein are antibodies, or antigen-binding portions thereof, that specifically bind to TREM-1 and inhibit TREM-1 signaling, which do not bind to one or more FcγRs and do not induce myeloid cells to produce proinflammatory cytokines. Use of such antibodies, or antigen-binding portions thereof, in therapeutic applications, such as the treatment of autoimmune diseases, is also provided.

Description

Technical Field

[0001] Reference to a Sequence Listing Electronically Submitted via EFS-Web The content of the Sequence Listing electronically submitted in an ASCII text file (name: 3338_092PC01_SeqListing.txt; size: 106,162 bytes; and creation date: March 27, 2019) submitted together with this application is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] TREM-1 is an activating receptor expressed on monocytes, macrophages, and neutrophils. These cells play a central role in chronic inflammatory diseases by releasing cytokines and other mediators that drive inflammation. The mRNA and protein expression of TREM-1 are upregulated in patients with rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and TREM-1-positive cells accumulate at the sites of inflammation in correlation with the severity of the disease. See Bouchon et al., Nature 410:1103-1107 (2001); Schenk et al., Clin Invest 117:3097-3106 (2007); and Kuai et al., Rheumatology 48:1352-1358 (2009). Peptidoglycan recognition protein 1 (PGLYRP1), which is mainly expressed by activated neutrophils, is a ligand of TREM-1 and mediates TREM-1 signaling upon binding. In vitro, binding of TREM-1 induces secretion of pro-inflammatory cytokines including TNF, IL-8, and monocyte chemoattractant protein-1. Further, TREM-1 signaling synergizes with multiple Toll-like receptors (TLRs) to further boost pro-inflammatory signals. This then results in an adverse cycle of upregulating the expression of TREM-1 to amplify inflammation. See Bouchon et al., J Immunol 164:4991-4995 (2000). Increasing evidence indicates that TLRs contribute to the development and progression of chronic inflammatory diseases such as RA and IBD.

[0003] Humanized anti-TREM-1 mAbs that inhibit the function of TREM-1 in both humans and cynomolgus monkeys are disclosed elsewhere. See WO2013 / 120553 and WO2016 / 009086. However, such antibodies either have a viscosity profile that can interfere with the manufacturing process or have other issues (such as cytokine storms and ADCC) that can limit their therapeutic potential. See Shire et al., J. Pharm. Sci. 93:1390-1402 (2004); and Warncke et al., J Immunol. 188:4405-11 (2012). Accordingly, there is a need for anti-TREM-1 antibodies that can specifically bind to TREM-1 and inhibit TREM-1 function without the problems of the initial anti-TREM-1 antibodies. SUMMARY OF THE INVENTION

[0004] Isolated antibodies, e.g., monoclonal antibodies, particularly human (e.g., monoclonal) antibodies, that specifically bind to Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) and have desirable functional properties are provided herein. In some embodiments, the antibody comprises a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, and the IgG1 heavy chain constant region comprises one or more amino acid substitutions as compared to the wild-type IgG1 heavy chain constant region (SEQ ID NO: 9). In some embodiments, the antibody cross-competes with mAb 0318 for binding to block TREM-1, and comprises a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, and the IgG1 heavy chain constant region comprises one or more amino acid substitutions as compared to the wild-type IgG1 heavy chain constant region (SEQ ID NO: 9). In some embodiments, the antibody binds to the same TREM-1 epitope as mAb 0318. In some embodiments, the antibody specifically binds to a TREM-1 epitope comprising one or more amino acid residues selected from the group consisting of D38, V39, K40, C41, D42, Y43, T44, L45, E46, K47, F48, A49, S50, S51, Q52, K53, A54, W55, Q56, Y90, H91, D92, H93, G94, L95, and L96 of SEQ ID NO: 1. In some embodiments, the antibody specifically binds to a TREM-1 epitope comprising amino acids D38-L45, E46-Q56, and / or Y90-L96 of SEQ ID NO: 1.

[0005] In some embodiments, the IgG1 heavy chain constant region of the antibodies disclosed herein comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, and L358M according to EU numbering. In some embodiments, this IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M according to EU numbering. In some embodiments, this IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S according to EU numbering. In some embodiments, this IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S according to EU numbering.

[0006] In some embodiments, the antibodies disclosed herein comprise heavy chain CDR1, CDR2, and CDR3 as well as light chain CDR1, CDR2, and CDR3, and this heavy chain CDR3 comprises DMGIRRQFAY (SEQ ID NO: 26) or DMGIRRQFAY (SEQ ID NO: 26) with one or two substitutions removed. In some embodiments, this heavy chain CDR3 comprises DQGIRRQFAY (SEQ ID NO: 72). In some embodiments, the antibodies disclosed herein comprise heavy chain CDR1, CDR2, and CDR3 as well as light chain CDR1, CDR2, and CDR3, and this heavy chain CDR2 comprises RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) or RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) with one or two substitutions removed. In some embodiments, the antibodies disclosed herein comprise heavy chain CDR1, CDR2, and CDR3 as well as light chain CDR1, CDR2, and CDR3, and this heavy chain CDR1 comprises TYAMH (SEQ ID NO: 24) or TYAMH (SEQ ID NO: 24) with one or two substitutions removed.

[0007] In some embodiments, the antibodies disclosed herein include heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, and this light chain CDR1 includes RASQSVDTFDYSFLH (SEQ ID NO: 27) or RASQSVDTFDYSFLH (SEQ ID NO: 27) excluding one or two substitutions. In some embodiments, this light chain CDR2 includes RASNLES (SEQ ID NO: 28) or RASNLES (SEQ ID NO: 28) excluding one or two substitutions. In some embodiments, this light chain CDR3 includes QQSNQDPYT (SEQ ID NO: 29) or QQSNQDPYT (SEQ ID NO: 29) excluding one or two substitutions.

[0008] In some embodiments, the VH of the antibodies disclosed herein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the VL of the antibodies disclosed herein includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, VH and VL include SEQ ID NOs: 14 and 15, respectively. In some embodiments, the antibodies of the present disclosure include a heavy chain and a light chain, and this heavy chain includes SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 53. In some embodiments, this light chain includes SEQ ID NO: 54.

[0009] An isolated antibody that specifically binds to TREM-1 and comprises heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2 and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively; the light chain CDR1, CDR2 and CDR3 comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively; and the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E and G237A according to EU numbering, is provided herein.

[0010] An isolated antibody that specifically binds to TREM-1 and comprises heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2 and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively; the light chain CDR1, CDR2 and CDR3 comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively; and the IgG1 heavy chain constant region comprises amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S and P331S according to EU numbering, is provided herein.

[0011] An isolated antibody that specifically binds to TREM-1 and comprises heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2, and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; the light chain CDR1, CDR2, and CDR3 comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; and the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S according to EU numbering, is provided herein.

[0012] An isolated antibody that specifically binds to TREM-1 and comprises heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2, and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; the light chain CDR1, CDR2, and CDR3 comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; and the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S according to EU numbering, is provided herein. In some embodiments, TREM-1 comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. In some embodiments, the antibody of the present disclosure has a reduced binding affinity for FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof, as compared to an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, this antibody has a reduced binding affinity for FcγRI (CD64) that is 1 / 2 or less, 1 / 3 or less, 1 / 4 or less, 1 / 5 or less, 1 / 6 or less, 1 / 7 or less, 1 / 8 or less, 1 / 9 or less, or 1 / 10 or less, as compared to an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54.

[0013] In some embodiments, the antibody disclosed herein has lower immunogenicity as compared to an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, this antibody does not agonize TREM-1 signaling in the absence of a stimulator upon binding to TREM-1. In some embodiments, this antibody does not induce the expression of inflammatory cytokines in immature dendritic cells (iDCs) when incubated in the presence of this antibody and in the absence of a stimulator, as compared to an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the antibody disclosed herein blocks the production of inflammatory cytokines in a cell when the cell is activated in the presence of both this antibody and a stimulator. In some embodiments, this stimulator is a TREM-1 ligand. In some embodiments, this inflammatory cytokine is selected from the group consisting of IL-6, TNF-α, IL-8, IL-1β, IL-12, chitinase-3-like protein 1 (CHI3L1), and combinations thereof.

[0014] In some embodiments, the antibodies of the present disclosure bind to human FcRn, cynomolgus FcRn, and / or mouse FcRn in a pH-dependent manner. In some embodiments, the antibodies disclosed herein are more thermally stable compared to a reference antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54 when measured by capillary differential scanning calorimetry (CAP-DSC). In some embodiments, about 10% to 20%, about 20% to 30% (e.g., 24%), or about 30% to 40% of this antibody is reversible when heated to 77°C. In some embodiments, this antibody has a higher melting temperature (Tm) compared to an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54.

[0015] In some embodiments, the antibodies disclosed herein have a viscosity of less than 5 cP, less than 4 cP, less than 3 cP, less than 2.5 cP, less than 2.4 cP, less than 2.3 cP, less than 2.2 cP, less than 2.1 cP, less than 2 cP, less than 1.9 cP, less than 1.8 cP, less than 1.7 cP, less than 1.6 cP, less than 1.5 cP, less than 1.4 cP, less than 1.3 cP, less than 1.2 cP, less than 1.1 cP, less than 1.0 cP, less than 0.9 cP, less than 0.8 cP, less than 0.7 cP, less than 0.6 cP, less than 0.5 cP, less than 0.4 cP, less than 0.3 cP, less than 0.2 cP, or less than 0.1 cP at a concentration of 80 mg / mL. In some embodiments, this antibody has a viscosity of less than 10 cP (e.g., 9 cP) at a concentration of 130 mg / mL. In some embodiments, this antibody has a K D of less than 4 nM (e.g., 3.4 nM) when binding to human TREM-1 as measured by Biacore. In some embodiments, this antibody has a K D of less than 1 nM (e.g., 0.91 nM) when binding to cynomolgus TREM-1.

[0016] In some embodiments, the antibody is monomeric as observed by size exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, the antibody exhibits minimal risk of fragmentation as observed by intact mass analysis using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC / MS) or liquid chromatography-tandem mass spectrometry (LC / MS). In some embodiments, the antibody has an isoelectric point of 8-9 (e.g., 8.75).

[0017] In some embodiments, the antibody is stable in a formulation containing histidine, sucrose, arginine and NaCl. In some embodiments, the antibody is stable for at least two months in a formulation containing 20 mM histidine, 150 mM sucrose, 25 mM arginine and 50 mM NaCl. In some embodiments, the formulation has a pH of 6.0 and / or the formulation is stored at 4°C, 25°C or 40°C. Also provided herein are bispecific molecules comprising an anti-TREM-1 antibody of the disclosure linked to a molecule having a second binding specificity. Also provided herein are nucleic acids encoding the antibodies disclosed herein, vectors comprising the nucleic acids, and cells transformed with the vectors. Also provided herein are immunoconjugates comprising an anti-TREM-1 antibody disclosed herein linked to a drug.

[0018] Also provided herein are compositions comprising an anti-TREM-1 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate described herein, and a carrier. Also provided herein are kits comprising an anti-TREM-1 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate described herein, and instructions for use. Provided herein is a method of inhibiting TREM-1 activity in a subject in need thereof, comprising administering an anti-TREM-1 antibody, bispecific molecule, nucleic acid, vector, cell or immunoconjugate of the present disclosure.

[0019] Provided herein is a method of treating an inflammatory disease or an autoimmune disease in a subject in need thereof, comprising administering an anti-TREM-1 antibody, bispecific molecule, nucleic acid, vector, cell or immunoconjugate of the present disclosure. In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, vasculitis, sepsis, systemic inflammatory response syndrome (SIRS), type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogrens's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, and other autoimmune diseases that are a result of either acute inflammation or chronic inflammation, and any combination thereof. In some embodiments, the method further comprises administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an anti-IP-10 antibody or an anti-TNF-α antibody.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] To make the description easier to understand, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0022] Note that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to indicate one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein.

[0023] Furthermore, "and / or" as used herein should be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" when used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in a phrase such as "A, B and / or C" is intended to encompass each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). When an aspect is described in this specification using the word "comprising", it is understood that other aspects similar in other respects are also provided, described in the turn of phrase "consisting of" and / or "consisting essentially of". Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide many common dictionaries for those of ordinary skill in the art with respect to terms used in this disclosure.

[0024] Units, prefixes, and symbols are denoted in their internationally recognized SI forms. Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in a 5' to 3' orientation. Amino acid sequences are written left to right in an amino to carboxy orientation. The headings provided in this specification are not limitations of the various aspects of the disclosure that can be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. The term "about" is used in this specification to mean approximately, roughly, around, or thereabouts. When the term "about" is used in conjunction with a numerical range, this modifies the range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify the numerical values above and below the stated value by, for example, a difference of up to or down 10 percent (higher or lower).

[0025] The term "triggering receptor expressed on myeloid cells 1" (also known as TREM1, TREM-1, and CD354) refers to a receptor expressed on monocytes, macrophages, and neutrophils. The main ligands of TREM-1 include peptidoglycan recognition protein 1 (PGLYRP1), which belongs to the family of peptidoglycan (PGN)-binding proteins (PGRPs). When activated, TREM-1 associates with the ITAM-containing signaling adapter protein DAP12. Downstream signaling can include activation of the NFAT transcription factor, which causes upregulation of pro-inflammatory cytokine production. The term "TREM-1" includes any variant or isoform of TREM-1 that is naturally expressed by cells. Thus, in some embodiments, the antibodies described herein can cross-react with TREM-1 from species other than human (e.g., cynomolgus monkey TREM-1).

[0026] Three isoforms of human TREM-1 have been identified. Isoform 1 (accession number NP_061113.1; SEQ ID NO: 1) consists of 234 amino acids and exhibits the canonical sequence. Isoform 2 (accession number NP_001229518.1; SEQ ID NO: 2) consists of 225 amino acids and differs from the canonical sequence at amino acid residues 201 - 234. These amino acid residues encode part of the transmembrane domain and the cytoplasmic domain. Isoform 3 (accession number NP_001229519; SEQ ID NO: 3) consists of 150 amino acids and is soluble. It lacks amino acid residues 151 - 234, which encode part of the transmembrane domain, the cytoplasmic domain, and part of the extracellular domain. Amino acid residues 138 - 150 also differ from the above canonical sequence.

[0027] The following are the amino acid sequences of the three known human TREM-1 isoforms. (A) Human TREM-1 isoform 1 (accession number NP_061113.1; SEQ ID NO: 1; nucleotide sequence having accession number NM_018643; encoded by SEQ ID NO: 4): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP (The signal sequence is underlined); (B) Human TREM-1 Isoform 2 (Accession number NP_001229518.1; SEQ ID NO: 2; nucleotide sequence having accession number NM_001242589; encoded by SEQ ID NO: 5): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRYSFQVPGPLVWTLSPLFPSLCAERM (The signal sequence is underlined); (C) Human TREM-1 Isoform 3 (Accession number NP_001229519; SEQ ID NO: 3; nucleotide sequence having accession number NM_001242590; encoded by SEQ ID NO: 6): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFRCSTLSFSWLVDS (The signal sequence is underlined).

[0028] The cynomolgus monkey TREM-1 protein (Accession number XP_001082517; SEQ ID NO: 7) is predicted to have the following amino acid sequence: MRKTRLWGLLWMLFVSELRA TTELTEEKYEYKEGQTLEVKCDYALEKYANSRKAWQKMEGKMPKILAKTERPSENSHPVQVGRITLEDYPDHGLLQVQMTNLQVEDSGLYQCVIYQHPKESHVLFNPICLVVTKGSSGTPGSSENSTQNVYRTPSTTAKALGPRYTSPRTVTQAPPESTVVVSTPGSEINLTNVTDIIRVPVFNIVIIVAGGFLSKSLVFSVLFAVTLRSFGP (The signal sequence is underlined). The present disclosure relates to antibodies that specifically bind to TREM-1 and block its function. These antibodies block TREM-1 function by reducing / blocking TREM-1 activation and downstream signaling.

[0029] The anti-TREM-1 antibodies of the present disclosure block TREM-1 signaling by one or a combination of several different mechanisms that directly or indirectly block TREM-1. In one embodiment, these antibodies prevent the peptide glycan recognition protein 1 (PGLYRP1), a natural ligand of TREM-1, from creating a functional complex with TREM-1. In another embodiment, these antibodies block TREM-1 by preventing individual TREM-1 molecules from forming either dimers or multimers. In some embodiments, dimerization or multimerization of TREM-1 is reduced or prevented by anti-TREM-1 antibodies that can bind to a portion of TREM-1 that would otherwise be present at the interface of the TREM-1 dimer, thereby preventing individual TREM-1 molecules from associating with each other. In other embodiments, dimerization or multimerization of TREM-1 is reduced or prevented by anti-TREM-1 antibodies that interfere with the interaction between TREM-1 and its ligand.

[0030] In some embodiments, the anti-TREM-1 antibody can block the PGLYRP1-induced activation of TREM-1. PGLYRP1 is a highly conserved 196-amino acid-long protein consisting of a signal peptide and a peptidoglycan-binding domain, which is expressed in neutrophils and released upon their activation. The amino acid sequence of PGLYRP1 (accession number NP_005082.1; SEQ ID NO: 8) is provided below: MSRRSMLLAWALPSLLRLGAA QETEDPACCSPIVPRNEWKALASECAQHLSLPLRYVVVSHTAGSSCNTPASCQQQARNVQHYHMKTLGWCDVGYNFLIGEDGLVYEGRGWNFTGAHSGHLWNPMSIGISFMGNYMDRVPTPQAIRAAQGLLACGVAQGALRSNYVLKGHRDVQRTLSPGNQLYHLIQNWPHYRSP (the signal sequence is underlined).

[0031] Accordingly, in some embodiments, the anti-TREM-1 antibodies of the present disclosure downregulate or block the release of pro-inflammatory cytokines from myeloid cells (e.g., dendritic cells and monocytes). In some embodiments, these anti-TREM-1 antibodies block the release of TNF-α, MIP-1 beta, MCP-1, IL-1 beta, GM-CSF, IL-6 and / or IL-8 from macrophages, neutrophils, synovial tissue cells and / or reporter cells, as disclosed herein.

[0032] The controlled release of inflammatory cytokines in response to foreign antigens can be beneficial (e.g., initiating an effective adaptive immune response), but excessive inflammatory cytokine release can have tragic consequences. For example, one common toxic clinical complication observed with the in vivo administration of certain antibodies to cell surface immune receptors (e.g., anti-human CD3 antibodies, such as OKT3) is cytokine release syndrome (CRS) associated with the excessive release of various cytokines (e.g., TNF-alpha, IFN-gamma, and IL-2) into the circulation. CRS can result from the simultaneous binding of the antibody to its cognate antigen (e.g., CD3 on T cells) (via the variable region of the antibody) and Fc receptors (e.g., FcγR) and / or complement receptors on accessory cells (e.g., antigen-presenting cells) (via the constant region of the antibody). This interaction results in the activation of cells (e.g., T cells and / or accessory cells) and the release of various cytokines that characterize a systemic inflammatory response, including hypotension, pyrexia, and rigors. Other symptoms of CRS include fever, chill, nausea, vomiting, and dyspnea.

[0033] In addition to blocking the PGLYRP1-induced production of inflammatory cytokines, in one embodiment, the anti-TREM-1 antibodies of the present disclosure reduce, or do not cause, the incidence of cytokine release syndrome when administered to a subject in need thereof. In some embodiments, these anti-TREM-1 antibodies do not induce the expression of inflammatory cytokines by these cells when the cells (e.g., dendritic cells) are incubated in the presence of the antibody alone, as compared to an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, these anti-TREM-1 antibodies have reduced binding to one or more FcγRs, which may assist in reducing the incidence of CSR.

[0034] In some embodiments, the anti-TREM-1 antibodies of the present disclosure bind to both human TREM-1 and TREM-1 from another species. Thus, the term "TREM-1" as used herein encompasses any naturally occurring form of TREM-1 that may be derived from any suitable organism. For example, TREM-1 for use as described herein can be vertebrate TREM-1, such as mammalian TREM-1, such as primate (e.g., human, chimpanzee, cynomolgus or rhesus monkey); rodent (e.g., mouse or rat), rabbit (e.g., rabbit) or artiodactyl (e.g., cow, sheep, pig or camel) derived TREM-1. In certain embodiments, TREM-1 is SEQ ID NO: 1 (human TREM-1, isoform 1). This TREM-1 can be the mature form of TREM-1, e.g., a TREM-1 protein that has undergone post-translational processing in a suitable cell. Such a mature TREM-1 protein can be glycosylated, for example. This TREM-1 can be a full-length TREM-1 protein.

[0035] In some embodiments, the anti-TREM-1 antibodies of the present disclosure are monoclonal antibodies in the sense that they are directly or indirectly derived from a single clone of B lymphocytes. In some embodiments, these anti-TREM-1 antibodies are produced, screened and purified using, for example, the methods described in the examples of International Application Publication No. WO2013 / 120553. Briefly, a suitable mouse, such as a TREM-1 or TREM-1 / TREM-3 knockout (KO) mouse, is immunized with TREM-1, TREM-1 expressing cells, or a combination of both. In another embodiment, these anti-TREM-1 antibodies are polyclonal antibodies in the sense that they are a mixture of monoclonal antibodies disclosed herein.

[0036] In some embodiments, the anti-TREM-1 antibodies of the present disclosure are recombinantly expressed in prokaryotic or eukaryotic cells. In some embodiments, the prokaryotic cell is E. coli. In certain embodiments, the eukaryote is a cell derived from an organism such as yeast, insect or mammalian cells, such as primates (e.g., human, chimpanzee, cynomolgus or rhesus monkey), rodents (e.g., mouse or rat), lagomorphs (e.g., rabbit) or artiodactyls (e.g., cow, sheep, pig or camel). Suitable mammalian cell lines include, but are not limited to, HEK293 cells, CHO cells and HELA cells. The anti-TREM-1 antibodies disclosed herein can also be produced by other methods known to those skilled in the art, such as phage display or yeast display. Once produced, the antibodies can be screened, for example, for binding to full-length TREM-1 or variants thereof, using the methods described in the examples of International Application Publication No. WO2013 / 120553.

[0037] As used herein, the term "antibody" refers to a protein derived from a germline immunoglobulin sequence that is capable of specifically binding to an antigen (TREM-1) or a portion thereof. This term includes full-length antibodies of any class or isotype (i.e., IgA, IgE, IgG, IgM, and / or IgY) and any single chain or fragment thereof. An antibody that specifically binds to an antigen or a portion thereof can bind exclusively to that antigen or portion thereof, or can bind to a limited number of homologous antigens or portions thereof. A full-length antibody typically includes at least four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains. One immunoglobulin subclass of particular pharmaceutical interest is the IgG family. In humans, the IgG class can be subdivided into four subclasses: IgG1, IgG2, IgG3, and IgG4, based on the sequence of their heavy chain constant regions. The light chains can be divided into two types, kappa and lambda, based on differences in their sequence composition. An IgG molecule is composed of two heavy chains linked by two or more disulfide bonds, and two light chains each linked to a heavy chain by one disulfide bond. The heavy chains can include one heavy chain variable region (VH) and up to three heavy chain constant (CH) regions: CH1, CH2, and CH3. The light chains can include one light chain variable region (VL) and one light chain constant region (CL). The VH region and the VL region can be further subdivided into hypervariable regions called complementarity determining regions (CDR) interspersed with more conserved regions called framework regions (FR). The VH region and the VL region typically consist of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy and light chains form binding domains capable of interacting with an antigen, while the constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors including, but not limited to, various cells of the immune system (effector cells), Fc receptors, and the first component of the classical complement system (C1q). The antibodies of the present invention can be isolated.The term "isolated antibody" refers to an antibody that has been separated and / or recovered from other components in the environment in which it was produced and / or purified from a mixture of components present in the environment in which it was produced. Since it has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody, certain antigen-binding fragments of an antibody may be appropriate in the context of the present invention.

[0038] The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen, such as TREM-1, as described herein. Examples of antigen-binding fragments include Fab, Fab’, F(ab)2, F(ab’)2, F(ab)S, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv; see, e.g., Bird et al., Science 242:42S-426 (1988); Huston et al., PNAS 85: 5879-5883 (1988)), dsFv, Fd (typically the VH and CH1 domains), and dAb (typically the VH domain) fragments; VH, VL, VhH and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetra-bodies and kappa bodies (see, e.g., Ill et al., Protein Eng 10:949-57 (1997)); camel IgG; IgNAR; and one or more isolated CDRs or functional paratopes, isolated CDRs or antigen-binding residues or polypeptides that can be associated or linked together to form a functional antibody fragment. Various types of antibody fragments are described or reviewed, e.g., in Holliger and Hudson, Nat Biotechnol 2S :1126-1136 (2005); International Publication No. WO2005 / 040219, and U.S. Patent Application Publication Nos. 2005 / 0238646 and 2002 / 0161201. These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and these fragments can be screened for utility in the same manner as intact antibodies.

[0039] A "human" antibody (HuMAb) refers to an antibody having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Further, when the antibody includes a constant region, this constant region is also derived from human germline immunoglobulin sequences. The anti-TREM-1 antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0040] A "humanized" antibody refers to a human / non-human chimeric antibody that contains one or more sequences (CDR regions or parts thereof) derived from non-human immunoglobulins. Thus, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues derived from the hypervariable regions of the recipient are replaced, at least, by the residues derived from the hypervariable regions of an antibody (donor antibody) from a non-human species, such as a mouse, rat, rabbit or non-human primate, that has the desired specificity, affinity, sequence composition and functionality. In some cases, the FR residues of the human immunoglobulin are replaced by the corresponding non-human residues. Examples of such modifications are typically the introduction of one or more so-called back mutations, which are amino acid residues derived from the donor antibody. Humanization of antibodies can be carried out using recombinant techniques known to those skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. C. Lo). Suitable human recipient frameworks for both the variable domains of the light and heavy chains can be identified, for example, by sequence or structural homology. Alternatively, a fixed recipient framework can be used, for example, based on knowledge of the structure, biophysical and biochemical properties. The recipient framework can be derived from the germline or from mature antibody sequences. The CDR regions derived from the donor antibody can be transferred by CDR grafting. CDR-grafted humanized antibodies can be further optimized, for example, with respect to affinity, functionality and biophysical properties, by the identification of important framework positions where the reintroduction of amino acid residues (back mutations) derived from the donor antibody has a beneficial effect on the properties of the humanized antibody. In addition to back mutations derived from the donor antibody, humanized antibodies can be engineered by the introduction of germline residues in the CDR or framework regions, the elimination of immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0041] Furthermore, a humanized antibody can contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. Generally, a humanized antibody contains at least one, typically two, variable domains, where all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are of human immunoglobulin sequence. A humanized antibody can also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The term "humanized antibody derivative" refers to any modified form of a humanized antibody, for example, a conjugate of the antibody with another agent or the antibody.

[0042] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created or isolated by recombinant means, e.g., (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express antibodies, e.g., transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, created or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by the germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg Nature Biotech. 23(9): 1117-1125 (2005)), the variable regions include antigen-binding domains encoded by various genes that are rearranged to form antibodies specific for foreign antigens. In addition to rearrangement, the variable regions can be further modified by multiple single amino acid changes (referred to as somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant regions change in response to further antigen (i.e., isotype switching). Thus, nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides that are rearranged and somatically mutated in response to antigen may not have sequence identity to the original nucleic acid molecules, but instead are substantially identical or similar (i.e., have at least 80% identity). A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, e.g., an antibody in which the variable regions are derived from a murine antibody and the constant regions are derived from a human antibody.

[0043] In one embodiment, the anti-TREM-1 antibody of the present disclosure is an IgG antibody. "IgG antibody", for example, human IgG1, as used herein, in certain embodiments, has the structure of a naturally occurring IgG antibody, i.e., has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, a TREM-1 IgG1 antibody consists of two heavy chains (HC) and two light chains (LC), and these two heavy and light chains are linked by the same number and position of disulfide bridges as the disulfide bridges present in a naturally occurring IgG1 antibody (unless the antibody has been mutated to modify the disulfide bridges). As used herein, "isotype" refers to antibody classes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

[0044] "Allotype" refers to naturally occurring variants within a particular isotype group, and these variants differ in some amino acids (see, e.g., Jefferis et al., mAbs 1:1 (2009)). The anti-TREM-1 antibodies described herein can be of any allotype. In some embodiments, these anti-TREM-1 antibodies are of the "IgG1.3f" allotype, which includes one or more amino acid substitutions selected from the group consisting of L234A, L235E, and G237A according to EU numbering, as compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In other embodiments, these anti-TREM-1 antibodies are of the "IgG1.1f" allotype, which includes one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S according to EU numbering, as compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In certain embodiments, these anti-TREM-1 antibodies are of the "IgG1-Aba" allotype, which includes one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S according to EU numbering, as compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In further embodiments, these anti-TREM-1 antibodies are of the "IgG4-Aba" allotype, which includes the CH1 domain of the wild-type IgG4 isotype (e.g., SEQ ID NO: 10) and the CH2 and CH3 domains of IgG1. In some embodiments, this IgG4-Aba allotype antibody includes one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S according to EU numbering, as compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen". "Isolated antibody," as used herein, is intended to refer to an antibody that has been separated and / or recovered from other components in the environment in which it was produced and / or purified from a mixture of components present in the environment in which it was produced.

[0045] "Effector function" refers to the interaction of the antibody Fc region with an Fc receptor or ligand, or the biochemical events resulting therefrom. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., the antibody variable domain). In one embodiment, the anti-TREM-1 antibodies of the present disclosure include an Fc region that does not bind to one or more FcγRs and thus lack effector function (i.e., effectorless).

[0046] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced forms of those receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). The various properties of human FcγRs are known in the art. Most natural effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, but natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice, FcγRIIIA in humans) and do not express the inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the type of activating Fc receptor to which it binds. The "Fc region" (crystalline fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component of the classical complement system (C1q). Thus, the Fc region includes the constant region of the antibody, excluding the first constant region immunoglobulin domain (e.g., CH1 or CL).

[0047] In IgG, the Fc region includes the immunoglobulin domains CH2 and CH3 and the hinge between the CH1 domain and the CH2 domain. The definition of the boundary of the Fc region of the immunoglobulin heavy chain can vary as defined herein, but the human IgG heavy chain Fc region is defined to extend from amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxy terminus of the heavy chain, where the numbering follows the Kabat EU index. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is positioned on the C-terminal side of the CH2 domain in the Fc region, i.e., the CH3 domain extends from amino acid 341 to amino acid 447 or 446 (if no C-terminal lysine residue is present) or 445 (if no C-terminal glycine and lysine residues are present) of IgG. As used herein, the Fc region can be a native sequence Fc including any allotype variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region in the context of a cleaved, or "Fc fusion protein" as it is also called, an Fc-containing protein polypeptide, e.g., a "binding protein comprising an Fc region" (e.g., an antibody or an immunoadhesion).

[0048] The "native sequence Fc region" or "native sequence Fc" contains an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. Native sequence human Fc regions include the native sequence human IgG1 Fc region; the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes Fc of various allotypes (see, e.g., Jefferis et al., mAbs 1:1 (2009)).

[0049] The "variant sequence Fc region" or "non-naturally occurring Fc" includes modifications, typically to alter one or more of its functional properties such as serum half-life, complement binding, Fc receptor binding, protein stability, and / or antibody-dependent cell-mediated cytotoxicity, or, in particular, the absence thereof. In some embodiments, the anti-TREM-1 antibodies of the present disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation to re-alter one or more functional properties of the antibody. In one embodiment, the anti-TREM-1 antibody is of the IgG1 isotype and has a modified Fc domain that includes one or more, and perhaps all, of the mutations (L234A, L235E, and G237A) that result in a reduced affinity for a particular Fc receptor and the mutations (A330S and P331S) that result in reduced C1q-mediated complement binding (residue numbering according to the EU index).

[0050] The terms "hinge", "hinge domain", "hinge region" and "antibody hinge region" refer to the domain of the heavy chain constant region that joins the CH1 domain to the CH2 domain, and includes the upper, middle and lower parts of the hinge (Roux et al., J Immunol 161:4083 (1998)). The hinge provides a variable level of mobility between the binding and effector regions of the antibody and also provides a site for intermolecular disulfide bonds between the two heavy chain constant regions. As used herein, the hinge begins at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., J Immunol 161:4083 (1998)). The sequences of wild-type IgG1, IgG2, IgG3 and IgG4 hinges are known in the art (e.g., International PCT Publication No. WO2017 / 087678). In one embodiment, the hinge region of the CH1 of the anti-TREM-1 antibody is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is further described, for example, in U.S. Patent No. 5,677,425.

[0051] The constant region can be modified to stabilize the antibody, for example, to reduce the risk of a bivalent antibody separating into two monovalent VH-VL fragments. For example, in the IgG4 constant region, residue S228 (residue numbering according to the EU index) can be mutated to a proline (P) residue to stabilize the formation of inter-heavy chain disulfide bridges in the hinge (see, for example, Angal et al., Mol Immunol. 30: 105-8 (1995)). Antibodies or fragments thereof can also be defined in terms of their complementarity determining regions (CDRs). The term "complementary determining region" or "hypervariable region", as used herein, refers to the region of the antibody where the amino acid residues involved in antigen binding are located. The hypervariable regions or CDRs can be identified as the regions having the highest variability in the amino acid alignment of the antibody variable domains. Databases, such as the Kabat database, can be used for CDR identification, and CDRs are defined to include, for example, amino acid residues 24-34 (CDR1), 50-59 (CDR2) and 89-97 (CDR3) of the light chain variable domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, CDRs can be defined as residues from "hypervariable loops" (residues 26-33 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk, J. Mol. Biol 196: 901-917 (1987)). Typically, the numbering of amino acid residues in this region is carried out by the method described by Kabat et al., supra.The terms "Kabat position," "Kabat residue," and "according to Kabat" in this specification refer to this numbering system for the heavy-chain variable domain or the light-chain variable domain. Using this Kabat numbering system, the actual linear amino acid sequence of a peptide can include fewer or additional amino acids corresponding to shortening of the framework (FR) or CDR of the variable domain, or insertions therein. For example, the heavy-chain variable domain can include amino acid insertions (residues 52a, 52b, and 52c according to Kabat) after residue 52 of CDR H2, and can include inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after heavy-chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment in the region of homology of the sequence of that antibody with the "standard" Kabat-numbered sequence.

[0052] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., TREM-1) to which an immunoglobulin or antibody specifically binds, defined, for example, by the specific method used to identify it. Epitopes can be formed from both contiguous amino acids (usually linear epitopes) or non-contiguous amino acids (usually conformational epitopes) that are adjacent due to the tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in their unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, where overlapping or contiguous peptides (e.g., from TREM-1) are tested for reactivity with a given antibody (e.g., an anti-TREM-1 antibody). Methods for determining the spatial conformation of an epitope include techniques known in the art and described herein, such as x-ray crystallography, antigen mutagenesis analysis, two-dimensional nuclear magnetic resonance and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

[0053] When referring to two or more antibodies, the term "bind to the same epitope" means that those antibodies bind to the same segment of amino acid residues when determined by a given method. Techniques for determining whether an antibody binds to the "same epitope" on TREM-1 as the antibodies described herein include, for example, epitope mapping methods such as x-ray analysis of crystals of antigen:antibody complexes that provide atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to antigen fragments or variant variations of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is often considered an indication of the epitope component. Additionally, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from a combinatorial phage display peptide library. Antibodies having the same VH and VL or the same CDR1, 2, and 3 sequences are predicted to bind to the same epitope.

[0054] An antibody that "competes for binding to a target with another antibody" refers to an antibody that (partially or completely) inhibits the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits the binding of the other antibody to the target and to what extent, can be determined using known competition assays, such as BIACORE® surface plasmon resonance (SPR) analysis. In certain embodiments, the antibody competes with the binding of another antibody to the target and inhibits it by at least 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). The competition assay can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc ; 2006; doi: 10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if they block each other by at least 50% in both directions, i.e., regardless of whether one antibody or the other antibody first contacts the antigen in the competition assay.

[0055] As used herein, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to antibody binding to an epitope on a given antigen. Typically, an antibody has an affinity constant (Kd) of approximately 10 -7 M or less, e.g., approximately 10 -8 M, 10 -9 M or 10-10 Less than M or even lower equilibrium dissociation constant (K D ), and (ii) binds to a predetermined antigen with an affinity that is at least two-fold greater than its affinity for non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). Thus, an antibody that "specifically binds to human TREM-1" has a K -7 of 10 M or less, for example, approximately 10 -8 M, 10 -9 M or 10 -10 M or less or even lower K D and refers to an antibody that binds to soluble or cell-bound human TREM-1. An antibody that "cross-reacts with cynomolgus TREM-1" has a K -7 of 10 M or less, for example, approximately 10 -8 M, 10 -9 M or 10 -10 M or less or even lower K D and refers to an antibody that binds to cynomolgus TREM-1. In certain embodiments, such antibodies that do not cross-react with TREM-1 from non-human species exhibit essentially undetectable binding to these proteins in a standard binding assay.

[0056] As used herein, the term "binding specificity" refers to the interaction of a molecule, such as an antibody or a fragment thereof, with a single exclusive antigen or a limited number of highly homologous antigens (or epitopes). In contrast, an antibody capable of specifically binding to TREM-1 is unable to bind to dissimilar molecules. The antibodies according to the present invention may be unable to bind to the natural killer cell p44-related protein Nkp44. The specificity of the interaction and the values of the equilibrium binding constants can be directly determined by well-known methods. Standard assays for evaluating the ability of a ligand (e.g., an antibody) to bind to its target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as SPR.

[0057] Competitive binding assays for determining whether two antibodies compete or cross-compete for binding include, for example, competition for binding to myeloid cells expressing TREM-1 by flow cytometry as described in the Examples. Other methods include: SPR (e.g., BIACORE®), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeled RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0058] As used herein, the term "bin" is defined using a reference antibody. If a second antibody cannot bind to the antigen simultaneously with the reference antibody, this second antibody is said to belong to the same "bin" as the reference antibody. In this case, the reference antibody and the second antibody competitively bind to the same portion of the antigen and are referred to as "competitive antibodies". If a second antibody can bind to the antigen simultaneously with the reference antibody, this second antibody is said to belong to a separate "bin". In this case, the reference antibody and the second antibody do not competitively bind to the same portion of the antigen and are referred to as "non-competitive antibodies".

[0059] Antibody "binning" does not provide direct information about the epitope. Competing antibodies, i.e., antibodies belonging to the same "bin", may have the same epitope, overlapping epitopes or even distinct epitopes. The latter applies when the reference antibody bound to its epitope on the antigen occupies the space required for the second antibody to contact its epitope on the antigen ("steric hindrance"). Non-competing antibodies generally have distinct epitopes. As used herein, the term "binding affinity" refers to a measure of the strength of non-covalent interaction between two molecules, e.g., between an antibody or a fragment thereof and an antigen. The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity). The binding affinity between two molecules, e.g., between an antibody or a fragment thereof and an antigen, via a monovalent interaction can be quantified by determination of the equilibrium dissociation constant (K D ). Next, K D can be determined, for example, by measurement of the kinetics of complex formation and dissociation by SPR method. The rate constants corresponding to the association and dissociation of the monovalent complex are the association rate constant k a (or k on ) and the dissociation rate constant k d (or k 0ff ), respectively. K D is related to k D and k d via the equation K a = k a / k d . According to the above definition, the comparison of binding affinities associated with different molecular interactions, e.g., the binding affinities of different antibodies for a given antigen, can be compared by comparison of the K D values for individual antibody / antigen complexes.

[0060] As used herein, the term "high affinity" for an IgG antibody refers to a K -8 of 10 -9 M or less, 10 -10 M or less or 10 Drefers to an antibody having [specific binding property]. However, the binding of "high affinity" can vary for other antibody isotypes. For example, the binding of "high affinity" for the IgM isotype refers to an antibody having a K of 10 -10 M or less or 10 -8 M or less. D In the context of in vitro or in vivo assays using an antibody or its antigen-binding fragment, the term "EC " refers to the concentration of the antibody or its antigen-binding portion that induces a response that is 50% of the maximum response, i.e., a response that is intermediate between the maximum response and the baseline. 50 The term "naturally occurring", as used herein in reference to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including a virus) that can be isolated from a natural source and has not been deliberately modified by humans in the laboratory is naturally occurring. The term "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, and there is no upper limit on the length of the chain. One or more amino acid residues in a protein can include modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" can include one or more polypeptides.

[0061] The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be cDNA.

[0062] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, predicted non-essential amino acid residues in an anti-TREM-1 antibody are replaced with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0063] For nucleic acids, the term "substantial homology" indicates that two nucleic acids or their designated sequences, when optimally aligned and compared, are at least about 80% identical, at least about 90%-95% identical or at least about 98%-99.5% identical in nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when a segment hybridizes to the complement of its strand under selective hybridization conditions. For polypeptides, the term "substantial homology" indicates that two polypeptides or their designated sequences, when optimally aligned and compared, have at least about 80%, at least about 90%-95% or at least about 98%-99.5% of the amino acids identical with appropriate amino acid insertions or deletions.

[0064] The percent identity between two sequences is a function of the number of identical positions shared by those sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the following non-limiting examples.

[0065] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using the NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) incorporated in the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Further, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) incorporated in the GAP program in the GCG software package (available at worldwideweb.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

[0066] The nucleic acid and protein sequences described herein can further be used, for example, as "query sequences" to perform searches against public databases to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0067] The nucleic acid can be present in whole cells, in cell lysates, or in a partially purified or substantially pure form. The nucleic acid is "isolated" or "substantially purified" when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other portions of the chromosome) or proteins, by standard techniques including, but not limited to, alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0068] Nucleic acids, such as cDNA, can be mutated according to standard techniques to provide gene sequences. For coding sequences, these mutations can, if desired, affect the amino acid sequence. In particular, DNA sequences that are substantially homologous to or derived from the native V, D, J, constant, switch, and other such sequences described herein are contemplated (wherein "derived from" indicates that the sequence is identical to or modified from another sequence).

[0069] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby being replicated along with the host genome. Further, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, the terms "plasmid" and "vector" can be used interchangeably herein. However, other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0070] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that does not naturally occur in the cell and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such term is intended to refer not only to a particular target cell but also to the progeny of such a cell. Since a particular modification can occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein. As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinantly fused). Such linkage can be achieved using a wide variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0071] As used herein, "administering" refers to the physical introduction of a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Administration by different routes for the anti-TREM-1 antibodies described herein includes, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, intraspinal or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein can be administered via non-parenteral routes, such as topical, epidermal or mucosal routes of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0072] As used herein, the terms "inhibit" or "block" (e.g., referring to inhibition / blockade of the binding of a TREM-1 ligand to TREM-1 on a cell) are used interchangeably and encompass both partial and complete inhibition / blockade. In some embodiments, an anti-TREM-1 antibody inhibits the binding of a TREM-1 ligand to TREM-1 by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99% or 100%, as determined, for example, as further described herein. In some embodiments, an anti-TREM-1 antibody inhibits the binding of a TREM-1 ligand to TREM-1 by 50% or less, e.g., about 40%, 30%, 20%, 10%, 5% or 1%, as determined, for example, as further described herein.

[0073] The terms "treat", "treating" and "treatment", as used herein, refer to any type of intervention or process performed on a subject, or administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting or slowing or preventing the progression, onset, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease, or enhancing overall survival. Treatment can be of a subject having a disease or a subject not having a disease (e.g., for prevention).

[0074] The terms "effective amount" or "effective dosage" are defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent, when used alone or in combination with another therapeutic agent, is any amount of the drug that promotes a demonstrable regression of the disease, as evidenced by a decrease in the severity of the disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or the prevention of functional or disability impairments resulting from the pain of the disease. The therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent, to a subject at risk of developing a disease or a subject at risk of disease recurrence, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or to inhibit the development or recurrence of a disease can be evaluated, for example, in human subjects during clinical trials, in animal model systems that predict human efficacy, using various methods known to those of skill in the art, or by assaying the activity of the agent in in vitro assays. The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject having cancer. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ", respectively. The various aspects described herein are described in further detail in the following subsections.

[0075] I. Anti-TREM-1 Antibodies Antibodies characterized by certain functional features or characteristics, such as fully human antibodies, are described herein. For example, the antibodies of the present disclosure specifically bind to human TREM-1, more specifically, to certain domains (e.g., functional domains) within the extracellular domain of human TREM-1. In one embodiment, these antibodies specifically bind to the site on TREM-1 to which the TREM-1 ligand (e.g., PGLYRP1) binds. In certain embodiments, these antibodies are antagonist antibodies, i.e., they inhibit or suppress the activity of TREM-1 on cells, such as monocytes, macrophages, and neutrophils (i.e., do not agonize upon binding). In some embodiments, these anti-TREM-1 antibodies cross-react with TREM-1 from one or more non-human primates, such as cynomolgus monkey TREM-1. In some embodiments, these anti-TREM-1 antibodies block the production of inflammatory cytokines (e.g., IL-6, TNF-α, IL-8, IL-1β, IL-12, and combinations thereof) by cells (e.g., macrophages, dendritic cells, neutrophils) upon activation. In other embodiments, these anti-TREM-1 antibodies comprise an Fc region that does not bind to one or more FcγRs. In further embodiments, these anti-TREM-1 antibodies do not induce the release of pro-inflammatory cytokines by myeloid cells (e.g., dendritic cells), thereby reducing or preventing the occurrence of a cytokine storm after administration of the anti-TREM-1 antibody to a subject in need thereof.

[0076] In some embodiments, certain anti-TREM-1 antibodies described herein are antibodies, such as monoclonal, recombinant, and / or human antibodies, that cross-compete with mAb 0318 for binding to human TREM-1. In some embodiments, these anti-TREM-1 antibodies also cross-compete with mAb 0318 for binding to cynomolgus monkey TREM-1. In other words, the anti-TREM-1 antibodies of the present disclosure belong to the same "bin" as mAb 0318 in certain embodiments. The mAb 0318 antibody has a heavy chain variable region (VH) containing SEQ ID NO: 14 and a light chain variable region (VL) containing SEQ ID NO: 15. See International Application Publication No. 2016 / 009086. mAb 0318 also has heavy chain CDR1, CDR2, and CDR3 corresponding to amino acids 31-35, 50-68, and 101-110 of SEQ ID NO: 14, respectively. The light chain CDR1, CDR2, and CDR3 of the mAb 0318 antibody correspond to amino acids 24-38, 54-60, and 93-101 of SEQ ID NO: 15, respectively.

[0077] Accordingly, in some embodiments, the anti-TREM-1 antibody of the present disclosure comprises VH and VL of SEQ ID NOs: 14 and 15, respectively. In another embodiment, the VH of this anti-TREM-1 antibody comprises the CDR1 sequence of amino acids 31-35 (TYAMH) of SEQ ID NO: 14, and one of these amino acids may be substituted by a different amino acid. In certain embodiments, the VH of this anti-TREM-1 antibody comprises the CDR2 sequence of amino acids 50-68 (RIRTKSSNYATYYAASVKG) of SEQ ID NO: 14, and one, two, or three of these amino acids may be substituted by different amino acids. In some embodiments, the VH of this anti-TREM-1 antibody comprises the CDR3 sequence of amino acids 101-110 (DMGIRRQFAY) of SEQ ID NO: 14, and one, two, or three of these amino acids may be substituted by different amino acids.

[0078] In some embodiments, the VL of this anti-TREM-1 antibody comprises the CDR1 sequence of amino acids 24-38 (QQSNQDPYT) of SEQ ID NO: 15, and one, two, or three of these amino acids may be substituted by different amino acids. In other embodiments, the VL of this anti-TREM-1 antibody comprises the CDR2 sequence of amino acids 54-60 (RASNLES) of SEQ ID NO: 15, and one or two of these amino acids may be substituted by different amino acids. In some embodiments, the VL of this anti-TREM-1 antibody comprises the CDR3 sequence of amino acids 93-101 (QQSNQDPYT) of SEQ ID NO: 15, and one or two of these amino acids may be substituted by different amino acids.

[0079] Methionine residues in the CDRs of antibodies can be oxidized, resulting in potential chemical degradation of the antibody and a consequent reduction in its potency. Thus, the anti-TREM-1 antibodies disclosed herein may have one or more methionine residues in the heavy and / or light chain CDRs replaced with amino acid residues that are not subject to oxidative degradation. In some embodiments, the methionine residues within heavy chain CDR1 and CDR3 are replaced with amino acid residues (e.g., glutamine or leucine) that are not subject to oxidative degradation. Thus, in one embodiment, the VH of this anti-TREM-1 antibody comprises the CDR3 sequence of amino acids 101-110 (DQGIRRQFAY) of SEQ ID NO: 81 or amino acids 101-110 (DLGIRRQFAY) of SEQ ID NO: 82. In other embodiments, the VH of this anti-TREM-1 antibody comprises the CDR1 sequence of amino acids 31-35 (TYAQH) of SEQ ID NO: 83 or amino acids 31-35 (TYALH) of SEQ ID NO: 84. Similarly, in some embodiments, deamidation sites can be removed from the anti-TREM-1 antibody, particularly within the CDRs.

[0080] In some embodiments, the VH and VL of this anti-TREM-1 antibody comprise the VH and VL sequences of the anti-TREM-1 antibodies disclosed in International Application Publication No. WO2017 / 152102, which is hereby incorporated by reference in its entirety. In some embodiments, the VL of this anti-TREM-1 antibody comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 9-27 of WO2017 / 152102, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 28-40 of WO2017 / 152102, and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 41-119 of WO2017 / 152102. In one embodiment, the VH of this anti-TREM-1 antibody comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 120-143 of WO2017 / 152102, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 144-172 of WO2017 / 152102, and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 173-247 of WO2017 / 152102. In some embodiments, the anti-TREM-1 antibodies of the present disclosure include CDR and / or variable region sequences having at least 80% identity (e.g., at least 85%, at least 95%, at least 95% or at least 99% identity) to the CDR and / or variable region sequences of the mAb 0318 antibody.

[0081] In some embodiments, these anti-TREM-1 antibodies include a heavy chain variable region (VH) and a light chain variable region (VL) each containing SEQ ID NOs: 14 and 15. In some embodiments, these anti-TREM-1 antibodies include a heavy chain (HC) and a light chain (LC), and this HC contains SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 53. In some embodiments, this LC contains SEQ ID NO: 54. In some embodiments, the anti-TREM-1 antibodies of the present disclosure include a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 396-475 of WO2017 / 152102 and / or a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 316-395 of WO2017 / 152102.

[0082] In some embodiments, this anti-TREM-1 antibody includes a heavy chain and a light chain, and these heavy chain and light chain contain the amino acid sequences shown in Table 7. In some embodiments, this anti-TREM-1 antibody includes a heavy chain and a light chain, this heavy chain contains the amino acid sequence shown in SEQ ID NO: 50, and the light chain contains the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, this anti-TREM-1 antibody includes a heavy chain and a light chain, this heavy chain contains the amino acid sequence shown in SEQ ID NO: 51, and the light chain contains the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, this anti-TREM-1 antibody includes a heavy chain and a light chain, this heavy chain contains the amino acid sequence shown in SEQ ID NO: 52, and the light chain contains the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, this anti-TREM-1 antibody includes a heavy chain and a light chain, this heavy chain contains the amino acid sequence shown in SEQ ID NO: 53, and the light chain contains the amino acid sequence shown in SEQ ID NO: 54.

[0083] Heavy or light chains as shown in this specification, for example, heavy and light chains containing an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to SEQ ID NOs: 50-54, can be used to form anti-TREM-1 antibodies having the desired characteristics, such as those further described herein. In some embodiments, the anti-TREM-1 antibodies of the present disclosure include a heavy chain and a light chain, the heavy chain of which contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 50, 51, 52 or 53, and the light chain of which contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 54.

[0084] In some embodiments, this anti-TREM1 antibody comprises a heavy chain constant region, and this heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, L358M, and any combination thereof according to EU numbering. In some embodiments, this anti-TREM-1 antibody comprises a heavy chain constant region, and this heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, L358M, and any combination thereof according to EU numbering. In some embodiments, this anti-TREM-1 antibody comprises a heavy chain constant region, and this heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, P238S, and any combination thereof according to EU numbering. In some embodiments, this anti-TREM-1 antibody comprises a heavy chain constant region, and this heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, P238S, and any combination thereof according to EU numbering. In one embodiment, this anti-TREM-1 antibody is capable of binding to variants of human TREM-1 (e.g., TREM-1 isoforms 2 and 3, SEQ ID NO: 2 and 3, respectively) when determined using, for example, surface plasmon resonance. In another embodiment, this anti-TREM-1 antibody is capable of binding to cynomolgus TREM-1 (SEQ ID NO: 7) when determined using, for example, surface plasmon resonance.

[0085] In some embodiments, the anti-TREM-1 antibodies described herein have high affinity, for example, 10 -7 M or less, 10 -8 M or less, 10 -9 M (1 nM) or less, 10 -10 M or less, 10 -11 M or less, 10 -12Less than M, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M or 10 -9 M to 10 -7 K of M D and binds to human TREM-1. In some embodiments, the anti-TREM-1 antibodies described herein bind to, for example, 10 as determined by BIACORE™ (e.g., as described in the Examples), -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11 Less than M, 10 -12 Less than M, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M or 10 -9 M to 10 -7 K of M D and binds to cynomolgus TREM-1.

[0086] In one embodiment, the antibody of the present disclosure binds to anti-TREM-1 at one or more of the same epitopes as the mAb 0318 antibody. In some embodiments, this anti-TREM-1 antibody binds specifically to at least one amino acid residue selected from the group consisting of (i) A21, T22, K23, L24, T25, E26, and any combination thereof, (ii) A49, S50, S51, Q52, K53, A54, W55, Q56, I57, I58, R59, D60, G61, E62, M63, P64, K65, T66, L67, A68, C69, T70, E71, R72, P73, S74, K75, N76, S77, H78, P79, V80, Q81, V82, G83, R84, I85, and any combination thereof, and (iii) C113, V114, I115, Y116, Q117, P118, P119, and any combination thereof of human TREM-1 (e.g., isoform 1, SEQ ID NO: 1). See WO2016 / 009086.

[0087] In one embodiment, this anti-TREM-1 antibody can specifically bind to amino acids D38 - F48 of SEQ ID NO: 1 (human TREM-1) when determined using, for example, HX-MS or X-ray diffraction. In some embodiments, this anti-TREM-1 antibody has an epitope that includes one, two, three, four, five, six, seven, or all of the amino acid residues D38, V39, K40, C41, D42, Y43, T44, and L45 of SEQ ID NO: 1 (human TREM-1) and one, two, or all of the amino acid residues selected from the group consisting of E46, K47, and F48 of SEQ ID NO: 1 (human TREM-1) when determined using, for example, HX-MS or X-ray diffraction. In certain embodiments, this anti-TREM-1 antibody has an epitope that includes one, two, three, or all of the amino acid residues selected from the group consisting of D42, E46, D92, and H93 of SEQ ID NO: 1 (human TREM-1) when determined using variants of TREM-1 and surface plasmon resonance.

[0088] In one embodiment, the anti-TREM-1 antibody of the present disclosure has an epitope that includes at least amino acid residues E46 and / or D92 of SEQ ID NO: 1 (human TREM-1) when determined using variants of TREM-1 and surface plasmon resonance. In another embodiment, this anti-TREM-1 antibody includes one, two, or all of the amino acid residues selected from the group consisting of L31, 186, and V101 of SEQ ID NO: 1 (human TREM-1). In certain embodiments, this anti-TREM-1 antibody is capable of specifically binding to a polypeptide that includes amino acid residues E19-L26 of cynomolgus TREM-1 (SEQ ID NO: 7) when determined using, for example, HX-MS or X-ray diffraction. In one embodiment, this anti-TREM-1 antibody is capable of specifically binding to human TREM-1, and the epitope of this antibody includes one, two, three, four, five, six, seven, eight, nine, or all of the amino acid residues selected from the group consisting of V39, K40, C41, D42, Y43, L45, E46, K47, F48, and A49 of SEQ ID NO: 1.

[0089] In one embodiment, this anti-TREM-1 antibody is capable of specifically binding to human TREM-1, and the epitope of this antibody includes D42 of SEQ ID NO: 1. In other embodiments, this anti-TREM-1 antibody is capable of specifically binding to human TREM-1, and the epitope of this antibody includes E46 of SEQ ID NO: 1. In some embodiments, the epitope of this antibody may include V39, C41, D42, Y43, and L45 of SEQ ID NO: 1. In further embodiments, the epitope of this antibody may include E46, K47, and A49 of SEQ ID NO: 1. In specific embodiments, the epitope of this anti-TREM-1 antibody may further include F48 of SEQ ID NO: 1.

[0090] In some embodiments, this anti-TREM-1 antibody has a viscosity profile similar to that of the mAb 0318 antibody. In some embodiments, the anti-TREM-1 antibody of the present disclosure has a viscosity of less than 5 cP, less than 4 cP, less than 3 cP, less than 2.5 cP, less than 2.4 cP, less than 2.3 cP, less than 2.2 cP, less than 2.1 cP, less than 2 cP, less than 1.9 cP, less than 1.8 cP, less than 1.7 cP, less than 1.6 cP, less than 1.5 cP, less than 1.4 cP, less than 1.3 cP, less than 1.2 cP, less than 1.1 cP, less than 1.0 cP, less than 0.9 cP, less than 0.8 cP, less than 0.7 cP, less than 0.6 cP, less than 0.5 cP, less than 0.4 cP, less than 0.3 cP, less than 0.2 cP or less than 0.1 cP at a concentration of 80 mg / mL. In some embodiments, this anti-TREM-1 antibody has a viscosity of less than 10 cP (e.g., 9 cP) at a concentration of 130 mg / mL.

[0091] In some embodiments, the anti-TREM-1 antibody of the present disclosure comprises a mutation in which one or more negatively charged residues in the light chain CDR1 and CDR3 regions of the antibody are replaced with uncharged residues. In some embodiments, this anti-TREM-1 antibody comprises a substitution with an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine at one or more of the amino acid residues D1, D30, D33, D74, D98, E27 and E97 of SEQ ID NO: 15. These mutations are referred to herein as "charge patch" mutations.

[0092] In some embodiments, the anti-TREM-1 antibodies of the present disclosure contain mutations in the Fab-Fab interaction region of SEQ ID NO: 14 to reduce Fab-Fab dimerization. Since an antibody contains two Fabs, it has previously been shown with the mAb 0318 antibody that multimerization can affect viscosity. These mutations are referred to as "Fab-Fab interaction" mutations. In certain embodiments, this anti-TREM-1 antibody has a mutation by an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine, and tyrosine at any one of residues Y32, R52, S55, S56, N57, A59, M102, I104, and R106 of SEQ ID NO: 14 or F32, D33, Y34, Y53, R54, and D98 of SEQ ID NO: 15.

[0093] In one embodiment, the anti-TREM-1 antibodies disclosed herein contain a mutation at position 32 of SEQ ID NO: 15, where phenylalanine is mutated to an amino acid selected from the amino acid residues glycine, serine, threonine, cysteine, alanine, valine, leucine, isoleucine, and methionine. Such a mutation is based on the observation that the Ala substitution at position Y90 of SEQ ID NO: 1 is improved in affinity for TREM-1 compared to the parent. Y90 was found to interact with the phenylalanine residue of SEQ ID NO: 15. The mutation of SEQ ID NO: 15 to improve the Fab-TREM-1 interaction is referred to as the "Fab-TREM-1 interaction" mutation. Anti-TREM-1 antibodies are provided herein that are linked (e.g., covalently linked or fused) to an IgG1, IgG2, IgG3, or IgG4 Fc, such as an Fc whose variable region can be for Fc, e.g., any allotype or isoallotype, e.g., for IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Km1, Km2, Km3 (see, e.g., Jeffries et al. (2009) mAbs 1:1). In some embodiments, the variable region of the anti-TREM-1 antibodies disclosed herein is linked to an effectorless or substantially effectorless Fc, e.g., linked to IgG1. In some embodiments, the variable regions of these anti-TREM-1 antibodies are linked to an Fc that has reduced binding to one or more FcγRs or is incapable of binding to one or more FcγRs.

[0094] In one embodiment, the VH domain of the anti-TREM-1 antibody described herein can be fused to the constant domain (i.e., Fc) of a naturally occurring or modified human IgG, such as IgG1, IgG2, IgG3, or IgG4, further described herein. For example, the VH domain can be fused to a human IgG, such as IgG1, constant region, such as the following wild-type human IgG1 constant domain amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) or the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E MIt may comprise the amino acid sequence of any VH domain described herein, fused to the amino acid sequence of an allotype variant of SEQ ID NO: 9 having TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77; allotype-specific amino acid residues are in bold and underlined).

[0095] In one embodiment, the VH domain of the anti-TREM-1 antibody described herein is a constant region without effector, for example, the following constant domain amino acid sequence of human IgG1 without effector: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPE AE G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP SS IEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78; "IgG1.1f" including the underlined substitutions L234A, L235E, G237A, A330S and P331S according to EU numbering) or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPE AE G APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP AP IEKTISKAKGQPREPQVYTLPPSR E E M It may contain the amino acid sequence of any VH domain described herein, fused to TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 79; "IgG1.3f" including the underlined substitutions L234A, L235E and G237A according to EU numbering).

[0096] For example, allotype variants of IgG1 are numbered according to those in SEQ ID NOs: 77 - 79 and include K97R, D239E and / or L241M (underlined and in bold above). Within the full - length heavy - chain region, according to EU numbering, these amino acid substitutions are numbered K214R, D356E and L358M. In some embodiments, the constant region of the anti - TREM - 1 antibody further includes one or more mutations or substitutions at amino acids L117, A118, G120, A213 and P214 (underlined above), or L234, A235, G237, A330 and P331 according to EU numbering, as numbered in SEQ ID NOs: 77 - 79. In further embodiments, the constant region of this anti - TREM - 1 antibody includes one or more mutations or substitutions at amino acids L117A, A118E, G120A, A213S and P214S, or L234A, L235E, G237A, A330S and P331S according to EU numbering, as numbered in SEQ ID NOs: 77 - 79. The constant region of this anti - TREM - 1 antibody may also include one or more mutations or substitutions at L117A, A118E and G120A of SEQ ID NO: 9, or L234A, L235E and G237A according to EU numbering.

[0097] In some embodiments, the VH domain of the anti - TREM - 1 antibody described herein has the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHT S PP S PAPELLGG S SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11; “IgG1-Aba” including the underlined substitutions K214R, C226S, C229S and P238S according to EU numbering); or ASTKGPSVFPLAP C S R STS ES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT K TY T CNV D HKPSNTKVDK R VEPKSCDKTHT S PP S PAPELLGG SIt comprises the amino acid sequence of any VH domain described herein, fused to an IgG1 constant domain comprising SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12; "IgG4-Aba" including the underlined substitutions S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S according to EU numbering).

[0098] The VL domains described herein can be fused to the constant domains of human kappa or lambda light chains. For example, the VL domain of an anti-TREM-1 antibody has the following human IgG1 kappa light chain amino acid sequence: It may comprise the amino acid sequence of any VL domain described herein, fused to RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13).

[0099] In certain embodiments, this heavy chain constant region contains a lysine or another amino acid at the C-terminus, for example, the following last amino acids: including LSPGK (SEQ ID NO: 48) in the heavy chain. In certain embodiments, this heavy chain constant region lacks one or more amino acids at the C-terminus and has, for example, the C-terminal sequence LSPG (SEQ ID NO: 49) or LSP. In one embodiment, the variable region of this anti-TREM-1 antibody is linked to an effectorless or substantially effectorless Fc. In certain embodiments, the variable region of this anti-TREM-1 antibody is linked to an Fc selected from the group consisting of IgG1.1f, IgG1.3f, IgG1-Aba and IgG4-Aba, as described herein.

[0100] Generally, the variable regions described herein can be linked to an Fc that typically includes one or more modifications to alter one or more functional properties of the antibody, such as Fc receptor binding, inflammatory cytokine release, serum half-life, complement binding, and / or antibody-dependent cell-mediated cytotoxicity. Additionally, the antibodies described herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation to change one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the Kabat EU index. The Fc region encompasses domains derived from the constant region of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, as well as other classes such as IgA, IgD, IgE, and IgM), including fragments, analogs, variants, mutants, or derivatives of the constant region. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide that is homologous to the immunoglobulin C-terminal region and can include the CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain, either separately or in combination.

[0101] Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγRs) that are specific for antibodies of the IgG class, namely, FcγRI, FcγRII, and FcγRIII. Sequences important for the binding of IgG to FcγR receptors have been reported to be located in the CH2 and CH3 domains. The serum half-life of an antibody is affected by its ability to bind to Fc receptors (FcRs). In one embodiment, the Fc regions of these anti-TREM-1 antibodies are variant Fc regions, e.g., Fc sequences that have been modified (e.g., by amino acid substitution, deletion, and / or insertion) compared to a parental Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate the variant) to provide desirable structural features and / or biological activities.

[0102] For example, modifications can be made in the Fc region to generate Fc variants that have (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors compared to the parental Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. It is particularly desirable to combine amino acid modifications. For example, a variant Fc region can contain, therein, for example, two, three, four, five, etc. substitutions at specific Fc region positions identified herein.

[0103] Variant Fc regions can also include sequence modifications in which amino acids involved in disulfide bond formation have been removed or replaced with other amino acids. Such removal can avoid reaction with other cysteine-containing proteins present in the host cells used to produce the anti-TREM-1 antibodies described herein. Even when cysteine residues are removed, the single-chain Fc domain can still form a dimeric Fc domain that is maintained non-covalently together. In other embodiments, the Fc region can be modified to be more compatible with the selected host cell. For example, the PA sequence, which can be recognized by digestive enzymes in E. coli, such as prolidase, near the N-terminus of a typical native Fc region can be removed. In other embodiments, one or more glycosylation sites within the Fc domain can be removed. Residues that are typically glycosylated (e.g., asparagine) can confer a cell lysis response. Such residues can be deleted or replaced with non-glycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement, such as the C1q binding site, can be removed from the Fc region. For example, the EKK sequence of human IgG1 can be deleted or replaced. In certain embodiments, sites that affect binding to Fc receptors, preferably sites other than the salvage receptor binding site, can be removed. In other embodiments, the Fc region can be modified to remove the ADCC site. The ADCC site is known in the art; see, e.g., Sarmay et al., Molec. Immunol. 29 (5): 633-9 (1992) for the ADCC site in IgG1. Specific examples of variant Fc domains are disclosed, for example, in WO97 / 34631 and WO96 / 32478.

[0104] In one embodiment, the hinge region of the Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of the Fc is altered, e.g., to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding as compared to native Fc-hinge domain SpA binding. This approach is described in more detail in U.S. Patent No. 6,165,745 by Ward et al.

[0105] In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and / or 331 may be replaced with different amino acid residues such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parental antibody. The effector ligand for which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter.

[0106] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 may be replaced with different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al. In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby change the ability of the antibody to fix complement. This approach is further described in PCT Publication No. WO94 / 29351 by Bodmer et al.

[0107] In yet another example, the Fc region is modified to reduce antibody-dependent cellular cytotoxicity (ADCC) and / or reduce the affinity for Fcγ receptors by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T and 267E / 268F / 324T. Other modifications to enhance the interaction with FcγR and complement include, but are not limited to, the substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0108] Other Fc modifications that can be made to Fc reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and / or 331 (e.g., positions 330 and 331), where the numbering follows the EU index. Exemplary substitutions include, but are not limited to, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S), where the numbering follows the EU index. The Fc variant can include 236R / 328R. Other modifications for reducing the interaction with FcγR and complement include the substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, and the removal of glycosylation at position 297 by mutagenic or enzymatic means or by production in organisms such as bacteria that do not glycosylate the protein. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0109] Optionally, the Fc region can contain non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; International Publication Nos. WO00 / 42072; WO01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752; WO04 / 074455; WO04 / 099249; WO04 / 063351; WO05 / 070963; WO05 / 040217, WO05 / 092925, and WO06 / 020114).

[0110] The affinity and binding characteristics of the Fc region for its ligand can be determined by various in vitro assay methods (biochemical or immunology-based assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE analysis), as well as other methods, such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize labels on one or more of the components being tested and / or can use various detection methods including, but not limited to, chromogenic, fluorescent, luminescent, or isotope labels. A detailed description of binding affinity and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0111] In certain embodiments, the anti-TREM-1 antibodies of the present disclosure comprise an Fc that has reduced binding to FcγR or is unable to bind to FcγR. In some embodiments, this anti-TREM-1 antibody has a reduced binding affinity for FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof, as compared to an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, this anti-TREM-1 antibody has a reduced binding affinity for FcγRI (CD64) that is less than or equal to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 of that of an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 54.

[0112] In some embodiments, these anti-TREM-1 antibodies comprise an IgG1 Fc variant comprising: (a) one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, and any combination thereof, according to EU numbering; (b) one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, P331S, and any combination thereof, according to EU numbering; (c) one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, P238S, and any combination thereof, according to EU numbering; or (d) one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, P238S, and any combination thereof, according to EU numbering.

[0113] In some embodiments, the anti-TREM-1 antibodies disclosed herein comprise (a) having an IgG1 isotype and containing one or more amino acid substitutions in the Fc region at amino acid residues selected from the group consisting of N297A, N297Q, D270A, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, L328E, P238D, S267E, L328F, E233D, G237D, H268D, P271G, A330R, and any combination thereof, wherein the residue numbering follows EU or Kabat numbering, or containing an amino acid deletion in the Fc region at the position corresponding to glycine 236; (b) having an IgG2 isotype and containing one or more amino acid substitutions in the Fc region at amino acid residues selected from the group consisting of P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, wherein the residue numbering follows EU or Kabat numbering; or (c) having an IgG4 isotype and containing one or more amino acid substitutions in the Fc region at amino acid residues selected from the group consisting of E233P, F234V, L234A / F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, wherein the residue numbering follows EU or Kabat numbering.In some embodiments, (a) the Fc region further comprises one or more additional amino acid substitutions at amino acid residues selected from the group consisting of A330L, L234F; L235E, P331S, and any combination thereof, wherein residue numbering follows EU or Kabat numbering; (b) the Fc region further comprises one or more additional amino acid substitutions at positions selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein residue numbering follows EU or Kabat numbering; or (c) the Fc region further comprises an S228P amino acid substitution according to EU or Kabat numbering. See WO2017 / 152102. In certain embodiments, an Fc having reduced complement binding is selected. Exemplary Fcs having reduced complement binding, such as IgG1 Fc, have the following two amino acid substitutions: A330S and P331S.

[0114] In certain embodiments, an Fc that is essentially devoid of effector function is selected, i.e., such Fc has reduced binding to FcγR and reduced complement binding. Exemplary effectorless Fcs, such as IgG1 Fc, contain the following five mutations: L234A, L235E, G237A, A330S and P331S.

[0115] II. Physical Characteristics of the Antibody Anti-TREM-1 antibodies, such as those described herein, have some or all of the physical characteristics of the specific anti-TREM-1 antibodies described herein, such as those described in the Examples. In particular, glycosylation sites within the variable regions can result in increased immunogenicity of the antibody, or a change in the pK of the antibody due to altered antigen binding (Marshall et al., (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J. Immunol 172:5489-94; Wallick et al., (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al., (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing the N-X-S / T sequence. In some embodiments, the anti-TREM-1 antibodies of the present disclosure do not contain variable region glycosylation or have reduced variable region glycosylation. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues within the glycosylation region. Thus, in some embodiments, the anti-TREM-1 antibodies disclosed herein have lower immunogenicity compared to antibodies comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, these anti-TREM-1 antibodies do not contain asparagine isomerization sites. Deamidation of asparagine can occur on N-G or D-G sequences, resulting in the creation of isoaspartic acid residues that introduce a kink into the polypeptide chain and reduce its stability (the isoaspartic acid effect).

[0116] Each antibody generally has an unique isoelectric point (pI) that falls within the pH range between 6 and 9.5. The pI for IgG1 antibodies typically falls within the pH range of 7 - 9.5, and the pI for IgG4 antibodies typically falls within the pH range of 6 - 8. Antibodies having a pI outside the normal range are inferred to have some degree of unfolding and instability under in vivo conditions. Thus, the anti-TREM-1 antibodies disclosed herein may include pI values that fall within the normal range (e.g., 8 - 9). This can be achieved by selecting antibodies having a pI within the normal range or by mutating charged surface residues.

[0117] Each antibody has a characteristic melting temperature, and a higher melting temperature indicates higher overall stability in vivo (Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71). Generally, T M i (temperature of initial unfolding) may be higher than 60°C, higher than 65°C, or higher than 70°C. In some embodiments, the anti-TREM-1 antibodies of the present disclosure have a higher melting temperature compared to an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. Thus, in some embodiments, the anti-TREM-1 antibodies of the present disclosure are thermally stable compared to a reference antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54, as measured, for example, by capillary differential scanning calorimetry (CAP-DSC). The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al., (2003) Pharm Res 20: 1952-60; Ghirlando et al.,(1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al., (2002) J. Chromatogr Sci 40:343-9). In some embodiments, about 10% - 20%, about 20% - 30% (e.g., 24%) or about 30% - 40% of the antibody is reversible when heated to 77°C. In some embodiments, the anti-TREM-1 antibodies of the present disclosure do not degrade rapidly. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

[0118] In some embodiments, the anti-TREM-1 antibodies disclosed herein have a minimal aggregation effect that can result in undesirable immune responses and / or induction of altered or undesirable pharmacokinetic properties. Generally, antibodies having 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less aggregation are acceptable. Aggregation can be measured by several techniques including size exclusion chromatography (SEC), high performance liquid chromatography (HPLC), and dynamic light scattering (DLS). In some embodiments, the anti-TREM-1 antibodies of the present disclosure are monomeric when observed by size exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, these anti-TREM-1 antibodies exhibit a minimal risk of fragmentation when observed by intact mass spectrometry using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC / MS), or liquid chromatography-tandem mass spectrometry (LC / MS).

[0119] III. Nucleic Acids, Vectors, and Cells Another aspect described herein relates to nucleic acid molecules encoding the anti-TREM-1 antibodies described herein. These nucleic acids can be present in whole cells, in cell lysates, or in a partially purified or substantially pure form. The nucleic acids are purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA that is virtually linked to the isolated DNA) or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art, and are then “isolated” or “substantially purified.” See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein can be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid is a cDNA molecule.

[0120] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas isolated from transgenic mice carrying human immunoglobulin genes, further described below), the cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding the antibodies can be recovered from the library. In some embodiments, the nucleic acids described herein are nucleic acids encoding the VH and VL sequences of the anti-TREM-1 antibodies of the present disclosure. Exemplary DNA sequences encoding the VH and VL sequences are shown in SEQ ID NOs: 58-61 and 62-65, respectively. The method for producing an anti-TREM-1 antibody disclosed herein may include the step of expressing a heavy chain and a light chain in a cell line that includes nucleotide sequences encoding the heavy chain and the light chain, for example, SEQ ID NOs: 58-61 and 62-65, respectively, together with a signal peptide. Host cells containing these nucleotide sequences are encompassed herein.

[0121] Once DNA fragments encoding the VH segment and the VL segment are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or a scFv gene. In these manipulations, the VL-encoding DNA fragment or the VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, for example, an antibody constant region or a flexible linker. The term "operably linked", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0122] Isolated DNA encoding the VH region can be converted into a full-length heavy-chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy-chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy-chain constant region genes are known in the art (see, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. This heavy-chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, e.g., an IgG2 and / or IgG4 constant region. For the Fab fragment heavy-chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy-chain CH1 constant region.

[0123] Isolated DNA encoding the VL region can be converted into a full-length light-chain gene (as well as a Fab light-chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light-chain constant region CL. The sequences of human light-chain constant region genes are known in the art (see, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. This light-chain constant region can be a kappa or lambda constant region.

[0124] Another aspect described herein relates to cells (e.g., host cells) that express (e.g., recombinantly) the anti-TREM-1 antibodies described herein, as well as related polynucleotides and expression vectors. Also provided herein are vectors comprising a polynucleotide that includes a nucleotide sequence encoding an anti-TREM-1 antibody or a fragment thereof. In some embodiments, these vectors can be used to recombinantly express the anti-TREM-1 antibodies described herein in a host cell, e.g., a mammalian cell. In some embodiments, these vectors can be used for gene therapy. Vectors suitable for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In some embodiments, the vector is a viral vector. As used herein, an expression vector refers to any nucleic acid construct that contains the elements necessary for transcription and translation of an inserted coding sequence when introduced into a suitable host cell, or, in the case of an RNA viral vector, the elements necessary for replication and translation. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

[0125] The expression vectors of the present disclosure may include a polynucleotide encoding an antibody or an antigen-binding portion thereof described herein. In some embodiments, the coding sequence of the antibody or an antigen-binding portion thereof is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked if they are covalently linked in such a way as to allow each of the component nucleic acid sequences to retain its functionality. A coding sequence and a gene expression control sequence are operably linked if they are covalently linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the gene expression control sequence. When induction of a promoter in a 5' gene expression sequence results in transcription of the coding sequence, and the nature of the linkage between the two DNA sequences is such that (1) it does not result in the introduction of a frameshift mutation, (2) it does not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) it does not interfere with the ability of the corresponding RNA transcript to be translated into a protein, these two DNA sequences are said to be operably linked. Thus, a gene expression sequence is operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of its coding nucleic acid sequence such that the resulting transcript is translated into the desired antibody or an antigen-binding portion thereof.

[0126] Viral vectors include, but are not limited to, nucleic acid sequences derived from the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesviruses; vaccinia virus; poliovirus; and RNA viruses such as retroviruses. Other vectors well known in the art can be readily used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA and subsequent proviral integration into host cellular DNA. Retroviruses are approved for human gene therapy trials. Replication-defective (i.e., capable of directing the synthesis of the desired protein but incapable of producing infectious particles) retroviruses are the most useful. Such genetically modified retroviral expression vectors have general utility for high-efficiency transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses (including steps of incorporation of exogenous gene material into a plasmid, transfection of a packaging cell line with the plasmid, production of recombinant retroviruses by the packaging cell line, collection of virus particles from tissue culture medium, and infection of target cells with the virus particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

[0127] In some embodiments, the virus is an adeno-associated virus, which is a double-stranded DNA virus. Adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. This further has advantages such as heat and lipid solvent stability; high transduction frequency in cells of diverse lineages including hematopoietic cells; and lack of superinfection inhibition allowing multiple rounds of transduction. According to reports, adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the potential for insertional mutagenesis characteristic of retroviral infection and the variability of expression of the inserted gene. Furthermore, wild-type adeno-associated virus infection has been followed in tissue culture for more than 100 passages in the absence of selection pressure, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated virus can also function in an episomal manner.

[0128] IV. Immunoconjugate The present disclosure also provides immunoconjugates comprising any of the anti-TREM-1 antibodies disclosed herein. In some embodiments, the immunoconjugate comprises an antibody or antigen-binding portion conjugated to a drug. In some embodiments, the immunoconjugate comprises a bispecific molecule disclosed herein conjugated to a drug (e.g., as a therapeutic or diagnostic agent).

[0129] For diagnostic purposes, suitable drugs are detectable labels including radioisotopes for whole body imaging, as well as radioisotopes, enzymes, fluorescent labels and other suitable antibody tags for sample testing. Detectable labels that can be conjugated to any of the anti-TREM-1 antibodies described herein include metal sols, such as particulate labels including colloidal gold, isotopes, such as I presented, for example, in N2S2, N3S or N4 type peptidic chelating agents 125 or Tc 99、 chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that become apparent after amplification by polymerase chain reaction or the like, and can be any of various types currently used in the field of in vitro diagnostics. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label can be a 1,2-dioxetane substrate, such as adamantylmethoxyphosphoryloxy phenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decane}-4-yl)phenyl phosphate (CSPD), and enzyme alkaline phosphatase detected by measuring the presence or formation of chemiluminescence after conversion of CDP and CDP-STAR (registered trademark), or other luminescent substrates well known to those skilled in the art, such as chelates of suitable lanthanides, such as terbium(III) and europium(III). The detection means is determined by the selected label. The appearance of the label or its reaction product can be achieved using the naked eye if the label is particulate and accumulates at an appropriate level, or can be achieved using equipment such as spectrophotometers, luminometers, fluorometers, etc. according to all standard practices.

[0130] In some embodiments, the conjugation method results in linkages that are substantially (or almost) non-immunogenic, such as peptide linkages (i.e., amide linkages), sulfide linkages, (sterically hindered), disulfide linkages, hydrazone linkages, and ether linkages. These linkages are almost non-immunogenic and exhibit reasonable stability in serum (see, for example, Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO2009 / 059278; WO95 / 17886).

[0131] Depending on the biochemical properties of the moiety and the antibody, different conjugation strategies can be used. If this moiety is between 50 and 500 amino acids, either naturally occurring or recombinant, standard procedures exist in textbooks that describe the chemistry for the synthesis of protein conjugates that can be readily followed by those skilled in the art (see, for example, Hackenberger, C. P. R., and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, the reaction of a maleinimido moiety with a cysteine residue within the antibody or moiety is used. This is a particularly suitable coupling chemistry, for example, when Fab or Fab’ fragments of the antibody are used. Alternatively, in some embodiments, coupling to the C-terminus of the antibody or moiety is performed. C-terminal modification of a protein, such as a Fab fragment, can be carried out as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0132] In general, site-specific reactions and covalent couplings are based on transforming natural amino acids into amino acids with reactivity orthogonal to the reactivity of other functional groups present. For example, certain cysteines within rare sequence contexts can be enzymatically converted to aldehydes (see Frese, M. A., and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid modifications can also be achieved by exploiting the specific enzymatic reactivity of a given enzyme with a natural amino acid in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; protease-catalyzed C-N bond formation is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site-specific reactions and covalent couplings can also be achieved by the selective reaction of a terminal amino acid with an appropriate modifying reagent. The reactivity of an N-terminal cysteine with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling. Native chemical ligation can also rely on a C-terminal cysteine residue (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96). U.S. Patent No. 6,437,095 describes a conjugation method based on the faster reaction of a cysteine within a stretch of negatively charged amino acids with a cysteine located within a stretch of positively charged amino acids.

[0133] This portion can also be a synthetic peptide or a peptidomimetic. When the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, for example, de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). A wide variety of orthogonal functional groups are relevant and can be introduced into the synthetic peptide, so conjugation of such a peptide to a linker is standard chemistry.

[0134] To obtain a monosubstituted polypeptide, a conjugate with a 1:1 stoichiometry can be separated from other conjugation by-products by chromatography. This procedure can be facilitated by using a dye-labeled binding partner member and a charged linker. By using this type of labeled and highly negatively charged binding partner member, the monoconjugated polypeptide is easily separated from the unlabeled polypeptide and the polypeptide carrying more than one linker, because the differences in charge and molecular weight can be used for separation. Fluorescent dyes can be useful for purifying the complex from unbound components such as labeled monovalent binders. In some embodiments, the portion bound to the anti-TREM-1 antibody is selected from the group consisting of a binding portion, a labeling portion, and a biologically active portion.

[0135] The anti-TREM-1 antibodies described herein may also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In an ADC, the antibody and the therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide or hydrazone linker. In some embodiments, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 80), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO02 / 096910; WO07 / 038658; WO07 / 051081; WO07 / 059404; WO08 / 083312; and WO08 / 103693; U.S. Patent Application Publication Nos. 20060024317; 20060004081; and 20060247295.

[0136] Anti-TREM-1 antibodies, such as those described herein, can also be used to detect TREM-1, such as human TREM-1, for example, in tissues or tissue samples. These antibodies can be used, for example, in ELISA assays or flow cytometry. In some embodiments, the anti-TREM-1 antibody is contacted with cells, such as cells in a tissue, for a time sufficient for specific binding to occur, and then a reagent, such as an antibody that detects the anti-TREM-1 antibody, is added. Exemplary assays are provided in the Examples. The anti-TREM-1 antibody can be a fully human antibody or can be a chimeric antibody, such as an antibody having human variable regions and mouse constant regions or portions thereof. Exemplary methods for detecting TREM-1, such as human TREM-1, in a sample (cell or tissue sample) include: (i) contacting the sample with an anti-TREM-1 antibody for a time sufficient to allow specific binding of the anti-TREM-1 antibody to TREM-1 in the sample; and (2) contacting the sample with a detection reagent, such as an antibody that specifically binds to the Fc region of the anti-TREM-1 antibody, thereby detecting TREM-1 to which the anti-TREM-1 antibody is bound. A washing step can be included after incubation with the antibody and / or detection reagent. Since separate detection agents can be used, the anti-TREM-1 antibody for use in these methods need not be linked to a label or detection agent. Other uses for anti-TREM-1 antibodies, for example, as monotherapy or combination therapy, are provided elsewhere herein, for example, in the section on combination treatments.

[0137] V. Bispecific Molecules The anti-TREM-1 antibodies described herein can be used to form bispecific molecules. The anti-TREM-1 antibody or its antigen-binding portion can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand to a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. For example, the anti-TREM-1 antibody can be linked to an antibody or scFv that specifically binds to any protein that can be used as a potential target for combination therapy, such as the proteins described herein (e.g., an antibody to IP-10 or TNF-α). The antibodies described herein can actually be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; as used herein, such multispecific molecules are also intended to be encompassed by the term "bispecific molecule." To create the bispecific molecules described herein, the antibodies described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other methods) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule results. Accordingly, bispecific molecules are provided herein that comprise at least one first binding specificity for TREM-1 and a second binding specificity for a second target epitope. In some embodiments described herein where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0138] In some embodiments, the bispecific molecules described herein comprise, as a binding specificity, at least one antibody or an antibody fragment thereof that comprises, for example, Fab, Fab’, F(ab’)2, Fv, or single-chain Fv (scFv). The antibody can also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single-chain construct as described in U.S. Patent No. 4,946,778 to Ladner et al. Human monoclonal antibodies are preferred, but other antibodies that can be used in the bispecific molecules described herein are mouse, chimeric, and humanized monoclonal antibodies.

[0139] The bispecific molecules described herein can be prepared by conjugating the binding specificities of the components using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include Protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83, and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Some conjugating agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL). When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region is modified to contain an odd number, preferably one sulfhydryl residue, prior to conjugation.

[0140] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is an mAb×mAb, mAb×Fab, mAb×(scFv)2, Fab×F(ab’)2 or ligand×Fab fusion protein. The bispecific antibody can include an antibody that contains an scFv at the C-terminus of each heavy chain. The bispecific molecules described herein can be single-chain molecules that include one single-chain antibody and one binding determinant, or single-chain bispecific molecules that include two binding determinants. The bispecific molecule can include at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[0141] Binding of the bispecific molecule to its specific targets can be confirmed using methods recognized in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition) or Western blot assay. Each of these assays generally detects the presence of the protein-antibody complex of interest, particularly by using a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0142] VI. Kit Kits are provided herein that include one or more anti-TREM-1 antibodies or antigen-binding portions thereof, bispecific molecules thereof, or immunoconjugates. In some embodiments, one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding portions thereof provided herein, and a pharmaceutical pack or kit including any instructions for use are provided herein. In some embodiments, these kits include the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein.

[0143] VII. Compositions and Formulations Compositions (e.g., pharmaceutical compositions) and formulations are further provided herein that include one or more of the anti-TREM-1 antibodies disclosed herein (including polynucleotides, vectors, and cells encoding and / or expressing the anti-TREM-1 antibodies). For example, in one embodiment, the disclosure provides a pharmaceutical composition comprising one or more anti-TREM-1 antibodies disclosed herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody, immunoconjugate, or bispecific molecule, may be coated with a material to protect the compound from the actions of acids and other natural conditions that may inactivate the compound.

[0144] Accordingly, one object of the present disclosure is to provide pharmaceutical formulations that improve the stability of anti-TREM-1 antibodies and thus enable their long-term storage. In some embodiments, the pharmaceutical formulations disclosed herein comprise: (a) an anti-TREM-1 antibody; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a bulking agent; and / or (f) a surfactant. In some embodiments, the pharmaceutical formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years or more. In some embodiments, the formulation is stable when stored at 4°C, 25°C or 40°C.

[0145] Buffer Buffers useful for the present invention can be weak acids or weak bases that are used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. Suitable buffers can maximize the stability of pharmaceutical formulations by maintaining pH control of the formulation. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties can also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) together with an isotonicity agent and potentially involves pH adjustment with acids or bases known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10 or between about 4 and about 8.

[0146] Stabilizer Stabilizers are added to pharmaceutical products to stabilize the product. Such agents can stabilize proteins in several different ways. Common stabilizers include amino acids such as glycine, alanine, lysine, arginine or threonine, carbohydrates such as glucose, sucrose, trehalose, raffinose or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrin or dextran of any type and molecular weight, or PEG, but are not limited thereto. In one aspect of the invention, this stabilizer is selected to maximize the stability of the FIX polypeptide in a lyophilized preparation. In certain embodiments, this stabilizer is sucrose and / or arginine. Bulking agent Bulking agents can be added to pharmaceutical products to add volume and mass to the product, thereby facilitating its accurate dosing and handling. Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate or magnesium stearate.

[0147] Surfactant Surfactants are amphiphilic substances having a lyophilic group and a lyophobic group. Surfactants can be anionic, cationic, zwitterionic or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethylamine oxide. In some embodiments, this surfactant is polysorbate 20 or polysorbate 80.

[0148] In some embodiments, the pharmaceutical formulation of the present disclosure (a) An anti-TREM-1 antibody at about 0.25 mg / mL to 250 mg / mL (e.g., 10 to 200 mg / mL); (b) About 20 mM of histidine; (c) About 150 mM of sucrose; (d) About 25 mM of arginine; and (e) About 50 mM of NaCl comprising. This formulation may further comprise one or more of a buffer system, a preservative, an isotonic agent, a chelating agent, a stabilizer and / or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well known to those skilled in the art. Remington: The Science and Practice of Pharmacy, 19 th edition, 1995 may be referred to.

[0149] In some embodiments, this pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions and multiphase materials. The term "aqueous formulation" is defined as a formulation containing at least 50% by weight of water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% by weight of water, and the term "aqueous suspension" is defined as a suspension containing at least 50% by weight of water. In some embodiments, this pharmaceutical formulation is a freeze-dried formulation to which a solvent and / or diluent is added by a physician or patient prior to use.

[0150] The pharmaceutical compositions described herein may also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy may comprise an anti-TREM-1 antibody described herein in combination with at least one other therapeutic agent. Examples of therapeutic agents that may be used in combination therapy may include other compounds, drugs and / or agents used for the treatment of a disease or disorder (e.g., an inflammatory disorder). Such compounds, drugs and / or agents may include, for example, anti-inflammatory drugs or antibodies that block or reduce the production of inflammatory cytokines. In some embodiments, the therapeutic agent may include an anti-IP-10 antibody, an anti-TNF-α antibody (e.g., adalimumab (HUMIRA®), golimumab (SIMPONI®), infliximab (REMICADE®), certolizumab pegol (CIMZIA®)), interferon beta-1a (e.g., AVONEX®, REBIF®), interferon beta-1b (e.g., BETASERON®, EXTAVIA®), glatiramer acetate (e.g., COPAXONE®, GLATOPA®), mitoxantrone (e.g., NOVANTRONE®), non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids, and combinations thereof.

[0151] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects (see, e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and those derived from non-toxic organic amines such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0152] The pharmaceutical compositions described herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include the following: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0153] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by both the above sterilization procedures and the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include in the composition isotonic agents such as sugars, sodium chloride, etc. Furthermore, prolonged absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption such as aluminum monostearate and gelatin.

[0154] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. The use thereof in the pharmaceutical compositions described herein is contemplated, except in cases where any conventional media or agent is incompatible with the active compound. The pharmaceutical compositions may or may not contain a preservative. Supplementary active compounds may be incorporated into the composition.

[0155] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coatings, such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, these compositions can contain isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition agents that delay absorption, such as monostearates and gelatin.

[0156] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in one or a combination of the above-mentioned ingredients in a suitable solvent and then, if necessary, sterilizing by aseptic filtration. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the necessary other ingredients described herein. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form varies depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01 percent to about 99 percent active ingredient, from about 0.1 percent to about 70 percent or from about 1 percent to about 30 percent active ingredient in combination with a pharmaceutically acceptable carrier.

[0157] The dosing regimen is adjusted to provide an optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased if indicated by the exigencies of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. A dosage unit form, as used herein, refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a calculated predetermined amount of the active compound in association with the required pharmaceutical carrier to produce the desired therapeutic effect. The specifications for the dosage unit forms described herein are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the art of compounding such active compounds for the treatment of sensitivity in individuals.

[0158] For administration of an anti-TREM-1 antibody, for example as described herein, the dosage is in the range of about 0.0001 to 100 mg, more usually 0.01 to 5 or 10 mg per kg of host body weight. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight, or may be within the range of 1 to 10 mg / kg. Exemplary treatment regimens require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Exemplary dosing regimens for the anti-TREM-1 antibody described herein include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, and this antibody is given using one of the following dosing schedules: (i) every four weeks for six doses, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once, then 1 mg / kg body weight every three weeks. In some embodiments, this anti-TREM-1 antibody is administered at a flat dose (flat dosing regimen). In other embodiments, this anti-TREM-1 antibody is administered at a fixed dose together with another antibody. In certain embodiments, this anti-TREM-1 antibody is administered at a dose based on body weight. In some methods, two or more monoclonal antibodies having different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the indicated range. Antibodies are typically administered on multiple occasions. The interval between single dosages can be, for example, weekly, monthly, every three months or annually. The interval can also be irregular if indicated by measuring the blood level of the antibody against the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1 - 1000 μg / ml, and in some methods, about 25 - 300 μg / ml.

[0159] Antibodies can be administered as sustained release formulations, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. Dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over a long period. Some patients continue treatment for the remainder of their lives. In therapeutic applications, relatively high dosages at relatively short intervals are sometimes required until the progression of the disease is reduced or terminated, or until the patient shows partial or complete remission of the symptoms of the disease. Thereafter, a prophylactic regimen can be administered to the patient.

[0160] The actual dosage level of the active ingredient in the pharmaceutical composition described herein can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors including the activity of the particular composition or its ester, salt or amide described herein, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition being used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and other factors well known in the medical arts.

[0161] The compositions described herein can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Routes of administration for the anti-TREM-1 antibodies described herein can include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, arterial, intracisternal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions. Alternatively, the antibodies described herein can potentially be administered via non-parenteral routes, such as topical, epidermal or mucosal routes of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0162] The active compound can be prepared using a carrier that protects the compound against rapid release, such as a controlled release formulation including a graft, a transdermal patch, and a microencapsulated delivery system. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for the adjustment of such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0163] The therapeutic composition can be administered using medical devices known in the art. For example, in certain embodiments, the therapeutic compositions described herein can be administered using a needleless subcutaneous injection device such as those disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known grafts and modules for use with the anti-TREM-1 antibodies described herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drug therapy at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Patent No. 4,447,233, which discloses a drug therapy infusion pump for delivering drug therapy at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are hereby incorporated by reference herein. Many other such grafts, delivery systems, and modules are known to those skilled in the art.

[0164] In some embodiments, the anti-TREM-1 antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (optionally, for example, for brain cancer), these can be formulated, for example, in liposomes. For methods of making liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibody (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273).

[0165] VIII. Uses and Methods The anti-TREM-1 antibodies of the present disclosure and compositions (e.g., pharmaceutical compositions, formulations, polynucleotides, vectors, and cells) containing such antibodies can be used for the treatment of inflammatory diseases (e.g., by inhibiting TREM-1 activity).

[0166] Accordingly, in one aspect, the present disclosure provides a method for treating an inflammatory disease in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective dose of an anti-TREM-1 antibody. Examples of inflammatory diseases that can be treated with the anti-TREM-1 antibodies of the present invention include, but are not limited to, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, and other autoimmune diseases that are a result of either acute or chronic inflammation.

[0167] In one embodiment, these anti-TREM-1 antibodies are suitable for use in the treatment of individuals having inflammatory bowel disease. Inflammatory bowel disease (IBD) is a disease that can affect any part of the gastrointestinal tract from the mouth to the anus and causes a wide variety of symptoms. IBD mainly causes abdominal pain, diarrhea (which may be bloody), vomiting or weight loss, but can also cause complications outside the gastrointestinal tract, such as rashes, arthritis, eye inflammation, fatigue and lack of concentration. Patients with IBD can be divided into two main classes: patients having ulcerative colitis (UC) and patients having Crohn's disease (CD). CD generally affects the ileum and colon and can affect any region of the intestine, but is often discontinuous (areas of concentrated disease spread throughout the intestine). UC always affects the rectum (colon) and is more continuous. In CD, the inflammation is transmural and results in abscesses, fistulas and strictures, while in UC, the inflammation is typically limited to the mucosa. For Crohn's disease, neither medical nor surgical cures are known, but some patients with UC can be cured by surgical removal of the colon. Treatment options are limited to symptom control, maintenance of remission and prevention of relapse. Efficacy in inflammatory bowel disease in the clinic can be measured as a reduction in the Crohn's Disease Activity Index (CDAI) score for CD, which is a scoring scale based on laboratory tests and quality of life questionnaires. In animal models, efficacy is mostly measured by weight gain and also by the Disease Activity Index (DAI), which is a combination of stool consistency, weight and bloody stools.

[0168] In one embodiment, the anti-TREM-1 antibodies of the present disclosure are suitable for use in the treatment of individuals having rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease that affects almost all but not all parts of the body and is one of the most common forms of arthritis. It is characterized by inflammation of the joints, which causes pain, stiffness, warmth, redness, and swelling. This inflammation is the result of inflammatory cells infiltrating the joints, and these inflammatory cells release enzymes that can digest bone and cartilage. As a result, this inflammation can lead to severe bone and cartilage damage, joint deterioration, and severe pain, among other physiological effects. The affected joints can lose their shape and alignment, resulting in pain and loss of movement. Several animal models of rheumatoid arthritis are known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop inflammatory arthritis similar to human rheumatoid arthritis. Since CIA shares immunological and pathological features similar to RA, this makes this model a suitable model for screening potential human anti-inflammatory compounds. Efficacy in this model is measured by a decrease in joint swelling. Efficacy in RA in the clinic is measured by the ability to reduce symptoms in patients as measured by a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein level, and serum factors such as the level of anti-citrullinated protein antibody.

[0169] In one embodiment, the anti-TREM-1 antibodies disclosed herein are suitable for use in the treatment of individuals having psoriasis. Psoriasis is a T cell-mediated inflammatory disorder of the skin that can cause considerable discomfort. It is a disease for which there is currently no cure and affects people of all ages. Individuals with mild psoriasis can often control their disease with topical agents, but more than one million patients worldwide require ultraviolet light treatment or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of ultraviolet irradiation and the toxicity of many therapeutic agents limit their long-term use. Furthermore, patients typically experience recurrences of psoriasis and, in some cases, rebound immediately after discontinuation of immunosuppressive therapy. A recently developed model of psoriasis based on the infiltration of CD4+ T cells mimics many aspects of human psoriasis and can therefore be used to identify compounds suitable for use in the treatment of psoriasis (Davenport et al., Internat. Immunopharmacol 2: 653-672, 2002). Efficacy in this model is measured by reduction in skin pathology using a scoring system. Similarly, efficacy in patients is measured by reduction in skin pathology.

[0170] In one embodiment, these anti-TREM-1 antibodies are suitable for use in the treatment of individuals having psoriatic arthritis. Psoriatic arthritis (PA) is one type of inflammatory arthritis that occurs in a subset of patients with psoriasis. In these patients, the skin pathology / symptoms are accompanied by joint swelling similar to that seen in rheumatoid arthritis. This is characterized by patchy raised red areas of skin inflammation with scaling. Psoriasis often affects the elbows and knees tips, scalp, umbilicus, and the area around the genitals or anus. Approximately 10% of patients with psoriasis also develop associated joint inflammation.

[0171] With respect to the present disclosure, prophylactic, palliative, symptomatic and / or curative treatments can represent separate aspects of the present disclosure. The antibodies of the invention can be administered parenterally, for example, intravenously, for example, intramuscularly, for example, subcutaneously. Alternatively, the antibodies of the invention can be administered via a non-parenteral route, for example, orally or topically. The antibodies of the invention can be administered prophylactically. The antibodies of the invention can be administered therapeutically (by request). The following examples are provided for illustrative purposes only and not for purposes of limitation. The contents of all references cited throughout this application are hereby expressly incorporated herein by reference. Another aspect of the present invention may be as follows. 〔1〕An isolated antibody that specifically binds to triggering receptor-1 (TREM-1) expressed on myeloid cells, comprising a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions compared to the wild-type IgG1 heavy chain constant region (SEQ ID NO: 9). 〔2〕An isolated antibody that cross-competes with mAb 0318 for binding to block TREM-1, comprising a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions compared to the wild-type IgG1 heavy chain constant region (SEQ ID NO: 9). 〔3〕The antibody according to 〔1〕 or 〔2〕 above, which binds to the same TREM-1 epitope as mAb 0318. 〔4〕The antibody according to 〔1〕 or 〔2〕 above, which specifically binds to a TREM-1 epitope comprising one or more amino acid residues selected from the group consisting of D38, V39, K40, C41, D42, Y43, T44, L45, E46, K47, F48, A49, S50, S51, Q52, K53, A54, W55, Q56, Y90, H91, D92, H93, G94, L95, and L96 of SEQ ID NO: 1. 〔5〕The antibody according to 〔1〕 or 〔2〕 above, which specifically binds to a TREM-1 epitope comprising amino acids D38-L45, E46-Q56, and / or Y90-L96 of SEQ ID NO: 1. 〔6〕The antibody according to any one of 〔1〕 to 〔5〕 above, wherein the IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E and L358M according to EU numbering. 〔7〕The antibody according to any one of 〔1〕 to 〔5〕 above, wherein the IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E and L358M according to EU numbering. 〔8〕The antibody according to any one of 〔1〕 to 〔5〕 above, wherein the IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S and P238S according to EU numbering. 〔9〕The isolated antibody according to any one of 〔1〕 to 〔5〕 above, wherein the IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S according to EU numbering. 〔10〕The antibody according to any one of 〔1〕 to 〔9〕 above, comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR3 comprises DMGIRRQFAY (SEQ ID NO: 26) or DMGIRRQFAY (SEQ ID NO: 26) with one or two substitutions removed. 〔11〕The antibody according to 〔10〕 above, wherein the heavy chain CDR3 comprises DQGIRRQFAY (SEQ ID NO: 72). 〔12〕The antibody according to any one of 〔1〕 to 〔11〕 above, comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR2 comprises RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) or RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) with one or two substitutions removed. 〔13〕The antibody according to any one of 〔1〕 to 〔12〕 above, comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR1 comprises TYAMH (SEQ ID NO: 24) or TYAMH (SEQ ID NO: 24) with one or two substitutions removed. The antibody according to any one of the above items [1] to

[13] , which comprises a heavy-chain CDR1, CDR2, and CDR3 and a light-chain CDR1, CDR2, and CDR3, wherein the light-chain CDR1 comprises RASQSVDTFDYSFLH (SEQ ID NO: 27) or RASQSVDTFDYSFLH (SEQ ID NO: 27) excluding one or two substitutions. The antibody according to any one of the above items [1] to

[14] , which comprises a heavy-chain CDR1, CDR2, and CDR3 and a light-chain CDR1, CDR2, and CDR3, wherein the light-chain CDR2 comprises RASNLES (SEQ ID NO: 28) or RASNLES (SEQ ID NO: 28) excluding one or two substitutions. The antibody according to any one of the above items [1] to

[15] , which comprises a heavy-chain CDR1, CDR2, and CDR3 and a light-chain CDR1, CDR2, and CDR3, wherein the light-chain CDR3 comprises QQSNQDPYT (SEQ ID NO: 29) or QQSNQDPYT (SEQ ID NO: 29) excluding one or two substitutions. The antibody according to any one of the above items [1] to

[16] , wherein the VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 14. The antibody according to any one of the above items [1] to

[17] , wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 15. The antibody according to any one of the above items [1] to

[18] , which comprises a heavy-chain variable region and a light-chain variable region, wherein the VH comprises SEQ ID NO: 14 and the VL comprises SEQ ID NO: 15. The antibody according to the above item

[19] , which comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. The antibody according to the above item

[20] , which comprises a heavy chain and a light chain, wherein the light chain comprises SEQ ID NO: 54. An isolated antibody that specifically binds to TREM-1 and comprises heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2, and CDR3 each comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26); the light chain CDR1, CDR2, and CDR3 each comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29); the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, and G237A according to EU numbering, an antibody. An isolated antibody that specifically binds to TREM-1 and comprises heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2, and CDR3 each comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26); the light chain CDR1, CDR2, and CDR3 each comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29); the IgG1 heavy chain constant region comprises amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S according to EU numbering, an antibody. An isolated antibody that specifically binds to TREM-1 and comprises heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2, and CDR3 each comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26); the light chain CDR1, CDR2, and CDR3 each comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29); The IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S according to EU numbering, antibody. 〔25〕An isolated antibody that specifically binds to TREM-1 and contains heavy chain CDR1, CDR2, CDR3; light chain CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2, and CDR3 each contain TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26); the light chain CDR1, CDR2, and CDR3 each contain RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29); the IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S according to EU numbering, antibody. 〔26〕The antibody according to any one of 〔1〕 to 〔25〕 above, wherein TREM-1 contains the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. 〔27〕The antibody according to any one of 〔1〕 to 〔26〕 above, which has a reduced binding affinity for FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof, as compared to an antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. 〔28〕The antibody according to any one of 〔1〕 to 〔26〕 above, which has a binding affinity reduced to 1 / 2 or less, 1 / 3 or less, 1 / 4 or less, 1 / 5 or less, 1 / 6 or less, 1 / 7 or less, 1 / 8 or less, 1 / 9 or less, or 1 / 10 or less for FcγRI (CD64) as compared to an antibody containing a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. The antibody according to any one of [1] to

[28] , which has lower immunogenicity as compared with an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. 〔30〕The antibody according to any one of [1] to

[29] , which does not agonize TREM-1 signal transduction in the absence of a stimulatory factor when binding to TREM-1. 〔31〕The antibody according to any one of [1] to

[30] , which does not induce the expression of inflammatory cytokines in the cells when immature dendritic cells (iDCs) are incubated in the presence of the antibody and in the absence of a stimulatory factor, as compared with an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. 〔32〕The antibody according to any one of [1] to

[31] , which blocks the production of inflammatory cytokines in the cells when the cells are activated in the presence of both the antibody and a stimulatory factor. 〔33〕The antibody according to any one of

[30] to

[31] , wherein the stimulatory factor is a TREM-1 ligand. 〔34〕The antibody according to

[31] or

[32] , wherein the inflammatory cytokine is selected from the group consisting of IL-6, TNF-α, IL-8, IL-1β, IL-12, chitinase-3-like protein 1 (CHI3L1), and combinations thereof. 〔35〕The antibody according to any one of [1] to

[34] , which binds to human FcRn, cynomolgus FcRn and / or mouse FcRn in a pH-dependent manner. 〔36〕The antibody according to any one of [1] to

[35] , which is more thermally stable as compared with a reference antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54, when measured by capillary differential scanning calorimetry (CAP-DSC). 〔37〕The antibody according to any one of [1] to

[36] , wherein about 10% to 20%, about 20% to 30% (e.g., 24%) or about 30% to 40% of the antibody is reversible when heated to 77°C. 〔38〕The antibody according to

[36] or

[37] , which has a higher melting temperature (Tm) as compared with an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 54. The antibody according to any one of [1] to

[38] above, having a viscosity of less than 5 cP, less than 4 cP, less than 3 cP, less than 2.5 cP, less than 2.4 cP, less than 2.3 cP, less than 2.2 cP, less than 2.1 cP, less than 2 cP, less than 1.9 cP, less than 1.8 cP, less than 1.7 cP, less than 1.6 cP, less than 1.5 cP, less than 1.4 cP, less than 1.3 cP, less than 1.2 cP, less than 1.1 cP, less than 1.0 cP, less than 0.9 cP, less than 0.8 cP, less than 0.7 cP, less than 0.6 cP, less than 0.5 cP, less than 0.4 cP, less than 0.3 cP, less than 0.2 cP or less than 0.1 cP at a concentration of 80 mg / mL. 〔40〕The antibody according to any one of [1] to

[38] above, having a viscosity of less than 10 cP (for example, 9 cP) at a concentration of 130 mg / mL. 〔41〕When measured by Biacore, K less than 4 nM (for example, 3.4 nM) D The antibody according to any one of [1] to

[40] above, which binds to human TREM-1 at. 〔42〕When measured by Biacore, K less than 1 nM (for example, 0.91 nM) D The antibody according to any one of [1] to

[41] above, which binds to cynomolgus TREM-1 at. 〔43〕The antibody according to any one of [1] to

[42] above, which is monomeric when observed by size exclusion high performance liquid chromatography (SE-HPLC). 〔44〕The antibody according to any one of [1] to

[43] above, which shows a minimum risk of fragmentation when observed by intact mass spectrometry using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC / MS) or liquid chromatography-tandem mass spectrometry (LC / MS). 〔45〕The antibody according to any one of [1] to

[44] above, having an isoelectric point of 8 to 9 (for example, 8.75). 〔46〕The antibody according to any one of [1] to

[45] above, which is stable in a formulation containing histidine, sucrose, arginine and NaCl. 〔47〕The antibody according to

[46] above, which is stable for at least two months in a formulation containing 20 mM histidine, 150 mM sucrose, 25 mM arginine and 50 mM NaCl. 〔48〕The antibody according to

[46] or

[47] above, wherein the formulation has a pH of 6.0 and / or the formulation is stored at 4 °C, 25 °C or 40 °C. A bispecific molecule comprising the antibody according to any one of [1] to

[48] linked to a molecule having a second binding specificity. 〔50〕A nucleic acid encoding the antibody according to any one of [1] to

[48] . 〔51〕A vector comprising the nucleic acid according to

[50] . 〔52〕A cell comprising the vector according to

[51] . 〔53〕An immunoconjugate comprising the antibody according to any one of [1] to

[48] linked to a drug. 〔54〕A composition comprising the antibody according to any one of [1] to

[48] , the bispecific molecule according to

[49] , the nucleic acid according to

[50] , the vector according to

[51] , the cell according to

[52] , or the immunoconjugate according to

[53] , and a carrier. 〔55〕A kit comprising the antibody according to any one of [1] to

[48] , the bispecific molecule according to

[49] , the nucleic acid according to

[50] , the vector according to

[51] , the cell according to

[52] , or the immunoconjugate according to

[53] , and instructions for use. 〔56〕A method for inhibiting TREM-1 activity in a subject in need thereof, comprising administering to the subject the antibody according to any one of [1] to

[48] , the bispecific molecule according to

[49] , the nucleic acid according to

[50] , the vector according to

[51] , the cell according to

[52] , or the immunoconjugate according to

[53] . 〔57〕A method for treating an inflammatory disease or an autoimmune disease in a subject in need thereof, comprising administering to the subject the antibody according to any one of [1] to

[48] , the bispecific molecule according to

[49] , the nucleic acid according to

[50] , the vector according to

[51] , the cell according to

[52] , or the immunoconjugate according to

[53] . 〔58〕The method according to 〔57〕, wherein the inflammatory disease or the autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, vasculitis, sepsis, systemic inflammatory response syndrome (SIRS), type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma, other autoimmune diseases that are the result of either acute inflammation or chronic inflammation, and any combination thereof. 〔59〕The method according to any one of 〔56〕 to 〔58〕, further comprising the step of administering one or more additional therapeutic agents. 〔60〕The method according to 〔59〕, wherein the additional therapeutic agent is an anti-IP-10 antibody or an anti-TNF-α antibody.

Example

[0172] (Example 1) Analysis of the kinetics of interaction of 318 antibody variants against TREM-1 in both humans and cynomolgus monkeys by surface plasmon resonance The binding kinetics of mAb 0318 variants to human TREM-1-Fc (hTREM-1) and cynomolgus monkey TREM-1-Fc (cTREM-1) were determined. Binding studies were performed using a ProteOn Analyzer (BioRad) that measures molecular interactions in real time via surface plasmon resonance. Experiments were carried out at 25 °C, and samples were stored at 15 °C in the sample compartment. The signals (RU, response units) reported by ProteOn are directly correlated with the mass on the surface of individual sensor chip in six parallel flow cells. Anti-human Fc monoclonal antibody or anti-mouse Fc polyclonal antibody from Biacore's human or mouse Fc capture kit was immobilized horizontally on the flow cells of the GLM sensor chip according to the manufacturer's instructions. The final immobilization level of the capture antibody was approximately 2600 - 6000 RU in each experiment. The capture of purified monoclonal mouse or recombinantly expressed anti-hTREM-1 antibody was carried out by diluting the antibody to 5 - 10 nM in running buffer (10 mM Hepes 0, 15 M NaCl, 5 mM EDTA, 0.05% surfactant P20, pH 7.4), and then injecting vertically at 30 μl / min for 60 seconds to create a reference interspot adjacent to all flow cells where only the anti-Fc antibody was immobilized. This typically resulted in a final capture level of approximately 100 - 300 RU of the test antibody and an Rmax value of 30 - 90 RU of the analyte. The binding of hTREM-1 or cTREM-1 protein was carried out by injecting the analyte (antigen) horizontally into all flow cells and comparing the binding to different captured anti-TREM-1 antibodies with the binding to the reference interspot, enabling a comparative analysis of binding. hTREM-1 or cTREM-1 protein was serially diluted 1:3 to 1.2 - 100 nM in running buffer or in the running buffer and injected at 100 μl / min for 250 seconds and dissociated for 600 seconds. The GLM surface was regenerated after each injection cycle of the analyte via two 18-second injections of 10 mM glycine, pH 1.7 and 50 mM NaOH at 100 μl / min.This regeneration step removed the anti-TREM-1 antibody and any bound TREM-1 protein from the immobilized capture antibody surface, allowing subsequent binding of the next interaction sample pair. The regeneration procedure did not remove the directly immobilized anti-Fc capture antibody from the chip surface.

[0173] The binding affinity between the antibody and antigen was quantified by determination of the equilibrium dissociation constant (K D ) determined by measurement of the kinetics of complex formation and dissociation. The rate constants corresponding to the association and dissociation of the monovalent complex, e.g., k a (association rate) and k d (dissociation rate), were retrieved by fitting the data to a 1:1 Langmuir model using ProteOn evaluation software for data analysis. K D is related to k D and k d via the equation K a = k a / k d . The binding curves were processed by double referencing (subtraction of the reference surface signal and blank buffer injection for the captured anti-TREM-1 antibody) prior to data analysis. This enabled correction for instrument noise, bulk shift, and drift during sample injection. As shown in FIGS. 1A and 1B, all of the mAb 0318 variants (i.e., 318-IgG1.1f, 318-IgG1.3f, 318-IgG4-Aba, and 318-IgG1-Aba) were found to have an affinity for human TREM-l-Fc similar to that of mAb0318-IgG4. The 0318-IgG1.3f variant bound to cynomolgus TREM-1 as well, although with a slightly reduced affinity compared to human TREM-1. See FIG. 1A.

[0174] (Example 2) Internalization analysis of mAb 0318-IgG1.3f upon binding to the TREM-1 receptor The mAb 0318-IgG1.3f variant antibody was tested for its internalization in primary human monocytes using both a laser scanning confocal microscope (data not shown) and Amnis ImageStream® Imaging Flow Cytometry Analysis. As shown in Figure 2A, at 0 hours, TREM-1 is mainly expressed on the surface of monocytes. However, by 24 hours after antibody binding, a significant percentage (about 36%) of TREM-1 receptors (revealed by 0318-IgG1.3f staining) were internalized, suggesting that the entire antibody-receptor complex is internalized into the cell upon mAb 0318-IgG1.3f antibody binding.

[0175] Next, to determine the fate of the TREM-1 receptor after internalization, the TREM26 antibody (Catalog No. 314902, Biolegend; see paragraph

[0005] of US Patent Application Publication No. 20150274825) was used for comparison. The TREM26 antibody does not compete with the 0318 antibody variant described in this disclosure. As shown in Figure 2B, there was a significant reduction (about 51%) in TREM26+ expression at the 20-hour time point compared to 0 hours. At the 20-hour time point (after mAb 0318-IgG1.3f treatment), there was also a significant reduction (one-fourth) in the TREM26+ MFI (mean fluorescence index), suggesting that the TREM-1 receptor is degraded upon internalization. However, once the antibody is removed, this loss of TREM-1 receptor expression upon antibody exposure is reversible (data not shown).

[0176] (Example 3) Analysis of mAb 0318 variants that efficiently block TREM-1 activation using the BWZ / hTREM-1 reporter cell assay The ability of the anti-TREM-1 mAb 0318 variant to inhibit human TREM-1 signaling was determined using the BWZ.36 / hTREM-1DAP12:NFAT-LacZ cell line (also referred to herein as the "BWZ / hTREM-1 reporter cell") assay as described, for example, in U.S. Patent No. 9,550,830 and International Application Publication No. WO2016 / 009086. Briefly, approximately 40,000 hTREM-1 / BWZ.36 cells / well were plated in clear-bottom black 96-well plates in the presence of 75 ng / ml of PGLYRP1 (SEQ ID NO: 8) with 2.5 μg / ml of PGN-ECndi (catalog number tlrl-kipgn, Invivogen San Diego, Calif., USA) to provide a maximal sub-optimal positive signal, or in the presence of a sub-maximal level (1 μg / ml) of plastic-adsorbed anti-TREM-1 monoclonal antibody (catalog number MAB1278, R&D Systems, Minneapolis, Minn., USA) to provide a positive signal.

[0177] The mAb 0318 variants (i.e., 0318-IgG1.3f; 0318-IgG1.1f; 0318-IgG1-Aba; and 0318-IgG4-Aba) were titrated during the assay in 5 serial two-fold dilutions starting at 10 μg / ml. The assay was incubated overnight at 37° C. and then developed with Beta Glo (catalog number E4740, Promega Madison, Wis., USA) according to the Beta Glo protocol, and luminescence was recorded. Data were plotted showing Beta Glo relative light units vs. test antibody concentration. Non-neutralizing negative control mIgG1 (catalog number MAB002, R&D Systems Minneapolis, Minn., USA) and neutralizing positive control polyclonal goat anti-hPGLYRP1 antibody (catalog number AF2590, R&D Systems, Minneapolis, Minn., USA) were run on each assay plate. MAB1278 antibody (catalog number MAB1278, R&D Systems; see paragraph

[0005] of U.S. Patent Application Publication No. 20150274825), a known agonist of TREM-1 signaling, was also used as a positive control (see the inserted boxed figure). As shown in FIG. 3, all of the mAb 0318 variants were potent in inhibiting human TREM-1 signaling, as previously observed with the mAb 0318 IgG4 antibody in International Application Publication No. WO2016 / 009086.

[0178] (Example 4) In vitro analysis of the potency of mAb 0318 antibody variants in inhibiting TREM-1-mediated production of inflammatory cytokines by different primary human cells To further evaluate the antagonist properties of the anti-TREM-1 mAb 0318 variants, their potency to block the release of various inflammatory cytokines (e.g., TNF-α, IL-6 or IL-8) from activated human primary cells was evaluated. Primary monocytes, neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood and stimulated with plate-bound PGRP1 and soluble peptidoglycan (PGN-ECndss; a form of peptidoglycan without TLR2 activity).

[0179] As shown in Figure 4, all mAb 0318 variants (i.e., IgG1.3f, IgG1.1f, IgG1-Aba and IgG4-Aba) were all potent in inhibiting the TREM-1-mediated release of TNF-α from PBMCs and monocytes (IC 50 values in the range of about 10 - 20 pM). The potency of these mAb 0318 variants was similar to that observed with the mAb 0318-IgG4 antibody. The mAb 0318-IgG1.3f antibody was also potent in inhibiting IL-6 production (IC 50 value of about 32 pM). For neutrophils, the mAb 0318-IgG1.3f variant appeared to be better than the mAb 0318-IgG4 antibody in blocking the TREM-1-mediated IL-8 production from neutrophils (see Figure 4).

[0180] As a further demonstration of the antagonist properties of the anti-TREM-1 mAb 0318 antibody variants, a monocyte-neutrophil co-culture assay was also used. When co-cultured with monocytes, neutrophil-associated PGRP1 can bind to the TREM-1 receptor and result in monocyte-derived TNF-α production. As shown in Figure 4, all mAb 0318 antibody variants effectively blocked this endogenous activation (IC 50 values in the range of 19 - 44 pM). Similar results were observed in RBC sedimented whole blood (see Figure 4).

[0181] (Example 5) In vitro analysis of the potency of mAb 0318-IgG1.3f to block IL-8 production from stimulated whole blood One of the major challenges in developing a whole blood pharmacodynamics (PD) assay to measure the antagonist properties of anti-TREM-1 antibody variants is the high background arising from PGN stimulation. To help address this problem, whole blood was stimulated with pre-complexed PGRP1+PGN in the presence of an NOD2 inhibitor (to block background cytokines generated by NOD2 stimulation by PGN). IL-8 levels were measured using a standard ligand-binding pharmacodynamic assay (HTRF®) (Figure 5A) or an intracellular cytokine staining (ICS) assay (Figure 5B).

[0182] As shown in Figures 5A and 5B, the 0318-IgG1.3f antibody effectively blocked TREM-1-mediated IL-8 production with percent inhibition in the range of approximately 60-90% (see Figure 5A). The observed IC50 values (average of 12 pM in HTRF and 19.6 pM in ICS) were similar to those observed in other functional assays (see, for example, Example 4).

[0183] (Example 6) In vitro analysis of the potency of mAb 0318-IgG1.3f to block mRNA expression of different inflammatory mediators in stimulated whole blood To further demonstrate the antagonist properties of mAb 0318-IgG1.3f, the expression levels of selected inflammatory mediators (i.e., chitinase-3-like protein 1 (“CHI3L1”), IL1β, and IL6) were measured by real-time PCR (qPCR). Briefly, human whole blood collected in EDTA tubes from three normal healthy volunteers (donor numbers 126, 290, and 322) was pre-complexed with hPGRP1 (50 μg / ml) and PGN-ECndss (10 μg / ml) (Invivogen tlrl-ksspgn) and stimulated overnight in the presence of varying concentrations of mAb 0318-IgG1.3f (0 to 1 nM). After stimulation, plasma was collected and frozen for cytokine measurement. mRNA was isolated from the samples using the MagMax-96 Blood Isolation Kit (ThermoFisher AM1837) according to the manufacturer's protocol. The isolated mRNA was then converted to cDNA using SuperScript VILO Master Mix (Thermo Fisher 11755250). Next, qPCR was performed using the following probes: HPRT1 (Hs99999909_m1) (Thermo Fisher 4351370), CHI3L1 (Hs01072228_m1) (Thermo Fisher 4331182), IL1β (Hs00174097_m1) (Thermo Fisher 4331182), and IL6 (Hs00985639_m1) (Thermo Fisher 4331182) as well as TaqMan Fast Universal Master Mix (2×) (Thermo Fisher 4366072). Gene expression values were normalized to HPRT1 and ΔΔCT values were generated. The results were then plotted and IC 50 values were determined.

[0184] As shown in FIGS. 6A-6C and in agreement with the above-described examples, mAb 0318-IgG1.3f was able to inhibit the TREM-1-mediated expression of different inflammatory mediators in human whole blood. This inhibition appeared to be dose-dependent. The IC50 values are shown in Table 1 below. Collectively, the above results demonstrate that the mAb 0318 antibody variant described in the present disclosure is antagonistic and can effectively block the TREM-1-mediated production of inflammatory cytokines from various human cells.

[0185] [Table 1]

[0186] (Example 7) Viscosity of mAb 0318 variant Samples were buffer-exchanged and dialyzed into the optimal formulation (20 mM histidine, 150 mM sucrose, 25 mM arginine, 50 mM sodium chloride, pH 6.0), and then concentrated using an Amicon Ultra centrifugal molecular weight cut-off filter. The aggregation state of the samples was measured by size exclusion chromatography to control for potential changes in monomericity during concentration, and no such changes were observed. Concentration-dependent viscosity was determined using a RheoSense m-VROC solution viscometer with a 3-point upward shear sweep for each concentration measured. Concentration was determined by measuring absorbance at 280 nm using a nanoDrop with dilution series and extrapolation.

[0187] As shown in Figure 7, both the 318-IgG1.1f variant and the 318-IgG1.3f variant had similar viscosity profiles. At a concentration of approximately 130 mg / mL, the 0318-IgG1.3f variant had a viscosity value of approximately 9 cP. Such a viscosity profile was very similar to that previously observed with mAb 0318-IgG4 (see International Application Publication No. WO2016 / 009086).

[0188] (Example 8) Immunogenic potential of mAb 0318 variant As shown in Figure 8 and Table 2 (below), the 318-IgG1.1f and 318-IgG1.3f variants had a low to intermediate risk of immunogenicity in human patients. Only about 22 - 30% of donors had an immunogenic response (measured by in vitro CD4 + T cell proliferation) to these antibodies. The immunogenicity to the 0318-IgG1-Aba and 0318-IgG4-Aba variants was 10% and 42.5% respectively. See Table 2 (below). In contrast, mAb 0318-IgG4 was more immunogenic (55%) in human patients. KLH (keyhole limpet hemocyanin) and VL6 (IL-21R mAb) used as positive controls were highly immunogenic in human patients (100% and 40% respectively). [Table 2]

[0189] (Example 9) Binding analysis of mAb 0318 variants to FcRn using surface plasmon resonance (SPR) To determine whether different mAb 0318 variants can bind to FcRn, FcRn receptors (mouse, human, and cynomolgus monkey) were immobilized onto a BIAcore CM5-biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) via amine coupling to a level of 400 response units (RU). This assay was performed at room temperature using PBS, 0.05% Tween-20™ pH 6.0 (GE Healthcare Bioscience) as running and dilution buffer. Different mAb 0318 variants (200 nM) were injected at room temperature at a flow rate of 50 μL / min at pH 6.0. The association time was 180 seconds and the dissociation phase (also at pH 6.0) took 360 seconds. Regeneration of the chip surface to return to baseline was achieved by a short injection of 50 mM Tris, pH 8.0 and 150 mM NaCl. Evaluation of the SPR data was performed by comparing the height of the biological response signal at 180 seconds after injection and 300 seconds after injection. The corresponding parameters are the RU max level (180 seconds after injection) and late stability (300 seconds after the end of injection).

[0190] As shown in FIGS. 9A and 9B, all mAb 0318 antibody variants (IgG1-Aba mod, IgG4-Aba mod, IgG1.1f, and IgG1.3f) were able to bind to human, mouse, and cynomolgus monkey FcRn in a pH-dependent manner. This was also true for the mAb 0318 antibody (IgG4).

[0191] (Example 10) Binding analysis of mAb 0318 variants to one or more FcγRs As previously discussed, in vivo administration of antibodies to certain cell surface immunoreceptors has the potential to induce cytokine release, which can result in the induction of a common toxic clinical complication known as cytokine release syndrome (CRS). Due to concerns that FcγR binding could lead to potential TREM-1 agonistic activity via cross-linking, mAb 0318-IgG4 was re-engineered into one of the variant formats described in this disclosure (i.e., IgG1.1f, IgG1.3f, IgG1-Aba or IgG4-Aba). The ability of the 0318 antibody variants to bind to different FcγRs was then evaluated.

[0192] As shown in FIGS. 10A and 10B, the 318-IgG1.1f and 318-IgG1.3f variants had minimal binding to all of the FcγRs (i.e., FcγRI (CD64), FcγRIIA (CD32a-H131 and CD32a-R131 variants), FcγRIIB (CD32b), FcγRIIIA (CD16a-V158 variant) and FcγRIIIB (CD16b-NA2 variant)). The 318-IgG1-Aba and 318-IgG4-Aba variants did not bind to FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB, but did bind to FcγRI. In contrast, mAb 0318 (IgG4) showed significant binding to all of the FcγRs.

[0193] (Example 11) Analysis of the induction of inflammatory cytokines by mAb 0318 variants To further determine whether in vivo treatment with mAb 0318 variants poses a risk of cytokine release syndrome, whole blood was collected from eight human donors and monocytes were isolated. The monocytes (4×10 6These monocytes were differentiated into immature dendritic cells by plating them (cells / well) and culturing them in a differentiation medium containing IL-4 and GM-CSF (100 ng / mL). Approximately 2 to 3 days after plating, approximately half of the differentiation medium was replaced with fresh medium. On day 7, the cells were collected and the differentiation efficiency was evaluated by analyzing CD14 expression on the cells using a flow cytometer. Next, the immature dendritic cells were plated on a flat-bottom plate (0.8×10 5 cells / well), and the mAb 0318 variants were added to each well (with or without CHO-CD32a). Immature dendritic cells stimulated with PGRP+PGN were used as a positive control. These cells were then incubated overnight at 37°C. The next day, the supernatant was collected from the wells and the amounts of TNF-α, IL-6, and IL-12 produced were evaluated using an ELISA assay.

[0194] Results from a representative donor are shown in FIGS. 11A-11I. The addition of different mAb 0318 variants (0318-IgG1.1f, 0318-IgG1.3f, and 0318-IgG1.1 Aba) resulted in minimal IL-6 (FIGS. 11A, 11B, and 11C), TNF-α (FIGS. 11D, 11E, and 11F), and IL-12 (FIGS. 11G, 11H, and 11I) production by immature dendritic cells. These results, together with the results of Example 9, demonstrate that the anti-TREM-1 antibodies disclosed herein have a low risk of inducing cytokine release syndrome when administered in vivo to patients.

[0195] (Example 12) Further Characterization of mAb 0318 Variants The biophysical characteristics of the 0318-IgG1.3f variant are provided in Table 3 (below). [Table 3]

[0196] The biophysical properties of the mAb 0318-IgG1.3f variant are beneficial for clinical development. The identity of the antibody was confirmed by mass spectrometry (intact mass spectrometry and peptide mapping). The antibody was >96% monomer when tested by size exclusion chromatography. A single N-glycosylation site was confirmed at N301 on the heavy chain with a glycan profile that matched the glycan profiles of the CHO-expressed monoclonal antibody (G0F, G1F, and G2F). The thermal stability (T m 1 = 66.2 °C; T m 2 = 78.4 °C; T m 3 = 83.2 °C) and thermal reversibility (24% at 77 °C) of mAb 0318-IgG1.3f were within the range for typical human IgG1.3 monoclonal antibodies.

[0197] The stability characteristics of the mAb 0318-IgG1.3f variant are provided in Table 4 (below).

Table 4

[0198] In the described formulation (20 mM histidine, 150 mM sucrose, 50 mM sodium chloride, 25 mM arginine, pH 6.0) at 150 mg / mL, no physical stability issues were observed during freeze-thaw stress (3 cycles). Forced degradation studies on the investigational formulation at 150 mg / mL were set at 4, 25, and 40 °C (for up to 3 months). The chemical modifications of CDR, as determined by SPR, remained low throughout all temperature conditions and had no effect on activity. VSNK deamidation was below prediction (an increase of 3% / month upon storage at 40 °C) compared to other monoclonal antibodies in the IgG1.3f framework, and the changes to it were time- and temperature-dependent. All other chemical modifications (oxidation, deamidation, isomerization) also remained low when monitored during the stability study. Notably, storage at 40 °C showed the formation of both HMW variants and LMW variants (demonstrating 1.2% / month and 2.4% / month respectively). The observed changes were time- and temperature-dependent, with HMW increasing by 0.25% / month and LMW remaining unchanged upon storage at 4 °C over the study period. The low LMW formation upon storage at 40 °C was characterized by 2D-LC / MS with high-resolution accurate mass measurement as the cumulative species of the loss of one Fab, as well as the Fab arm in the conserved sequence in the upper hinge region by estimation.

[0199] (Example 13) PK / TK / PD Study on mAb 0318-IgG1.3f Variant in Cynomolgus Monkeys A single-dose pharmacokinetic (PK), toxicokinetic (TK), and pharmacodynamic (PD) study of the anti-TREM-1 0318-IgG1.3f antibody was conducted in cynomolgus monkeys. Some of the animals received the 0318-IgG1.3f antibody intravenously at 2 mg / kg (n = 3). Other animals received one of the following doses of the 0318-IgG1.3f antibody subcutaneously: (i) 0 mg / kg (i.e., control) (n = 4), (ii) 0.1 mg / kg (n = 4), (iii) 0.5 mg / kg (n = 4), (iv) 2 mg / kg (n = 3), or (v) 10 mg / kg (n = 4). At the time of antibody administration, PK, anti-drug antibody (ADA), TREM-1 receptor occupancy (RO), and ex vivo pharmacodynamic responses were tested at predetermined time points.

[0200] Pharmacokinetics (PK) To evaluate PK, the serum concentration of the 0318-IgG1.3f antibody was evaluated in animals using a ligand-binding assay that used biotinylated recombinant TREM-1 protein as a capture reagent and a commercially available polyclonal goat anti-TREM-1 antibody as a detection reagent. As shown in Figure 12, the PK of the 0318-IgG1.3f antibody was determined to be non-linear between 0.1 mg / kg and 10 mg / kg, mainly due to target-mediated clearance (i.e., internalization and degradation of the antibody upon binding to the TREM-1 receptor; see Example 2).

[0201] Non-compartmental analysis-based PK parameters are shown in Table 5 (intravenous administration) and Table 6 (subcutaneous administration). Briefly, a 100-fold increase in dose resulted in an 830-fold increase in serum exposure (AUC). See Table 6. Clearance after a 2 mg / kg IV dose was 0.1 ± 0.02 mL / hour / kg in monkeys and was similar to other IgG1-based mAbs. See Table 4. Vss at 36 ± 5 mL / kg was similar to plasma volume, indicating limited extravascular distribution. The half-life of the 0318-IgG1.3f antibody increased from 2 days for the 0.1 mg / kg dose to 10 days at 2 and 10 mg / kg (single subcutaneous dose administration). See Table 6. The bioavailability of the 0318-IgG1.3f antibody after subcutaneous administration (2 mg / kg) was high at 84%.

[0202]

Table 5

[0203]

Table 6

[0204] Anti-drug antibody (ADA) Serum ADA was detected in most monkeys (16 out of 18) after a single dose (regardless of the route of administration), but the exposure of the 0318-IgG1.3f antibody and the TREM-1 receptor occupancy (RO) were not impaired in most of the ADA-positive animals. See Tables 4 and 5. Accelerated decay in the terminal exposure of the 0318-IgG1.3f antibody associated with ADA formation was observed in only 2 monkeys (1 in the 0.1 mg / kg dose group on day 7 and 1 in the 0.5 mg / kg dose group on day 21). Since ADA affected the exposure in these 2 monkeys at the end stage, the corresponding data points were excluded for PK analysis.

[0205] TREM-1 receptor occupancy (RO) and total TREM-1 receptor levels Next, the occupancy of the TREM-1 receptor expressed on peripheral blood monocytes and granulocytes was evaluated. As shown in FIGS. 15A and 15B, for all doses tested, the percentage of TREM-1 receptors that were occupied (i.e., bound to the anti-TREM-1 antibody) was similar between monocytes and granulocytes. Furthermore, the duration of receptor occupancy appeared to depend on the dose of 0318-IgG1.3f antibody administered to the animals. At 0.5 mg / kg, ≧85% RO was observed for up to 2 weeks after antibody administration. In contrast, at 2 and 10 mg / kg, ≧85% of the TREM-1 receptors remained occupied for at least 1 month after administration. After dosing with the 0318-IgG1.3f antibody, total TREM-1 receptor levels were reduced on both monocytes and granulocytes. See FIGS. 14A and 14B. As previously discussed, this reduction is likely due to increased receptor turnover after antibody binding. The decrease in surface TREM-1 receptor expression was reversible, and the duration of surface receptor loss correlated with the duration of receptor occupancy, at least at the dose levels tested.

[0206] Levels of soluble TREM-1 (sTREM-1) appeared to increase 10- to 50-fold after a single dose of the mAb 0318 mAb-IgG1.3f variant in all dose groups tested (data not shown). Whether the increase in sTREM-1 levels is related to anti-TREM-1 antibody administration or to the general handling of the animals during the course of the study is unclear. In either case, since the drug concentration far exceeded the sTREM-1 levels (>1000-fold), the soluble target is not expected to result in binding of the mAb 0318 mAb-IgG1.3f variant to cell surface TREM-1.

[0207] (Example 14) PK / TK / PD study for the mAb 0318-IgG1.3f variant in cynomolgus monkeys as described by a two-compartment PK model using TMDD in the central compartment The PK, RO, total receptor levels, and PD data in monkeys were described using a two-compartment PK model with saturable target-mediated drug disposition (TMDD) in the central compartment adapted to the observed non-linear PK (see Example 12 and Figure 13). A direct effect inhibitory model was used to describe the PD response. This model was able to effectively capture the time course of PK / RO / PD observed in monkeys. The observed (open circles) and model-predicted endpoints (solid lines) are provided in Figure 16A (subcutaneous dosing at 0.1 mg / kg) and Figure 16B (subcutaneous dosing at 10 mg / kg). Table 7 provides the estimated PK / PD parameters. Pooling the serum exposure of the mAb0318-IgG1.3f variant and RO data for TREM-1 from all monkeys, a concentration-dependent increase in RO was observed. See Figure 16A and Figure 16B. The Emax model used to describe the concentration-RO relationship estimated the in vivo RO EC 50 to be 1.6 ± 0.2 nM.

[0208]

Table 7

[0209]

Table 8

Claims

**Claim 1**: An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 85, and the light chain comprises SEQ ID NO:

54. **Claim 2** A bispecific molecule comprising the antibody according to claim 1, linked to a molecule having a second binding specificity. **Claim 3** A nucleic acid encoding the antibody according to claim 1. **Claim 4** A vector comprising the nucleic acid according to claim 3. **Claim 5** A host cell comprising the vector according to claim 4. **Claim 6** An immunoconjugate comprising the antibody according to claim 1, linked to a drug. **Claim 7** A composition comprising the antibody according to claim 1, the bispecific molecule according to claim 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5, or the immunoconjugate according to claim 6, and a carrier. **Claim 8** A kit comprising the antibody according to claim 1, the bispecific molecule according to claim 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5, or the immunoconjugate according to claim 6, and instructions for use. **Claim 9** An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 85 and the light chain comprises SEQ ID NO:

54. **Claim 10** An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50 and the light chain comprises SEQ ID NO:

54. **Claim 11** An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 51 and the light chain comprises SEQ ID NO:

54. **Claim 12** A pharmaceutical composition for blocking TREM-1 in a human, comprising the antibody according to claim 1, or any one of claims 9 to 11, the bispecific molecule according to claim 2, or the immunoconjugate according to claim 6. **Claim 13** A pharmaceutical composition for treating an inflammatory disease or an autoimmune disease, comprising the antibody according to claim 1, or any one of claims 9 to 11, the bispecific molecule according to claim 2, or the immunoconjugate according to claim 6. **Claim 14** The pharmaceutical composition according to claim 13, wherein the inflammatory disease or the autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, vasculitis, sepsis, systemic inflammatory response syndrome (SIRS), type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma, and other autoimmune diseases that are the result of either acute inflammation or chronic inflammation, and any combination thereof.

15. The pharmaceutical composition according to claim 12, further comprising one or more additional therapeutic agents.

16. The pharmaceutical composition according to claim 14, wherein the inflammatory disease or the autoimmune disease is Crohn's disease.

17. The pharmaceutical composition according to claim 14, wherein the inflammatory disease or the autoimmune disease is ulcerative colitis.

18. A pharmaceutical composition for treating Crohn's disease, comprising an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 85 and the light chain comprises SEQ ID NO:

54.

19. A pharmaceutical composition for treating ulcerative colitis, comprising an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 85 and the light chain comprises SEQ ID NO:

54.

20. A pharmaceutical composition for treating Crohn's disease, comprising an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50 and the light chain comprises SEQ ID NO:

54.

21. A pharmaceutical composition for treating ulcerative colitis, comprising an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50 and the light chain comprises SEQ ID NO: 54.

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