Anti-TREM-1 antibody and its use

Abstract

To provide an anti-TREM-1 antibody that can specifically bind to TREM-1 and inhibit the TREM-1 function but does not have the issues of the earlier anti-TREM-1 antibodies.SOLUTION: Provided herein are antibodies, or antigen-binding portions thereof, that specifically bind to TREM-1 and inhibit TREM-1 signaling, where the antibodies do not bind to one or more FcγRs and do not induce myeloid cells to produce inflammatory cytokines. Also provided are uses of such antibodies, or antigen-binding portions thereof, in therapeutic applications, such as treatment of autoimmune diseases.SELECTED DRAWING: None

Description

Technical Field

[0001] Reference to a Sequence Listing Electronically Filed via EFS-Web The content of the sequence listing electronically filed as an ASCII text file (name: 3338_092PC01_SeqListing.txt; size: 106,162 bytes; and creation date: March 27, 2019) submitted 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. TREM-1 mRNA and protein expression are upregulated in patients with rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and TREM-1-positive cells accumulate at sites of inflammation in correlation with disease severity. 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, TREM-1 binding induces the secretion of pro-inflammatory cytokines, including TNF, IL-8, and monocyte chemotactic protein-1. Furthermore, TREM-1 signaling synergistically boosts pro-inflammatory signaling with multiple Toll-like receptors (TLRs). This, in turn, leads to a vicious cycle of upregulating TREM-1 expression and amplifying 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 viscosity profiles that can interfere with the manufacturing process or have other problems (e.g., cytokine storms and ADCC) that can limit their therapeutic potential; see Shire et al., J. Pharm. Sci. 93:1390-1402 (2004); and Warnkke et al., J Immunol. 188:4405-11 (2012). Therefore, there is a need for an anti-TREM-1 antibody that can specifically bind to TREM-1 and inhibit TREM-1 function, but without the problems of earlier anti-TREM-1 antibodies. [Overview of the project]

[0004] Isolated antibodies, such as monoclonal antibodies, particularly human (e.g., monoclonal) antibodies, that specifically bind to trigger receptor-1 (TREM-1) expressed on myeloid cells and possess 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, wherein the IgG1 heavy chain constant region includes one or more amino acid substitutions 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, wherein the IgG1 heavy chain constant region includes one or more amino acid substitutions compared to the wild-type IgG1 heavy chain constant region (SEQ ID NO: 9). In some embodiments, this antibody binds to the same TREM-1 epitope as mAb 0318. In some embodiments, this antibody specifically binds to a TREM-1 epitope containing 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, this antibody specifically binds to a TREM-1 epitope containing 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 antibody disclosed herein includes 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 includes 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 includes one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S, according to EU numbering. In some embodiments, the IgG1 heavy chain constant region 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.

[0006] In some embodiments, the antibodies disclosed herein include heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR3 includes DMGIRRQFAY (SEQ ID NO: 26) or DMGIRRQFAY (SEQ ID NO: 26) with one or two substitutions removed. In some embodiments, the heavy chain CDR3 includes DQGIRRQFAY (SEQ ID NO: 72). In some embodiments, the antibodies disclosed herein comprise 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 omitted. In some embodiments, the antibodies disclosed herein comprise 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.

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

[0008] In some embodiments, the VH of the antibody disclosed herein contains 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 antibody disclosed herein contains 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 antibody of this disclosure comprises a heavy chain and a light chain, the heavy chain comprising SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. In some embodiments, the light chain comprises SEQ ID NO: 54.

[0009] An isolated antibody that specifically binds to TREM-1, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, wherein the heavy chains CDR1, CDR2, and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; the light chains 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, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, wherein the heavy chains CDR1, CDR2, and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; the light chains 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, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, wherein the heavy chains CDR1, CDR2, and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; the light chains 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, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, wherein the heavy chains CDR1, CDR2, and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; and the light chains CDR1, CDR2, and CDR3 comprise, respectively Antibodies comprising RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29); wherein 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, are provided herein. In some embodiments, TREM-1 includes 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 this disclosure has a reduced binding affinity to FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), FcγRIIIB(CD16b), or any combination thereof, 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 to FcγRI(CD64) of 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 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 antibodies disclosed herein exhibit lower immunogenicity compared to antibodies 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 upon binding to TREM-1 in the absence of a stimulating factor. 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 stimulating factor, compared to antibodies 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 antibodies disclosed herein block the production of inflammatory cytokines in cells when the cells are activated in the presence of both the antibody and a stimulating factor. In some embodiments, the stimulating factor is a TREM-1 ligand. In some embodiments, 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.

[0014] In some embodiments, the antibodies of this disclosure bind to human FcRn, cynomolgus monkey FcRn, and / or mouse FcRn in a pH-dependent manner. In some embodiments, the antibodies disclosed herein are more thermally stable than a reference antibody comprising a heavy chain of the amino acid sequence shown in SEQ ID NO: 76 and a light chain of the amino acid sequence shown in SEQ ID NO: 54, as measured by capillary differential scanning calorimeter (CAP-DSC). In some embodiments, about 10% to 20%, about 20% to 30% (e.g., 24%), or about 30% to 40% of this antibody are reversible when heated to 77°C. In some embodiments, this antibody has a higher melting temperature (Tm) than an antibody comprising a heavy chain of the amino acid sequence shown in SEQ ID NO: 76 and a light chain 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 130 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 content of less than 4 nM (e.g., 3.4 nM) when measured by Biacore. D It binds to human TREM-1. In some embodiments, this antibody has a K content of less than 1 nM (e.g., 0.91 nM) as measured by Biacore. D It then binds to the cynomolgus monkey TREM-1.

[0016] In some embodiments, the antibody is monomeric when observed by size exclusion high-performance liquid chromatography (SE-HPLC). In some embodiments, the antibody exhibits a minimal risk of fragmentation when observed by two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC / MS) or intact mass analysis using 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 is stored at 4°C, 25°C, or 40°C. Also provided herein are bispecific molecules, including the anti-TREM-1 antibody of this disclosure linked to a molecule having a second binding specificity. Nucleic acids encoding antibodies disclosed herein, vectors containing such nucleic acids, and cells transformed with such vectors are provided herein. An immunoconjugate comprising an anti-TREM-1 antibody, as disclosed herein, conjugated to a drug is provided herein.

[0018] Compositions comprising an anti-TREM-1 antibody or its antigen-binding moiety, a bispecific molecule or immunoconjugate, and a carrier as described herein are provided herein. Kits comprising an anti-TREM-1 antibody or its antigen-binding moiety, a bispecific molecule or immunoconjugate, and instructions for use as described herein are also provided herein. Methods for inhibiting TREM-1 activity in a target subject requiring such inhibition are provided herein, comprising the step of administering an anti-TREM-1 antibody, a bispecific molecule, a nucleic acid, a vector, cells, or an immunoconjugate as described herein.

[0019] Methods for treating inflammatory or autoimmune diseases in subjects requiring such treatment are provided herein, comprising the step of administering an anti-TREM-1 antibody, a bispecific molecule, a nucleic acid, a vector, cells, or an immunoconjugate as described herein. In some embodiments, the inflammatory 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 1 diabetes mellitus, 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 syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma, other autoimmune diseases resulting from either acute or chronic inflammation, and any combination thereof. In some embodiments, the method further includes the step of administering one or more additional therapeutic agents. In some embodiments, these additional therapeutic agents are anti-IP-10 antibodies or anti-TNF-α antibodies. Another aspect of the present invention may be as follows: [1] An isolated antibody comprising a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, which specifically binds to trigger receptor-1 (TREM-1) expressed on myeloid cells, wherein the IgG1 heavy chain constant region contains one or more amino acid substitutions compared to the wild-type IgG1 heavy chain constant region (SEQ ID NO: 9). [2] An isolated antibody comprising a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, which cross-competes with mAb 0318 for binding to block TREM-1, 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 described in [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 containing 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], which specifically binds to the 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 the above [1] to [5], wherein the IgG1 heavy chain constant region comprises 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 the above [1] to [5], wherein the 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. [8] The antibody according to any one of the above [1] to [5], wherein 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. [9] The isolated antibody according to any one of the above [1] to [5], wherein 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.

[10] An antibody according to any one of the above items [1] to [9], 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] , wherein the heavy chain CDR3 comprises DQGIRRQFAY (SEQ ID NO: 72).

[12] An antibody according to any one of the above items [1] to

[11] , 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] An antibody according to any one of the above items [1] to

[12] , 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.

[14] An antibody according to any one of the above items [1] to

[13] , comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the light chain CDR1 comprises RASQSVDTFDYSFLH (SEQ ID NO: 27) or RASQSVDTFDYSFLH (SEQ ID NO: 27) with one or two substitutions removed.

[15] An antibody according to any one of the above items [1] to

[14] , comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the light chain CDR2 comprises RASNLES (SEQ ID NO: 28) or RASNLES (SEQ ID NO: 28) with one or two substitutions removed.

[16] An antibody according to any one of the above items [1] to

[15] , comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the light chain CDR3 comprises QQSNQDPYT (SEQ ID NO: 29) or QQSNQDPYT (SEQ ID NO: 29) with one or two substitutions removed.

[17] The antibody according to any one of the above [1] to

[16] , wherein the VH contains 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.

[18] The antibody according to any one of the above [1] to

[17] , wherein the VL contains 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.

[19] An antibody according to any one of the above items [1] to

[18] , comprising 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.

[20] An antibody according to

[19] , comprising 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.

[21] An antibody according to

[20] , comprising a heavy chain and a light chain, wherein the light chain comprises SEQ ID NO: 54.

[22] An isolated antibody that specifically binds to TREM-1, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, The heavy chains CDR1, CDR2, and CDR3 each contain TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; The light chains CDR1, CDR2, and CDR3 each contain RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; 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. antibody.

[23] An isolated antibody that specifically binds to TREM-1, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, The heavy chains CDR1, CDR2, and CDR3 each contain TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; The light chains CDR1, CDR2, and CDR3 each contain RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; The IgG1 heavy chain constant region includes an amino acid substitution selected from the group consisting of L234A, L235E, G237A, A330S, and P331S, according to EU numbering. antibody.

[24] An isolated antibody that specifically binds to TREM-1, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, The heavy chains CDR1, CDR2, and CDR3 each contain TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; The light chains CDR1, CDR2, and CDR3 each contain RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; 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. antibody.

[25] An isolated antibody that specifically binds to TREM-1, comprising heavy chains CDR1, CDR2, CDR3; light chains CDR1, CDR2, CDR3; and the IgG1 heavy chain constant region, The heavy chains CDR1, CDR2, and CDR3 each contain TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively; The light chains CDR1, CDR2, and CDR3 each contain RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; 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 the above items [1] to

[25] , wherein 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.

[27] The antibody according to any one of the above [1] to

[26] , which has reduced binding affinity to FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), FcγRIIIB(CD16b), or any combination thereof, 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.

[28] An antibody according to any one of the above [1] to

[26] , which has a binding affinity to FcγRI(CD64) that is reduced to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 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.

[29] An antibody according to any one of the above [1] to

[28] , which has lower immunogenicity 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.

[30] An antibody according to any one of the above items [1] to

[29] , which, upon binding to TREM-1, does not agonize TREM-1 signaling in the absence of a stimulating factor.

[31] The antibody according to any one of the above [1] to

[30] , which, when immature dendritic cells (iDCs) are incubated in the presence of the antibody and in the absence of a stimulating factor, does not induce the expression of inflammatory cytokines in the cells, 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.

[32] The antibody according to any one of items [1] to

[31] above, which blocks the production of inflammatory cytokines in the cells when the cells are activated in the presence of both the antibody and the stimulating factor.

[33] The antibody according to any one of the above

[30] to

[31] , wherein the stimulating 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] An antibody according to any one of the above [1] to

[34] , which binds to human FcRn, cynomolgus monkey FcRn and / or mouse FcRn in a pH-dependent manner.

[36] The antibody according to any one of the above [1] to

[35] , which is more thermally stable when measured by capillary differential scanning calorimeter (CAP-DSC) than 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.

[37] The antibody according to any one of the above items [1] to

[36] , wherein about 10% to 20%, about 20% to 30% (for example, 24%), or about 30% to 40% of the antibody is reversible when heated to 77°C.

[38] The antibody according to

[36] or

[37] , having 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.

[39] An antibody according to any one of the above [1] to

[38] , 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] An antibody according to any one of the above items [1] to

[38] , having a viscosity of less than 10 cP (e.g., 9 cP) at a concentration of 130 mg / mL.

[41] KnM less than 4nM (e.g., 3.4nM) when measured by Biacore D An antibody according to any one of the above items [1] to

[40] , which binds to human TREM-1.

[42] Kless than 1 nM (e.g., 0.91 nM) when measured by Biacore D An antibody according to any one of the above items [1] to

[41] , which binds to cynomolgus monkey TREM-1.

[43] An antibody according to any one of the above items [1] to

[42] , which is a monomer when observed by size exclusion high-performance liquid chromatography (SE-HPLC).

[44] An antibody according to any one of the above items [1] to

[43] , which exhibits the minimum risk of fragmentation when observed by two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC / MS) or intact mass spectrometry using liquid chromatography-tandem mass spectrometry (LC / MS).

[45] An antibody according to any one of the above items [1] to

[44] , having an isoelectric point of 8 to 9 (for example, 8.75).

[46] An antibody according to any one of the above items [1] to

[45] , which is stable in a preparation containing histidine, sucrose, arginine, and NaCl.

[47] The antibody described in

[46] , which is stable for at least two months in a preparation containing 20 mM histidine, 150 mM sucrose, 25 mM arginine, and 50 mM NaCl.

[48] ​​The antibody according to

[46] or

[47] , wherein the formulation has a pH of 6.0 and / or the formulation is stored at 4°C, 25°C or 40°C.

[49] A bispecific molecule comprising the antibody described in any one of the above items [1] to

[48] , linked to a molecule having a second binding specificity.

[50] A nucleic acid encoding an antibody as described in any one of the items [1] to

[48] above.

[51] A vector containing the nucleic acid described in

[50] above.

[52] A cell containing the vector described in

[51] above.

[53] An immunoconjugate comprising an antibody described in any one of the above items [1] to

[48] , which is linked to a drug.

[54] A composition comprising an antibody according to any one of items [1] to

[48] above, a bispecific molecule according to

[49] above, a nucleic acid according to

[50] above, a vector according to

[51] above, a cell according to

[52] above or an immunoconjugate according to

[53] above, and a carrier.

[55] A kit comprising an antibody according to any one of items [1] to

[48] above, a bispecific molecule according to

[49] above, a nucleic acid according to

[50] above, a vector according to

[51] above, a cell according to

[52] above or an immunoconjugate according to

[53] above, and instructions for use.

[56] A method for inhibiting TREM-1 activity in a subject requiring it, comprising the step of administering to a subject an antibody described in any one of items [1] to

[48] , a bispecific molecule described in

[49] , a nucleic acid described in

[50] , a vector described in

[51] , a cell described in

[52] , or an immunoconjugate described in

[53] .

[57] A method for treating an inflammatory disease or autoimmune disease in a subject requiring it, comprising the step of administering to a subject an antibody described in any one of items [1] to

[48] , a bispecific molecule described in

[49] , a nucleic acid described in

[50] , a vector described in

[51] , a cell described in

[52] , or an immunoconjugate described in

[53] .

[58] The method according to

[57] , wherein 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 1 diabetes mellitus, 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 syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma, other autoimmune diseases resulting from any acute or chronic inflammation, and any combination thereof.

[59] The method according to any one of the items

[56] to

[58] , further comprising the step of administering one or more additional therapeutic agents.

[60] The method according to

[59] , wherein the further therapeutic agent is an anti-IP-10 antibody or an anti-TNF-α antibody. [Brief explanation of the drawing]

[0020] [Figure 1A]Figures 1A and 1B show that all mAb 0318 variants bind to human and cynomolgus monkey TREM-1 with similar affinity to the original mAb 0318 antibody (IgG4). Figure 1A shows binding affinity data for the 318-IgG1.3f variant to both human (top row) and cynomolgus monkey (bottom row) TREM-1. [Figure 1B] Figures 1A and 1B show that all mAb 0318 variants bind to human and cynomolgus monkey TREM-1 with similar affinity to the original mAb 0318 antibody (IgG4). Figure 1B shows binding affinity data for several different mAb 0318 variants against human TREM-1. The different variants shown include: (i) 318-IgG1.1f, (ii) 318-IgG1.3f, (iii) 318-IgG4-Aba, (iv) 318-IgG1-Aba, and (v) 318-IgG1.1f (M to Q variant). Binding affinity data for mAb 0318 antibodies (IgG4 A and B) are provided in Figure 1B for comparison. [Figure 2A] Figures 2A and 2B show the internalization of mAb 0318-IgG1.3f upon binding to TREM-1 expressed on CD14+ monocytes at various time points after TREM-1 binding. In Figure 2A, TREM-1 receptor expression was analyzed at 0, 6, and 24 hours after binding. [Figure 2B] Figures 2A and 2B show the internalization of mAb 0318-IgG1.3f upon binding to TREM-1 expressed on CD14+ monocytes at various time points after TREM-1 binding. In Figure 2B, TREM-1 receptor expression was analyzed at 0, 4, and 20 hours after binding. Figure 2B also provides data using the TREM26 antibody, which does not compete with the 0318 antibody variant for binding to TREM-1. The TREM26 antibody was used to assess the fate of the TREM-1 receptor after its internalization (e.g., whether it is degraded or recycled back to the surface). [Figure 3]Figure 3 shows that the mAb 0318 variant does not agonize TREM-1 signaling when reporter cell lines are incubated with or without CHO-CD32 ("both") or without CHO-CD32 ("BWZ 36 only"), as measured by the BWZ / hTREM-1 reporter cell assay. The variant antibodies shown include: (i) 318-IgG1.1f ("IgG1.1f"), (ii) 318-IgG1.3f ("IgG1.3f"), (iii) 318-IgG4-Aba ("FcAba-4"), and (iv) 318-IgG1-Aba ("FcAba-1"). MAB1278 antibody, a known agonist of TREM-1 signaling, was used as the positive control antibody (see inserted box). 5C8 (isotype control) antibody was used as the negative control. [Figure 4] Figure 4 shows the potency of different mAb 0318 variants in blocking TREM-1-mediated production of inflammatory cytokines by different human cells. The mAb 0318 antibody variants shown in Figure 4 inhibit the TREM-1-mediated release of inflammatory cytokines (e.g., TNF-α, IL-6, or IL-8) from different human cell types: PBMCs, monocytes, neutrophils, and RBC-precipitated whole blood (i.e., the majority of red blood cells were removed using the dextran-based RBC precipitation protocol described in the examples). To produce inflammatory cytokines, cells were stimulated with either plate-bound soluble peptidoglycan lacking PGRP1 and TLR2 activity ("PGRP+PGN-Ecndss") or phorbol 12-myristate 13-acetate (PMA)-stimulated PGRP1-expressing neutrophils ("PMA-stimulated neutrophil endogenous PGRP"). The variant antibodies shown include: (i) 0318-IgG4, (ii) 0318-IgG1.3f, (iii) 0318-IgG1.1f, (iv) 0318-IgG1-Aba, and (v) 318-IgG4-Aba. "N / D" indicates that the expression level of a specific inflammatory cytokine could not be determined. [Figure 5A]Figures 5A and 5B show the potency of mAb 0318-IgG1.3f blocking IL-8 production from whole blood after PGN stimulation with and without PGRP1 in the presence of a NOD2 inhibitor. Figure 5A provides inhibition data generated using the whole blood pharmacodynamic assay described in the examples. [Figure 5B] Figures 5A and 5B show the potency of mAb 0318-IgG1.3f blocking IL-8 production from whole blood after PGN stimulation with and without PGRP1 in the presence of a NOD2 inhibitor. Figure 5B provides inhibition data generated using the whole blood intracellular cytokine assay described in the examples. [Figure 6A] Figures 6A–6C show the RNA expression levels of chitinase-3-like protein 1 ("CHI3L1") (Figure 6A), IL-1β (Figure 6B), and IL-6 (Figure 6C) from whole blood stimulated in the presence of varying concentrations of mAb 0318-IgG1.3f (0–1 nM). Whole blood was collected from three different donors (126, 290, and 322) and stimulated with soluble PGRP1 and soluble peptidoglycan lacking TLR2 activity ("soluble PGRP + soluble PGN-Ecndss"). In each of Figures 6A–6C, RNA expression levels (y-axis) are shown as both % inhibition (right column) and ΔΔCt (difference between the reference gene value and the test sample value) (left column). Different concentrations of 0318-IgG1.3f antibody are shown on the x-axis. [Figure 6B]Figures 6A–6C show the RNA expression levels of chitinase-3-like protein 1 ("CHI3L1") (Figure 6A), IL-1β (Figure 6B), and IL-6 (Figure 6C) from whole blood stimulated in the presence of varying concentrations of mAb 0318-IgG1.3f (0–1 nM). Whole blood was collected from three different donors (126, 290, and 322) and stimulated with soluble PGRP1 and soluble peptidoglycan lacking TLR2 activity ("soluble PGRP + soluble PGN-Ecndss"). In each of Figures 6A–6C, RNA expression levels (y-axis) are shown as both % inhibition (right column) and ΔΔCt (difference between the reference gene value and the test sample value) (left column). Different concentrations of 0318-IgG1.3f antibody are shown on the x-axis. [Figure 6C] Figures 6A–6C show the RNA expression levels of chitinase-3-like protein 1 ("CHI3L1") (Figure 6A), IL-1β (Figure 6B), and IL-6 (Figure 6C) from whole blood stimulated in the presence of varying concentrations of mAb 0318-IgG1.3f (0–1 nM). Whole blood was collected from three different donors (126, 290, and 322) and stimulated with soluble PGRP1 and soluble peptidoglycan lacking TLR2 activity ("soluble PGRP + soluble PGN-Ecndss"). In each of Figures 6A–6C, RNA expression levels (y-axis) are shown as both % inhibition (right column) and ΔΔCt (difference between the reference gene value and the test sample value) (left column). Different concentrations of 0318-IgG1.3f antibody are shown on the x-axis. [Figure 7] Figure 7 shows the viscosity-concentration profiles for both the 318-IgG1.1f (square) and 318-IgG1.3f (circle) variant antibodies. Viscosity profiles were generated from a dilution scheme using a Rheosense m-VROC dynamic viscometer with a 3-point shear sweep at each point at a constant temperature. Solid lines provide the best nonlinear curve fit for the data shown. Dotted lines provide the maximum acceptable viscosity level for potency. [Figure 8]Figure 8 shows that both 318-IgG1.1f and 318-IgG1.3f variant antibodies carry a low to intermediate risk of immunogenicity. VL6 (immunogenic IL-21R mAb) and KLH (keyhole limpet hemocyanin) were used as positive controls. Avastin was used as a negative control. [Figure 9A] Figures 9A and 9B show that all mAb 0318 variants are capable of binding to FcRn (human (black), cynomolgus monkey (white), and mouse (gray)) in a pH-dependent manner. Figure 9A provides FcRn binding as %Rmax (maximum FcRn binding capacity). [Figure 9B] Figures 9A and 9B show that all mAb 0318 variants are capable of binding to FcRn (human (black), cynomolgus monkey (white), and mouse (gray)) in a pH-dependent manner. Figure 9B provides FcRn binding as a sensorgram. The 0318 antibody variants shown include: (i) IgG1-Aba, (ii) IgG4-Aba, (iii) IgG1.1f, and (iv) IgG1.3f. The mAb 0318 (IgG4) antibody is also shown for comparison. [Figure 10A] Figures 10A and 10B demonstrate that all mAb 0318 antibody variants exhibit reduced binding to one or more human FcγRs (i.e., CD64, CD32a-H131 variant, CD32a-R131 variant, CD32b, CD16a-V158 variant, and CD16B-NA2 variant). Figure 10A shows the binding affinity as %Rmax (maximum FcγR activity). [Figure 10B]Figures 10A and 10B demonstrate that all mAb 0318 antibody variants exhibit reduced binding to one or more human FcγRs (i.e., CD64, CD32a-H131 variant, CD32a-R131 variant, CD32b, CD16a-V158 variant, and CD16B-NA2 variant). Figure 10B shows a sensorgram. The 0318 antibody variants shown include: (i) IgG1-Aba, (ii) IgG4-Aba, (iii) IgG1.1f, and (iv) IgG1.3f. The mAb 0318 (IgG4) antibody is shown for comparison purposes. The 1F4-hIgG1f antibody, known to bind to multiple FcγRs, was used as a control. [Figure 11A] Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11A, 11B, and 11C provide the amounts of IL-6 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11B]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11A, 11B, and 11C provide the amounts of IL-6 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11C]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11A, 11B, and 11C provide the amounts of IL-6 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11D]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11D, 11E, and 11F provide the amounts of TNF-α produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11E]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11D, 11E, and 11F provide the amounts of TNF-α produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11F]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11D, 11E, and 11F provide the amounts of TNF-α produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11G]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11G, 11H, and 11I provide the amounts of IL-12 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11H]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11G, 11H, and 11I provide the amounts of IL-12 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 11I]Figures 11A–11I show that the 0318 variant antibodies are not agonist by themselves (i.e., in the absence of stimulation) when cultured with immature dendritic cells, as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12). Figures 11G, 11H, and 11I provide the amounts of IL-12 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosage (μg / mL) of the antibodies used is indicated in parentheses. Immature dendritic cells were cultured with and without membrane CD32a-expressing CHO cells (to stimulate Fc crosslinking) with anti-TREM-1 antibody variants. PGRP+PGN, CD40L trimmer ("Trimmer"), and Pfizer's anti-CD40 agonist antibody ("CP870") were used as positive controls. An isotype antibody ("CHIL-6") was used as a negative control. The dotted line indicates the detection limit for the assay. [Figure 12] Figure 12 shows the pharmacokinetics of a single dose of the mAb 0318-IgG1.3f variant in cynomolgus monkeys after subcutaneous administration. Each animal received one of the following doses: (i) 0.1 mg / kg (n=4) ("1"), (ii) 0.5 mg / kg (n=4) ("2"), (iii) 2 mg / kg (n=3) ("3"), or (iv) 10 mg / kg (n=4) ("4"). Data are presented as mean ± standard deviation. [Figure 13] Figure 13 shows a schematic diagram of target-mediated pharmacokinetics used to describe pharmacokinetic (PK), pharmacodynamic (PD), and receptor occupancy (RO) data in cynomolgus monkeys. [Figure 14]Figures 14A and 14B show the total TREM-1 receptor density on monocytes (Figure 14A) and granulocytes (Figure 14B) after a single dose (subcutaneous (sc) or intravenous (iv) administration) of the mAb 0318-IgG1.3f variant in cynomolgus monkeys. TREM-1 receptor density is shown as a percentage of the TREM-1 receptor density before antibody administration. Each animal received one of the following doses: (a) 0.1 mg / kg (sc) (n=4) ("A"), (b) 0.5 mg / kg (n=4) (sc) ("B"), (c) 2 mg / kg (n=3) (sc) ("C"), (d) 2 mg / kg (n=3) (iv) ("D"), or (e) 10 mg / kg (n=4) (sc) ("E"). Data are shown as mean ± standard deviation. [Figure 15] Figures 15A and 15B show the TREM-1 receptor occupancy on monocytes (Figure 15A) and granulocytes (Figure 15B) after a single dose of the mAb 0318-IgG1.3f variant in cynomolgus monkeys. Receptor occupancy data are shown as the percentage of total TREM-1 receptors expressed on the cells. Each animal received one of the following doses: (a) 0.1 mg / kg (sc) (n=4) ("A"), (b) 0.5 mg / kg (n=4) (sc) ("B"), (c) 2 mg / kg (n=3) (sc) ("C"), (d) 2 mg / kg (n=3) (iv) ("D"), or (e) 10 mg / kg (n=4) (sc) ("E"). Data are shown as mean ± standard deviation. The dotted line indicates an 85% receptor occupancy. [Figure 16A] Figures 16A and 16B show observed (outlined circles) and model-predicted (solid lines) PK (upper left panel), PD (upper right panel), RO (lower left panel), and total surface TREM-1 receptor expression (lower right panel) after a single dose of the mAb 0318-IgG1.3f variant in cynomolgus monkeys, described by a two-compartment PK model using TMDD in the central compartment. In Figure 16A, each monkey received a single dose of 0.1 mg / kg of antibody subcutaneously. [Figure 16B]Figures 16A and 16B show observed (outlined circles) and model-predicted (solid lines) PK (upper left panel), PD (upper right panel), RO (lower left panel), and total surface TREM-1 receptor expression (lower right panel) after a single dose of the mAb 0318-IgG1.3f variant in cynomolgus monkeys, described by a two-compartment PK model using TMDD in the central compartment. In Figure 16B, animals received a single dose of 10 mg / kg antibody subcutaneously. [Modes for carrying out the invention]

[0021] To make this document easier to understand, certain terms are defined first. Further definitions are provided throughout the detailed explanation.

[0022] It should be noted that the term “one (a)” or “one (an)” entity refers to one or more of those entities; for example, “one (a) nucleotide sequence” is understood to refer to one or more nucleotide sequences. Thus, the terms “one (a)” (or “one (an)”), “one or more” and “at least one” may be used interchangeably herein.

[0023] Furthermore, when used herein, “and / or” should be interpreted as a specific disclosure of each of two identified features or components, with or without the other. Thus, when the term “and / or” is used in phrases such as “A and / or B” herein, it is intended to include “A and B,” “A or B,” “A” (alone) and “B” (alone). Similarly, when the term “and / or” is used in phrases such as “A, B and / or C,” it is intended to include each of the following embodiments: 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). Where an aspect is described herein using the word “including,” it is understood that other similar aspects are also provided, described using phrases such as “consisting of” and / or “essentially consisting of.” Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art relating to this disclosure. 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 those skilled in the art with many common dictionaries of the terms used herein.

[0024] Units, prefixes, and symbols are given in their SI-recognized forms. Numerical ranges include the number defining the range. Unless otherwise specified, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations on the various aspects of this disclosure that can be obtained by referring to this specification as a whole. Thus, the terms defined immediately below are more fully defined by referring to this specification as a whole. The term "approximately" is used herein to mean roughly, roughly, approximately, or around that. When the term "approximately" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the given number. Generally, the term "approximately" can modify numbers above and below a given value, for example, a difference of 10 percent above or below (higher or lower).

[0025] The term “Myeloid cell-expressed trigger receptor 1” (also known as TREM1, TREM-1, and CD354) refers to a receptor expressed on monocytes, macrophages, and neutrophils. The main ligand for TREM-1 includes peptidoglycan-recognizing protein 1 (PGLYRP1), which belongs to the family of peptidoglycan (PGN)-binding proteins (PGRPs). Upon activation, TREM-1 associates with the ITAM-containing signaling adapter protein DAP12. Downstream signaling may include activation of NFAT transcription factors, which leads to upregulation of pro-inflammatory cytokine production. The term “TREM-1” includes any variant or isoform of TREM-1 spontaneously expressed by cells. Therefore, in some embodiments, the antibodies described herein can cross-react with TREM-1 from non-human species (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 represents 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, cytoplasmic domain, and extracellular domain. Amino acid residues 138-150 also differ from the canonical sequence described above.

[0027] The following are the amino acid sequences of three known human TREM-1 isoforms. (A) Human TREM-1 isoform 1 (accession number NP_061113.1; SEQ ID NO: 1; nucleotide sequence with accession number NM_018643; encoded by SEQ ID NO: 4): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP (Signal sequence is underlined); (B) Human TREM-1 isoform 2 (nucleotide sequence having accession number NP_001229518.1; SEQ ID NO: 2; accession number NM_001242589; encoded by SEQ ID NO: 5): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRYSFQVPGPLVWTLSPLFPSLCAERM (Signal sequence is underlined); (C) Human TREM-1 isoform 3 (encoded by accession number NP_001229519; SEQ ID NO: 3; nucleotide sequence with accession number NM_001242590; SEQ ID NO: 6): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFRCSTLSFSWLVDS (Signal sequences are 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 (Signal sequence is underlined). This 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 this 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 peptidoglycan-recognizing protein 1 (PGLYRP1), the native ligand of TREM-1, from forming 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 an anti-TREM-1 antibody capable of binding to a portion of TREM-1 otherwise 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 an anti-TREM-1 antibody that interferes with the interaction of TREM-1 with its ligand.

[0030] In some embodiments, anti-TREM-1 antibodies can block the PGLYRP1-induced activation of TREM-1. PGLYRP1 is a highly conserved 196-amino acid 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 (Signal sequences are underlined).

[0031] Accordingly, in some embodiments, the anti-TREM-1 antibodies of this 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] While controlled release of inflammatory cytokines in response to exogenous antigens can be beneficial (e.g., initiating an effective adaptive immune response), excessive release of inflammatory cytokines can have disastrous consequences. For example, one common toxic clinical complication observed with in vivo administration of certain antibodies against cell surface immune receptors (e.g., anti-human CD3 antibodies, e.g., OKT3) is cytokine release syndrome (CRS), associated with the excessive release of various cytokines into circulation (e.g., TNF-alpha, IFN-gamma, and IL-2). CRS can result from the simultaneous binding of an antibody to its cognitive antigen (e.g., CD3 on T cells) (via the variable region of the antibody) as well as to Fc receptors (e.g., FcγR) and / or complement receptors (via the constant region of the antibody) on accessory cells (e.g., antigen-presenting cells). This interaction results in activation of cells (e.g., T cells and / or accessory cells) and the release of various cytokines, which produce a systemic inflammatory response characterized by hypotension, fever (pyrexia), and chills (rigors). Other symptoms of CRS include fever, chills, nausea, vomiting, and difficulty breathing.

[0033] In addition to blocking PGLYRP1-induced production of inflammatory cytokines, in one embodiment, the anti-TREM-1 antibodies of this disclosure reduce or prevent the occurrence of cytokine release syndrome when administered to subjects requiring it. In some embodiments, these anti-TREM-1 antibodies do not induce the expression of inflammatory cytokines by cells (e.g., dendritic cells) when incubated in the presence of the antibody alone, compared to antibodies comprising a heavy chain of the amino acid sequence shown in SEQ ID NO: 76 and a light chain of the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, these anti-TREM-1 antibodies have reduced binding to one or more FcγRs, which may help reduce the occurrence of CSR.

[0034] In some embodiments, the anti-TREM-1 antibody of this disclosure binds 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 originate from any suitable organism. For example, TREM-1 for use as described herein may be vertebrate TREM-1, e.g., mammalian TREM-1, e.g., primate (e.g., human, chimpanzee, cynomolgus macaque or rhesus macaque); rodent (e.g., mouse or rat); rabbit (e.g., rabbit); or artiodactyla (e.g., cattle, sheep, pig or camel). In certain embodiments, TREM-1 is SEQ ID NO: 1 (human TREM-1, isoform 1). This TREM-1 may be a 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 may, for example, be glycosylated. This TREM-1 could be the full-length TREM-1 protein.

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

[0036] In some embodiments, the anti-TREM-1 antibody of this disclosure is 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 that is a yeast, insect, or mammalian cell, e.g., a primate (e.g., human, chimpanzee, cynomolgus monkey, or rhesus monkey), a rodent (e.g., mouse or rat), a rabbit (e.g., rabbit), or an even-toed ungulate (e.g., cattle, 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 antibody disclosed herein may also be produced by other methods known to those skilled in the art, e.g., phage display or yeast display. Once produced, the antibody may be screened for binding to, for example, full-length TREM-1 or its variants 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. The 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 may bind exclusively to that antigen or portion, or to a limited number of homologous antigens or portions thereof. A full-length antibody typically contains at least four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains. One immunoglobulin subclass of particular pharmaceutically 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 subdivided into two types, kappa and lambda, based on differences in their sequence composition. An IgG molecule consists of two heavy chains linked by two or more disulfide bonds, and two light chains each linked to the heavy chains by one disulfide bond. The heavy chain may contain one heavy chain variable region (VH) and up to three heavy chain constant (CH) regions: CH1, CH2, and CH3. The light chain may contain one light chain variable region (VL) and one light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interposed by more conserved regions called framework regions (FRs). The VH and VL regions typically consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy and light chains form binding domains capable of interacting with antigens, while the constant region of the antibody can mediate the binding of immunoglobulins 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 a fragment of a full-length antibody, certain antigen-binding fragments of an antibody may be suitable in light of the present invention.

[0038] The term “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to 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 containing a single VH and a single VL chain; minibody, diabody, triabody, tetrabody, and kappa body (see, e.g., Ill et al., Protein Eng 10:949-57) (See 1997); camel IgG; IgNAR; and one or more isolated CDRs or functional paratopes, in which isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together to form a functional antibody fragment. Various types of antibody fragments are described or outlined, for example, in Holliger and Hudson, Nat Biotechnol 2S:1126-1136 (2005); International Patent Application Publication No. WO2005 / 040219, and U.S. Patent Applications Publications 2005 / 0238646 and 2002 / 0161201. These antibody fragments may be obtained using prior art known to those skilled in the art, and these fragments may 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 a human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, this constant region is also derived from a human germline immunoglobulin sequence. Anti-TREM-1 antibodies described herein may contain amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced in vitro by random or site-directed mutagenesis, or in vivo by somatic mutation). However, as used herein, the term “human antibody” is intended not to include antibodies in which a CDR sequence derived from the germline of another mammalian species, e.g., mouse, is grafted onto a human framework sequence. 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 a non-human immunoglobulin. Therefore, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region have been replaced with residues from the hypervariable region of a non-human species antibody (donor antibody), such as mouse, rat, rabbit, or non-human primate antibody, which possess the desired specificity, affinity, sequence composition, and functionality. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. An example of such modification is the introduction of one or more so-called reverse mutations, typically amino acid residues derived from the donor antibody. Antibody humanization can be carried out using recombinant techniques known to those skilled in the art (see, e.g., Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny KC Lo). Suitable human recipient frameworks for both light and heavy chain variable domains can be identified, for example, by sequence or structural homology. Alternatively, a fixed recipient framework may be used, for example, based on knowledge of its structure, biophysical, and biochemical properties. The recipient framework may be germline-derived or derived from a mature antibody sequence. CDR regions derived from donor antibodies may be transferred by CDR grafting. CDR-grafted humanized antibodies may be further optimized, for example, in terms of affinity, functionality, and biophysical properties, by identifying key framework locations where the reintroduction (reverse mutation) of amino acid residues derived from the donor antibody has a beneficial effect on the properties of the humanized antibody. In addition to reverse mutation from donor antibodies, humanized antibodies may be manipulated by introducing germline residues in the CDR region or framework region, eliminating immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0041] Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further refine the antibody's performance. Generally, humanized antibodies contain at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR residues are from a human immunoglobulin sequence. Humanized antibodies may 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, e.g., a conjugate of the antibody with another drug or antibody.

[0042] The term “recombinant human antibody” as used herein includes all human antibodies prepared, expressed, created or isolated by recombinant means, for example: (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal with respect to human immunoglobulin genes or from hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express antibodies, for example, 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 germline genes, but also include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is well known in the field (see, for example, Lonberg Nature Biotech. 23(9): 1117-1125 (2005)), the variable region contains antigen-binding domains encoded by various genes that are rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (called somatic mutations or high-frequency mutations) to increase the affinity of antibodies to foreign antigens. The constant region changes in further response to the antigen (i.e., isotype switching). Thus, nucleic acid molecules encoding light-chain and heavy-chain immunoglobulin polypeptides that have been rearranged and somatically mutated in response to an antigen cannot have sequence identity to the original nucleic acid molecule, 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 region originates from one species and the constant region originates from another species; for example, an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0043] In one embodiment, the anti-TREM-1 antibody of this 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., it 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 positions of disulfide crosslinks as those present in a naturally occurring IgG1 antibody (unless the antibody has mutated to alter the disulfide crosslinks). As used herein, “isotype” refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0044] "Allotype" refers to a naturally occurring variant within a particular group of isotypes, which differ in several amino acids (see, e.g., Jefferis et al., mAbs 1:1 (2009)). The anti-TREM-1 antibodies described herein may be of any allotype. In some embodiments, these anti-TREM-1 antibodies are of the "IgG1.3f" allotype, which, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9), contains one or more amino acid substitutions selected from the group consisting of L234A, L235E, and G237A according to EU numbering. In other embodiments, these anti-TREM-1 antibodies are of the "IgG1.1f" allotype, which, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9), contains one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S according to EU numbering. In certain embodiments, these anti-TREM-1 antibodies are "IgG1-Aba" allotypes, comprising one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S, according to EU numbering, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In further embodiments, these anti-TREM-1 antibodies are "IgG4-Aba" allotypes, comprising 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 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, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). The terms “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably in this specification with the term “antibody that specifically binds to an antigen.” As used herein, “isolated antibody” 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 between an antibody Fc region and an Fc receptor or ligand, or the resulting biochemical events. Exemplary "effector functions" include C1q binding, complement-dependent cell-mediated 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 receptors; BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain). In one embodiment, the anti-TREM-1 antibody of this disclosure comprises one or more Fc regions that do not bind to FcγR, and therefore lacks effector function (i.e., no effector).

[0046] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include the FcγR family of receptors, which include 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). Various properties of human FcγR are known in the field. Most native effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIBs, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice, FcγRIIIA in humans) and do not express inhibitory FcγRIIB in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the type of activated Fc receptor it binds to. The "Fc region" (crystalline fragment region), "Fc domain," or "Fc" refers to the C-terminal region of the antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Therefore, 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, as well as the hinge between the CH1 and CH2 domains. While the definition of the boundary of the Fc region of the immunoglobulin heavy chain may vary as defined herein, the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxyl terminus of the heavy chain, where 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 located 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 the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of IgG. As used herein, the Fc region may be a native sequence Fc containing any allotype variant, or a variant Fc (e.g., an Fc that does not exist in nature). Fc may also refer to this region in relation to an Fc-containing protein polypeptide, such as an "Fc-region-containing binding protein" (e.g., an antibody or immunoadhesion), which is cleaved or also called an "Fc fusion protein."

[0048] A "native sequence Fc region" or "native sequence Fc" contains an amino acid sequence identical to that of a naturally occurring Fc region. Native sequence human Fc regions include native sequence human IgG1 Fc regions; native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as their naturally occurring variants. Native sequence Fc includes various allotypes of Fc (see, for example, Jefferis et al., mAbs 1: 1 (2009)).

[0049] A “variant sequence Fc region” or “Fc that does not exist naturally” typically involves modifications to alter one or more of its functional properties, such as serum half-life, complement binding, Fc receptor binding, protein stability, and / or antigen-dependent cell-mediated cytotoxicity, or, among other things, the absence of those properties. In some embodiments, the anti-TREM-1 antibody of this disclosure may be chemically modified (e.g., one or more chemical moieties may be bound to the antibody) or modified to alter its glycosylation in order to further alter one or more of the functional properties of the antibody. In one embodiment, the anti-TREM-1 antibody is an IgG1 isotype and each possesses a modified Fc domain (residue numbering according to the EU index) containing one or more, possibly all, of the following mutations: mutations resulting in reduced affinity for a particular Fc receptor (L234A, L235E, and G237A) and mutations resulting in reduced C1q-mediated complement binding (A330S and P331S).

[0050] The terms “hinge,” “hinge domain,” “hinge region,” and “antibody hinge region” refer to the domain of the heavy chain constant region that junctions the CH1 domain to the CH2 domain, including the upper, middle, and lower portions of the hinge (Roux et al., J Immunol 161:4083 (1998)). The hinge provides a fluctuating level of mobility between the antibody binding region and the effector region, and also provides a site for intermolecular disulfide bonding 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 the wild-type IgG1, IgG2, IgG3, and IgG4 hinges are publicly known in the art (e.g., International PCT Publication No. WO2017 / 087678). In one embodiment, the hinge region of CH1 of an anti-TREM-1 antibody is modified such that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased. This approach is further described, for example, in U.S. Patent No. 5,677,425.

[0051] The constant region may 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) may be mutated to a proline (P) residue to stabilize the formation of inter-heavy chain disulfide crosslinks at the hinge (see, e.g., Angal et al., Mol Immunol. 30: 105-8 (1995)). Antibodies or fragments may also be defined in terms of their complementarity-determining regions (CDRs). The terms “complementarity-determining region” or “hypervariable region,” as used herein, refer to the region of the antibody where amino acid residues involved in antigen binding are located. A hypervariable region or CDR can be identified as the region with the highest variability in the amino acid alignment of the antibody variable domain. A database, such as the Kabat database, may be used for CDR identification, and a CDR is defined as including, for example, amino acid residues 24-34 (CDR1), 50-59 (CDR2), and 89-97 (CDR3) in the light chain variable domain, as well as 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, US Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, the CDR can be defined as residues derived from the "hypervariable loop" (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 above by Kabat et al.In this specification, terms such as “Kabat position,” “Kabat residue,” and “according to Kabat” refer to this numbering system for heavy chain variable domains or light chain variable domains. Using this Kabat numbering system, the actual linear amino acid sequence of a peptide may include fewer or more amino acids, or insertions therein, corresponding to shortenings of the variable domain framework (FR) or CDR. For example, a heavy chain variable domain may include amino acid insertions (residues 52a, 52b, and 52c according to Kabat) after residue 52 of the CDR H2, and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment in regions of homology between the antibody sequence and a “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, as defined, for example, by the specific method used to identify it. Epitopes can be formed from both continuous amino acids (usually linear epitopes) or discontinuous amino acids adjacent by tertiary folding of the protein (usually conformational epitopes). Epitopes formed from continuous amino acids are typically retained, though not always, 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 own spatial conformation. Methods for determining which epitopes are bound to a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or sequential peptides (e.g., derived from TREM-1) are tested for reactivity with a given antibody (e.g., anti-TREM-1 antibody). Methods for determining the spatial conformation of epitopes include techniques described in the art and herein, such as X-ray crystallography, antigen mutation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0053] When referring to two or more antibodies, the term “binding to the same epitope” means that those antibodies bind to the same segment of amino acid residues, if 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, e.g., X-ray analysis of the antigen:antibody complex crystals that provide atomic dissolution 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 alteration of amino acid residues in the antigen sequence is often considered an appearance of an epitope component. Furthermore, computational combinatorial methods for epitope mapping may 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 with another antibody for binding to a target" refers to an antibody that (partially or completely) inhibits the binding of another antibody to the 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 experiments, such as BIACORE® surface plasmon resonance (SPR) analysis. In certain embodiments, an antibody competes with the binding of another antibody to the target, inhibiting it by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may differ depending on which antibody is a "blocking antibody" (i.e., the cold antibody that is initially incubated with the target). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harbor 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 comes into contact with the antigen first in the competitive assay or the other antibody comes into contact with the antigen first in the competitive assay.

[0055] As used herein, the terms “specific binding,” “selective binding,” “selectively binding,” and “specifically binding” refer to antibody binding to an epitope on a given antigen. Typically, the antibody binds approximately 10 times, as determined by (i) surface plasmon resonance (SPR) technology in a BIACORE® 2000 instrument using a given antigen, e.g., recombinant human TREM-1, as the analyte and the antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than M, for example, 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 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 incapable of binding to dissimilar molecules. Antibodies according to the present invention may be incapable of binding to natural killer cell p44-related protein Nkp44. The specificity of the interaction and the value of the equilibrium binding constant 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 binding 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 to determine whether two antibodies compete or cross-compete for binding include: competition for binding to TREM-1-expressing myeloid cells by flow cytometry, for example, 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 labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546) (1990)); and directly 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, the 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 called “competitive antibodies.” If a second antibody can bind to the antigen simultaneously with the reference antibody, the 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 called “non-competitive antibodies.”

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

[0060] As used herein, the term "high affinity" for IgG antibodies means 10% of the target antigen. -8 M or less, 10 -9 M or less or 10 -10 K below M DThis refers to antibodies that possess high affinity. However, "high affinity" binding can vary for other antibody isotypes. For example, the "high affinity" binding for IgM isotypes is 10 -10 M or less or 10 -8 K below M D This refers to antibodies that possess [a certain characteristic]. "EC" in association with in vitro or in vivo assays using antibodies or their antigen-binding fragments 50 The term "maximum response" refers to the concentration of the antibody or its antigen-binding moiety that induces 50% of the maximum response, i.e., an intermediate response between the maximum response and the baseline. The term "naturally occurring," when applied to an object and used herein, refers to the fact that the object can be found in nature. For example, polypeptides or polynucleotide sequences present in living organisms (including viruses) that can be isolated from a natural source and have not been deliberately modified by humans in a laboratory are naturally occurring.

[0061] A "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, with no upper limit on chain length. One or more amino acid residues in a protein may undergo modifications, such as glycosylation, phosphorylation, or disulfide bond formation, but are not limited to these. A "protein" may contain one or more polypeptides. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and may be cDNA.

[0062] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an 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 with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-loading side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted non-essential amino acid residue in an anti-TREM-1 antibody is replaced with another amino acid residue from the same side-chain family. Methods for identifying conserved nucleotide and amino acid substitutions that do not exclude antigen binding are well known in the field (see, for example, 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] With respect to nucleic acids, the term "substantial homology" indicates that, when two nucleic acids or their specified sequences are optimally aligned and compared, at least about 80% of the nucleotides, at least about 90%–95% of the nucleotides, or at least about 98%–99.5% of the nucleotides are identical, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when a segment hybridizes to its chain complement under selective hybridization conditions. With respect to polypeptides, the term "substantial homology" indicates that two polypeptides or their specified sequences are identical in at least about 80% of their amino acids, at least about 90%–95% of their amino acids, or at least about 98%–99.5% of their amino acids, with appropriate amino acid insertions or deletions, when optimally aligned and compared.

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

[0065] Percent identicality between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) with 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. Percent identicality 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 into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap-length penalty of 12, and a gap penalty of 4. Furthermore, the percentage identity between two amino acid sequences can be determined using either a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6, using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at worldwideweb.gcg.com).

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

[0067] Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids are considered "isolated" or "substantially purified" when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques including alkali / 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 may, if desired, affect the amino acid sequence. In particular, DNA sequences substantially homologous to or derived from the native V, D, J, stationary, switch, and other such sequences described herein are intended (where “derived” means that the sequence is identical to or modified from another sequence).

[0069] The term “vector,” as used herein, 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 further DNA segments can be ligated. Another type of vector is a viral vector, where further DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating together with the host genome. Furthermore, certain vectors are capable of directing the expression of a gene to which they are operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vectors, “plasmid” and “vector” may be used interchangeably herein. However, other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0070] The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell containing nucleic acids that are not naturally present within it, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such cell. Since a particular modification may occur in subsequent generations due to either mutation or environmental influence, such offspring may not actually be identical to the parent cell, but as used herein, they are still included within the scope of the term “host cell.” As used herein, the term “conjugated” refers to the association of two or more molecules. Conjugation can be covalent or noncovalent. Conjugation can also be genetic (i.e., recombinant fusion). Such conjugation can be achieved using a wide range of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0071] As used herein, “administer” refers to the physical delivery of a composition containing a therapeutic agent to a target using any of the various methods and delivery systems known to those skilled in the art. Different routes of administration for anti-TREM-1 antibodies described herein include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes by injection or infusion. As used herein, “parenteral administration” 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, subarachnoid, lymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via non-parenteral routes, such as topical, dermal, or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Administration may 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” (for example, referring to inhibition / blockage of the binding of TREM-1 ligand to TREM-1 on cells) are used interchangeably and encompass both partial and complete inhibition / blockage. In some embodiments, an anti-TREM-1 antibody inhibits the binding of TREM-1 ligand to TREM-1 by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as determined, e.g., as further described herein. In some embodiments, an anti-TREM-1 antibody inhibits the binding of TREM-1 ligand to TREM-1 by 50% or less, e.g., about 40%, 30%, 20%, 10%, 5%, or 1%, as determined, e.g., as further described herein.

[0073] The terms “to treat,” “to treat,” and “treatment” as used herein refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the aim of reversing, mitigating, relieving, inhibiting, slowing or preventing the progression, onset, severity or relapse of disease-related symptoms, complications, conditions or biochemical signs, or enhancing overall survival. Treatment may be performed on a subject with the disease or on a subject without the disease (e.g., for preventive purposes).

[0074] The term “effective dose” or “effective dosage” is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The “therapeutic effective dose” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug, used alone or in combination with another therapeutic agent, that promotes apparent disease regression by reducing the severity of disease symptoms, increasing the frequency and duration of disease-free periods, or preventing functional impairment or disability resulting from the distress of the disease. The therapeutic effective dose or dosage of a drug includes the “preventive effective dose” or “preventive effective dosage,” which is any amount of the drug, used alone or in combination with another therapeutic agent, that inhibits the onset or recurrence of the disease when administered to subjects at risk of developing the disease or at risk of disease recurrence. The ability of a therapeutic agent to promote disease regression or inhibit the onset or recurrence of the disease can be evaluated, for example, in human subjects during clinical trials, in animal model systems predicting human efficacy, using various methods known to those skilled in the art, or by assaying the activity of the drug in in vitro assays. The term "patient" includes human and other mammalian subjects receiving either preventive or therapeutic treatment. As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions described herein may be used to treat a subject having cancer. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc. As used herein, the terms "ug" and "uM" are interchangeable with "μg" and "μM," respectively. Various embodiments described herein are described in further detail in the following subsections.

[0075] I. Anti-TREM-1 antibody Antibodies characterized by specific functional features or properties, such as fully human antibodies, are described herein. For example, the antibodies of this disclosure specifically bind to human TREM-1, more specifically to a specific domain (e.g., a functional domain) within the extracellular domain of human TREM-1. In one embodiment, these antibodies specifically bind to a site on TREM-1 to which a 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, e.g., monocytes, macrophages, and neutrophils (i.e., they 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, e.g., cynomolgus monkey TREM-1. In some embodiments, these anti-TREM-1 antibodies, upon activation, 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). In other embodiments, these anti-TREM-1 antibodies include one or more Fc regions that do not bind to FcγR. 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 an inflammatory cytokine storm after administration of the anti-TREM-1 antibody to a target that requires it.

[0076] In some embodiments, the specific anti-TREM-1 antibodies described herein are antibodies that cross-compete with mAb 0318 for binding to human TREM-1, such as monoclonal, recombinant, and / or human antibodies. 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 this 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 Patent 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.

[0077] Accordingly, in some embodiments, the anti-TREM-1 antibody of this 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 NOs. 14, where one of these amino acids may be substituted with a different amino acid. In a particular embodiment, the VH of this anti-TREM-1 antibody comprises the CDR2 sequence of amino acids 50-68 (RIRTKSSNYATYYAASVKG) of SEQ ID NOs. 14, where one, two, or three of these amino acids may be substituted with 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 NOs. 14, where one, two, or three of these amino acids may be substituted with different amino acids.

[0078] In some embodiments, the VL of this anti-TREM-1 antibody contains 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 with different amino acids. In other embodiments, the VL of this anti-TREM-1 antibody contains 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 with different amino acids. In some embodiments, the VL of this anti-TREM-1 antibody contains 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 with different amino acids.

[0079] Methionine residues in the CDR of an antibody can be oxidized, leading to potential chemical degradation of the antibody and, consequently, a reduction in its potency. Therefore, in the anti-TREM-1 antibodies disclosed herein, one or more methionine residues in the heavy chain and / or light chain CDR may be replaced with amino acid residues that are not subject to oxidative degradation. In some embodiments, methionine residues in the heavy chain CDR1 and CDR3 are replaced with amino acid residues that are not subject to oxidative degradation (e.g., glutamine or leucine). Thus, in one embodiment, the VH of this anti-TREM-1 antibody contains 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 contains 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, the deamide site can be removed from the anti-TREM-1 antibody, particularly in the CDR.

[0080] In some embodiments, the VH and VL sequences of this anti-TREM-1 antibody include the VH and VL sequences of the anti-TREM-1 antibody disclosed in international application publication number WO2017 / 152102, which is incorporated herein by reference in its entirety. In some embodiments, the VL sequence of this anti-TREM-1 antibody includes a CDR1 sequence selected from the group consisting of SEQ ID NOs. 9 to 27 of WO2017 / 152102, a CDR2 sequence selected from the group consisting of SEQ ID NOs. 28 to 40 of WO2017 / 152102, and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs. 41 to 119 of WO2017 / 152102. In one embodiment, the VH of the anti-TREM-1 antibody includes a CDR1 sequence selected from the group consisting of sequence numbers 120 to 143 of WO2017 / 152102, a CDR2 sequence selected from the group consisting of sequence numbers 144 to 172 of WO2017 / 152102, and / or a CDR3 sequence selected from the group consisting of sequence numbers 173 to 247 of WO2017 / 152102. In some embodiments, the anti-TREM-1 antibody of the present disclosure comprises a CDR and / or variable region sequence having at least 80% identity (e.g., at least 85%, at least 95%, at least 95%, or at least 99%) to the CDR and / or variable region sequence 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), respectively, including SEQ ID NOs. 14 and 15. In some embodiments, these anti-TREM-1 antibodies include a heavy chain (HC) and a light chain (LC), where the HC includes SEQ ID NOs. 50, 51, 52, or 53. In some embodiments, the LC includes SEQ ID NOs. 54. In some embodiments, the anti-TREM-1 antibody of the present disclosure comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs. 396 to 475 of WO2017 / 152102 and / or a light chain variable region (VL) selected from the group consisting of SEQ ID NOs. 316 to 395 of WO2017 / 152102.

[0082] In some embodiments, the anti-TREM-1 antibody comprises a heavy chain and a light chain, the heavy chain and light chain comprising the amino acid sequence shown in Table 7. In some embodiments, the anti-TREM-1 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 50, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the anti-TREM-1 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 51, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the anti-TREM-1 antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 52, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 54.

[0083] Heavy and light chains, either those shown herein, for example, those containing amino acid sequences identical by at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% to SEQ ID NOs. 50-54, may be used to form anti-TREM-1 antibodies having desired characteristics, such as those further described herein. In some embodiments, the anti-TREM-1 antibody of the present disclosure comprises a heavy chain and a light chain, the heavy chain comprising 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 comprising 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, the anti-TREM1 antibody includes a heavy chain constant region, which contains 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, the anti-TREM-1 antibody includes a heavy chain constant region, which contains 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, the anti-TREM-1 antibody includes a heavy chain constant region, which contains 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, the anti-TREM-1 antibody comprises a heavy chain constant region, which 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 can bind to human TREM-1 variants (e.g., TREM-1 isoforms 2 and 3, SEQ ID NOs. 2 and 3, respectively) as determined, for example, using surface plasmon resonance. In another embodiment, this anti-TREM-1 antibody can bind to cynomolgus monkey TREM-1 (SEQ ID NO: 7) as determined, for example, using surface plasmon resonance.

[0085] In some embodiments, the anti-TREM-1 antibodies described herein have high affinity, for example, when determined by BIACORE® (as described in the examples), 10 -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -11 M or less, 10 -12M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M or 10 -9 M~10 -7 M's K D Then, it binds to human TREM-1. In some embodiments, the anti-TREM-1 antibody described herein is, for example, determined by BIACORE® (as described in the examples), 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M or 10 -9 M~10 -7 M's K D Then, it binds to the cynomolgus macaque TREM-1.

[0086] In one embodiment, the antibody of this 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 to (i) at least one amino acid residue selected from the group consisting of A21, T22, K23, L24, T25, E26 and any combination thereof, of human TREM-1 (e.g., isoform 1, SEQ ID NO: 1), and (ii) A49, S50, S51, Q52, K53, A54, W55, Q56, 157, 158, R59, D60, G61, E62, M63, P64, K65, T66, L67, A68, C6 It is possible to specifically bind to at least one amino acid residue selected from the group consisting of 9, T70, E71, R72, P73, S74, K75, N76, S77, H78, P79, V80, Q81, V82, G83, R84, 185, and any combination thereof, and (iii) at least one amino acid residue selected from the group consisting of C113, V114, 1115, Y116, Q117, P118, P119, and any combination thereof. See WO2016 / 009086.

[0087] In one embodiment, the anti-TREM-1 antibody can specifically bind to amino acids D38-F48 of SEQ ID NO: 1 (human TREM-1) as determined, for example, by HX-MS or X-ray diffraction. In some embodiments, the anti-TREM-1 antibody has an epitope comprising, for example, 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), as well as 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), as determined, for example, by HX-MS or X-ray diffraction. In certain embodiments, the anti-TREM-1 antibody has an epitope containing 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), as determined using TREM-1 variants and surface plasmon resonance.

[0088] In one embodiment, the anti-TREM-1 antibody of this disclosure has an epitope containing at least amino acid residues E46 and / or D92 of SEQ ID NO: 1 (human TREM-1) as determined using TREM-1 variants and surface plasmon resonance. In another embodiment, this anti-TREM-1 antibody contains 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 a particular embodiment, this anti-TREM-1 antibody can specifically bind to a polypeptide containing amino acid residues E19-L26 of cynomolgus monkey TREM-1 (SEQ ID NO: 7) as determined, for example, using HX-MS or X-ray diffraction. In one embodiment, the anti-TREM-1 antibody is capable of specifically binding to human TREM-1, and the epitope of this antibody contains 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, the 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 another embodiment, the 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 epitopes of this antibody may include V39, C41, D42, Y43, and L45 of SEQ ID NO: 1. In further embodiments, the epitopes of this antibody may include E46, K47, and A49 of SEQ ID NO: 1. In a specific embodiment, the epitope of this anti-TREM-1 antibody may further include F48 of SEQ ID NO: 1.

[0090] In some embodiments, the 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 this disclosure includes a mutation in which one or more negatively charged residues in the CDR1 and CDR3 regions of the antibody's light chain are replaced with uncharged residues. In some embodiments, this anti-TREM-1 antibody includes a substitution in one or more of the amino acid residues D1, D30, D33, D74, D98, E27, and E97 of SEQ ID NO: 15 with an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, and tyrosine. These mutations are referred to herein as “charge patch” mutations.

[0092] In some embodiments, the anti-TREM-1 antibody of this disclosure contains a mutation in the Fab-Fab interaction region of SEQ ID NO: 14 to reduce Fab-Fab dimerization. Since the antibody contains two Fabs, it has been previously 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 contains a mutation in any one of the 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, with an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine.

[0093] In one embodiment, the anti-TREM-1 antibody disclosed herein includes a mutation at position 32 of SEQ ID NO: 15, where phenylalanine is substituted for 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 an Ala substitution at position Y90 of SEQ ID NO: 1 improved the affinity of SEQ ID NO: 3 for TREM-1. Y90 was found to interact with the phenylalanine residue of SEQ ID NO: 15. Mutations in SEQ ID NO: 15 that improve the Fab-TREM-1 interaction are referred to as "Fab-TREM-1 interaction" mutations. The variable region can be Fc, for example 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, which can be IgG1, IgG2, IgG3, or IgG4. Anti-TREM-1 antibodies linked to Fc (e.g., covalently linked or fused) are provided herein (see, for example, 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 effector-less or nearly effector-less Fc, e.g., IgG1. In some embodiments, the variable region of these anti-TREM-1 antibodies is linked to an Fc having reduced binding to one or more FcγRs or being unable to bind to one or more FcγRs.

[0094] In one embodiment, the VH domain of the anti-TREM-1 antibody described herein may be fused to the constant domain (i.e., Fc) of naturally occurring or modified human IgG, e.g., IgG1, IgG2, IgG3, or IgG4, as further described herein. For example, the VH domain may be fused to the constant region of human IgG, e.g., IgG1, e.g., the following wild-type human IgG1 constant domain amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 9) or the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E MThis may include an 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 an effector-free constant region, for example, the following effector-free human IgG1 constant domain amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPE AE G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP SS IEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 78; following EU numbering, including underlined substitutions L234A, L235E, G237A, A330S and P331S for "IgG1.1f") or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPE AE G APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP AP IEKTISKAKGQPREPQVYTLPPSR E E M This may include an amino acid sequence of any VH domain described herein, fused to TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 79; "IgG1.3f" including underlined substitutions L234A, L235E, and G237A according to EU numbering).

[0096] For example, the allotype variant of IgG1 includes K97R, D239E and / or L241M (underlined and bold above), numbered according to SEQ ID NOs.77-79. 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 in amino acids L117, A118, G120, A213 and P214 (underlined above), numbered similarly to SEQ ID NOs.77-79, or in L234, A235, G237, A330 and P331 according to EU numbering. In further embodiments, the constant region of this anti-TREM-1 antibody includes one or more mutations or substitutions in the amino acids L117A, A118E, G120A, A213S and P214S of SEQ ID NOs. 77-79, or L234A, L235E, G237A, A330S and P331S according to EU numbering. The constant region of this anti-TREM-1 antibody may also include one or more mutations or substitutions in L117A, A118E and G120A of SEQ ID NOs. 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 (Sequence ID 11; "IgG1-Aba" including 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 SThe amino acid sequence of any VH domain described herein, fused to an IgG1 constant domain containing SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 12; "IgG4-Aba" according to EU numbering, containing underlined substitutions S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S).

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

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

[0100] In general, the variable regions described herein may be linked to Fc, typically involving 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 antigen-dependent cell-mediated cytotoxicity. Furthermore, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be linked to the antibody) or modified to alter their glycosylation in order to alter 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 according to the Kabat EU index. The Fc region encompasses domains derived from the constant region of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and other classes, e.g., 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 homologous to the immunoglobulin C-terminal region, and may contain a 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) specific to IgG class antibodies: FcγRI, FcγRII, and FcγRIII. Sequences crucial for IgG binding to FcγR receptors have been reported to be located within the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to Fc receptors (FcRs). In one embodiment, the Fc region of these anti-TREM-1 antibodies is a variant Fc region, for example, a modified Fc sequence (e.g., by amino acid substitution, deletion, and / or insertion) compared to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate the variant) in order to provide desirable structural features and / or biological activity.

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

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

[0104] In one embodiment, the hinge region of Fc is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, 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 Fc is changed, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to reduce 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 so that the antibody has impaired Staphylococcus aureus (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is further described in U.S. Patent No. 6,165,745 by Ward et al.

[0105] In other embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter 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 a different amino acid residue so that the antibody has a modified affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patents 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 so that the antibody has altered C1q binding and / or reduced or absent complement-dependent cell-mediated 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 modified to thereby alter the antibody's ability to fix complement. This approach is further described by Bodmer et al. in PCT publication number WO94 / 29351.

[0107] In another example, the Fc region is located 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, By modifying one or more amino acids in 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, the molecules may be modified to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or reduce affinity for the Fcγ receptor. 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 interaction with FcγR and complement include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are outlined in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

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

[0109] Optionally, the Fc region may contain amino acid residues that do not exist in nature at further and / or alternative positions known to those skilled in the art (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 No. See 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 / 0201 14).

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

[0111] In certain embodiments, the anti-TREM-1 antibody of this disclosure contains Fc that has reduced binding to FcγR or is incapable of binding to FcγR. In some embodiments, this anti-TREM-1 antibody has reduced binding affinity to FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), FcγRIIIB(CD16b), or any combination thereof, 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 anti-TREM-1 antibody has reduced binding affinity to FcγRI(CD64) to less than half, less than one-third, less than one-quarter, less than one-fifth, less than one-sixth, less than one-seventh, less than one-eighth, less than one-ninth, or less than one-tenth, 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.

[0112] In some embodiments, these anti-TREM-1 antibodies include IgG1 Fc variants 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 have (a) IgG1 isotypes, including 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, (b) an amino acid residue selected from the group consisting of P238D, S267E, L328F, E233D, G237D, H268D, P271G, A330R, and any combination thereof, which contains one or more amino acid substitutions in the Fc region, where the residue numbering follows EU or Kabat numbering, or contains an amino acid deletion in the Fc region at the position corresponding to glycine 236; (b) having the IgG2 isotype, and P238 In amino acid residues selected from the group consisting of S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, the Fc region contains one or more amino acid substitutions, where the residue numbering follows EU or Kabat numbering; or (c)IgG4 isota The amino acid residues have a type and are 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, and contain one or more amino acid substitutions in the Fc region, where the numbering of the residues follows EU or Kabat numbering.In some embodiments, (a) the Fc region further includes one or more further amino acid substitutions at amino acid residues selected from the group consisting of A330L, L234F;L235E, P331S, and any combination thereof, wherein the residue numbering follows EU or Kabat numbering; (b) the Fc region further includes one or more further amino acid substitutions at positions selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein the residue numbering follows EU or Kabat numbering; or (c) the Fc region further includes the S228P amino acid substitution according to EU or Kabat numbering. See WO2017 / 152102. In certain embodiments, Fc having reduced complement binding is selected. An exemplary Fc having reduced complement binding, for example, IgG1 Fc, has the following two amino acid substitutions: A330S and P331S.

[0114] In certain embodiments, Fc molecules that essentially lack effector function are selected, i.e., such Fc molecules have reduced binding to FcγR and reduced complement binding. An exemplary effector-free Fc molecule, e.g., IgG1 Fc, includes the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0115] II. Physical Properties of Antibodies 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 region can lead to increased immunogenicity of the antibody or alteration of the antibody's pK due to modified 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 NXS / T sequences. In some embodiments, the anti-TREM-1 antibodies of this disclosure have no variable region glycosylation or reduced variable region glycosylation. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region, or by mutating residues within the glycosylation region. Accordingly, 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 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, these anti-TREM-1 antibodies do not contain asparagine isomerized sites. Deamide of asparagine can occur on NG or DG sequences, introducing twists into the polypeptide chain and resulting in the creation of isoaspartic acid residues that reduce its stability (isoaspartic acid effect).

[0116] Each antibody has its own isoelectric point (pi), which generally falls within the pH range of 6 to 9.5. The pi for IgG1 antibodies typically falls within the pH range of 7–9.5, while the pi for IgG4 antibodies typically falls within the pH range of 6–8. It is inferred that antibodies with pi outside the normal range may exhibit some unfolding and instability under in vivo conditions. Therefore, the anti-TREM-1 antibodies disclosed herein may contain pi values ​​within the normal range (e.g., 8–9). This can be achieved by selecting antibodies with pi within the normal range or by mutating charged surface residues.

[0117] Each antibody has a characteristic melting temperature, and higher melting temperatures indicate greater overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). Generally, T M i (initial unfolding temperature) may be higher than 60°C, higher than 65°C, or higher than 70°C. In some embodiments, the anti-TREM-1 antibody of this disclosure has 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. Accordingly, in some embodiments, the anti-TREM-1 antibody of this disclosure is 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, for example, when measured by capillary differential scanning calorimeter (CAP-DSC). The melting point of the 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 this disclosure do not degrade rapidly. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0118] In some embodiments, the anti-TREM-1 antibodies disclosed herein have minimal agglutination effects that may result in an undesirable immune response and / or altered or undesirable pharmacokinetic properties. Generally, antibodies with agglutination of ≤25%, ≤20%, ≤15%, ≤10%, or ≤5% are acceptable. Agglutination can be measured by several techniques, including size exclusion column (SEC), high-performance liquid chromatography (HPLC), and dynamic light scattering (DLS). In some embodiments, the anti-TREM-1 antibodies of this disclosure are monomeric when observed by size exclusion high-performance liquid chromatography (SE-HPLC). In some embodiments, these anti-TREM-1 antibodies exhibit minimal risk of fragmentation when observed by two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC / MS) or intact mass spectrometry using 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 may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. A nucleic acid is considered "isolated" or "substantially purified" if it has been purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA substantially linked to isolated DNA) or proteins, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may 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 possessing human immunoglobulin genes, as 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 techniques), the nucleic acids encoding these 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 antibody of this disclosure. Exemplary DNA sequences encoding the VH and VL sequences are shown in SEQ ID NOs. 58-61 and 62-65, respectively. A method for producing an anti-TREM-1 antibody disclosed herein may include the step of expressing the heavy chain and light chain, along with a signal peptide, in a cell line containing nucleotide sequences encoding the heavy chain and light chain, for example, SEQ ID NOs. 58-61 and 62-65, respectively. Host cells containing these nucleotide sequences are included herein.

[0121] Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques to convert, for example, a variable region gene to a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the VL-coding DNA fragment or the VH-coding DNA fragment is operably ligated to another DNA fragment encoding another protein, such as an antibody constant region or a mobile linker. The term "operably ligated," as used in this context, is intended to mean that the two DNA fragments are joined in such a way 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 ligating the VH-coding DNA to another DNA molecule encoding the heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are publicly known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. This heavy chain constant region may be the IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, for example, the IgG2 and / or IgG4 constant region. For Fab fragment heavy chain genes, the VH-coding DNA can be operably ligated 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 (and Fab light chain gene) by operably ligating the VL-coding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are publicly known in the field (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. This light chain constant region may be a kappa or lambda constant region.

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

[0125] Expression vectors in this disclosure may comprise polynucleotides encoding an antibody or its antigen-binding moiety as described herein. In some embodiments, the coding sequence of an antibody or its antigen-binding moiety is operably ligated to an expression regulatory sequence. As used herein, two nucleic acid sequences are operably ligated if they are covalently linked in such a way that each component nucleic acid sequence retains its functionality. A coding sequence and a gene expression regulatory sequence are said to be operably ligated if they are covalently linked in such a way that the expression or transcription and / or translation of the coding sequence is placed under the influence or control of the gene expression regulatory sequence. Two DNA sequences are said to be operably ligated if the induction of a promoter in the 5' gene expression sequence results in the 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 the 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. Therefore, if a gene expression sequence is capable of producing a transcription of its coding nucleic acid sequence so that the resulting transcript is translated into the desired antibody or its antigen-binding portion, then this gene expression sequence is operably ligated to the coding nucleic acid sequence.

[0126] Viral vectors include, but are not limited to, nucleic acid sequences derived from the following viruses: retroviruses, e.g., Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary cancer virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40 virus; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses; and RNA viruses, e.g., retroviruses. Other vectors well known in the art can readily be used. Certain viral vectors are based on non-cellularly invasive eukaryotic viruses in which non-essential genes are replaced with genes of interest. Non-cellularly invasive viruses include retroviruses, whose life cycle involves the 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-deficient retroviruses (i.e., those that can be directed to synthesize the desired protein but are unable to produce infectious particles) are most useful. Such genetically modified retroviral expression vectors have general utility for highly efficient transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including steps of incorporating exogenous genetic material into a plasmid, transfection of a packaging cell line with the plasmid, production of recombinant retrovirus by the packaging cell line, collection of viral particles from tissue culture medium, and infection of target cells with the viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, EJ, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0127] In some embodiments, this virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication-deficient and capable of infecting a wide range of cell types and species. This offers further advantages such as thermal and lipid solvent stability; high transduction frequency in cells of diverse lineages, including hematopoietic cells; and the absence of re-infection inhibition, which allows for multiple transduction sequences. Reports indicate that adeno-associated viruses can be integrated into human cellular DNA in a site-specific manner, thereby minimizing the potential for insertional mutagenesis and variability in the expression of inserted genes characteristic of retroviral infections. Furthermore, wild-type adeno-associated virus infections have been tracked in tissue cultures for longer than 100 passages in the absence of selective pressure, suggesting that genomic integration of adeno-associated viruses is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

[0128] IV. Immunoconjugate This disclosure also provides an immunoconjugate comprising one of the anti-TREM-1 antibodies disclosed herein. In some embodiments, this immunoconjugate comprises an antibody or antigen-binding moiety linked to a drug. In some embodiments, this immunoconjugate comprises a bispecific molecule disclosed herein linked to a drug (e.g., as a therapeutic or diagnostic agent).

[0129] For diagnostic purposes, suitable agents include radioisotopes for whole-body imaging, as well as detectable labels that include radioisotopes, enzymes, fluorescent labels, and other suitable antibody tags for sample testing. Detectable labels that can be linked to any anti-TREM-1 antibody described herein include metal sols, e.g., granular labels containing colloidal gold, isotopes, e.g., peptide chelating agents of type N2S2, N3S, or N4, for example. 125 or Tc 99This can be any of the various types currently used in the field of in vitro diagnostics, including chromophores such as fluorescent markers, luminescent markers, and phosphorescent markers, as well as enzymatic 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. Suitable enzymatic labels include horseradish peroxidase and alkaline phosphatase. For example, the label may be a 1,2-dioxetane substrate, such as adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), 3-(4-(methoxyspiro{l,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decane}-4-yl)phenyl disodium phosphate (CSPD), and the enzyme alkaline phosphatase, which is detected by measuring the presence or formation of chemiluminescence after conversion of CDP and CDP-STAR®, or other luminescent substrates well known to those skilled in the art, such as suitable lanthanides, such as terbium(III) and europium(III) chelates. The means of detection 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 granular and accumulates at an appropriate level, or it can be achieved using instruments such as spectrophotometers, luminometers, and fluorophotometers, all in accordance with standard practice.

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

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

[0132] Generally, site-directed reactions and covalent couplings are based on transforming native amino acids into amino acids that have orthogonal reactivity to other functional groups present. For example, certain cysteines in rare sequence contexts can be enzymatically converted to aldehydes (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). Desired amino acid modifications can also be obtained by utilizing the specific enzymatic reactivity between a particular enzyme and native amino acids 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 CN bond formation is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site-directed reactions and covalent coupling can also be achieved by selective reactions between terminal amino acids and appropriate modification reagents. The reactivity of the 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-directed covalent coupling. Native chemical ligation may also depend on the 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 a faster reaction between cysteine ​​located in a stretch of negatively charged amino acids and cysteine ​​located in a stretch of positively charged amino acids.

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

[0134] To obtain monolabeled polypeptides, conjugates with a 1:1 stoichiometry can be separated from other conjugation byproducts by chromatography. This procedure can be facilitated by using dye-labeled binding pair members and charged linkers. By using this type of labeled and highly negatively charged binding pair member, monoconjugated polypeptides are readily separated from unlabeled polypeptides and polypeptides possessing more than one linker, because differences in charge and molecular weight can be used for separation. Fluorescent dyes may be useful for purifying the complex from unbound components such as labeled monovalent binders. In some embodiments, the portion that binds 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 therapeutic agents to form immunoconjugates such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA subgroove 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 ADCs, the antibody and 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 may 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, may also be used to detect TREM-1, such as human TREM-1, for example, in tissue or tissue samples. These antibodies may 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 tissue, for a period of time appropriate for specific binding to occur, and then a reagent, such as an antibody for detecting the anti-TREM-1 antibody, is added. Exemplary assays are provided in the Examples. This anti-TREM-1 antibody may be a fully human antibody or a chimeric antibody, such as an antibody having a human variable region and a mouse constant region or a portion thereof. Exemplary methods for detecting TREM-1 in a sample (cell or tissue sample), such as human TREM-1, include: (i) contacting the sample with an anti-TREM-1 antibody for a sufficient time 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 anti-TREM-1 antibody, 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 has bound. A washing step may 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 does not need to be labeled or bound to the detection agent. For example, other uses of anti-TREM-1 antibodies, either as monotherapy or in combination therapy, are provided elsewhere in this specification, for example, in the section on combination therapy.

[0137] V. Bispecific molecules The anti-TREM-1 antibodies described herein may be used to form bispecific molecules. The anti-TREM-1 antibody or its antigen-binding moiety may be derivatized or ligated to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand against a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. For example, an anti-TREM-1 antibody may be ligated to any protein that can be used as a potential target for concomitant treatment, e.g., an antibody or scFv that specifically binds to the proteins described herein (e.g., an antibody against IP-10 or TNF-α). The antibodies described herein may, in fact, be derivatized or ligated to more than one other functional molecule to produce a polyspecific molecule that binds to more than two different binding sites and / or target molecules; such polyspecific molecules, as used herein, are also intended to be encompassed by the term “bispecific molecule.” To create the bispecific molecules described herein, the antibodies described herein may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, so that the bispecific molecules are generated. Accordingly, a bispecific molecule comprising at least one first binding specificity to TREM-1 and a second binding specificity to a second target epitope is provided herein. In some embodiments described herein where the bispecific molecule is polyspecific, the molecule may further comprise a third binding specificity.

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

[0139] The bispecific molecules described herein can be prepared by conjugating the binding specificities of their constituent components using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then conjugated with one another. When the binding specificity is a protein or peptide, various coupling or crosslinking agents can be used for covalent conjugation. Examples of crosslinking binders 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 sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane (cyclohaxane)-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 conjugates are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL). When the binding specificity is an antibody, these can be conjugated via sulfhydryl linkages in the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region is modified before conjugation to contain an odd number, preferably one, sulfhydryl residue.

[0140] Alternatively, both binding specificities may 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 may comprise an antibody containing scFv at the C-terminus of each heavy chain. The bispecific molecules described herein may be a single-chain molecule comprising one single-chain antibody and one binding determinant, or a single-chain bispecific molecule comprising two binding determinants. The bispecific molecule may comprise at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0141] The binding of bispecific molecules to their specific targets can be confirmed using methods recognized in the field, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of the protein-antibody complex of interest by using a reagent (e.g., antibody) specifically labeled for the complex of interest.

[0142] VI. Kit Kits comprising one or more anti-TREM-1 antibodies or their antigen-binding moieties, their bispecific molecules, or immunoconjugates as described herein are provided herein. In some embodiments, pharmaceutical packs or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, for example, one or more antibodies or their antigen-binding moieties as provided herein, and optional instructions for use are provided herein. In some embodiments, these kits comprise the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, for example, those described herein.

[0143] VII. Compositions and Formulations Compositions (e.g., pharmaceutical compositions) and formulations comprising one or more anti-TREM-1 antibodies (including polynucleotides, vectors, and cells encoding and / or expressing anti-TREM-1 antibodies) disclosed herein are further provided herein. For example, in one embodiment, the disclosure provides a pharmaceutical composition comprising one or more anti-TREM-1 antibodies disclosed herein, formulated together with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc. 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., antibody, immunoconjugate, or bispecific molecule, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0144] Accordingly, one object of this disclosure is to provide a pharmaceutical formulation that improves the stability of anti-TREM-1 antibodies and thus enables their long-term storage. In some embodiments, the pharmaceutical formulation disclosed herein comprises: (a) anti-TREM-1 antibody; (b) buffer; (c) stabilizer; (d) salt; (e) bulking agent; and / or (f) surfactant. In some embodiments, the pharmaceutical formulation is stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years or longer. In some embodiments, the formulation is stable when stored at 4°C, 25°C, or 40°C.

[0145] cushioning agent A buffer useful for the present invention may be a weak acid or weak base used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. A suitable buffer can maximize the stability of a pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may 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, potentially accompanied by pH adjustment with an acid or base known in the art. In certain embodiments, this 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 them. Such agents can stabilize proteins in several different ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine; carbohydrates such as glucose, sucrose, trehalose, raffmose, or maltose; polyols such as glycerol, mannitol, sorbitol, cyclodextrin, or dextran of any type and molecular weight, or PEG. In one aspect of the present invention, the stabilizer is selected to maximize the stability of the FIX polypeptide in a lyophilized preparation. In certain embodiments, the stabilizer is sucrose and / or arginine. Bulking agent Volume extenders may be added to pharmaceutical products to increase their volume and mass, thereby facilitating their accurate measurement and handling. Common volume extenders include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0147] surfactant A surfactant is an amphiphilic substance having a hydrophilic group and a hydrophobic 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 oxides, polypropylene oxides, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0148] In some embodiments, the pharmaceutical formulations of this disclosure are (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 histidine; (c) About 150 mM sucrose; (d) About 25 mM arginine; and (e) About 50 mM NaCl It contains. This formulation may further contain 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 can 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 mass of water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% by mass of water, and the term "aqueous suspension" is defined as a suspension containing at least 50% by mass 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 before 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 include 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 contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” is defined as a salt that retains the desired biological activity of the parent compound and does not impose any undesirable toxicological effects (see, for example, Berge, SM, 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, and phosphorous acid, as well as those derived from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanoates, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

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

[0153] These compositions may also contain auxiliary agents, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by both the sterilization procedure described above and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, extended absorption of injectable pharmaceutical forms can be achieved by including absorption-delaying agents, 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. Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is intended. The pharmaceutical compositions may or may not contain preservatives. Complementary active compounds may be incorporated into these compositions.

[0155] Therapeutic compositions are typically sterile and stable under manufacturing and storage conditions. These compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by coating, e.g., the use of lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Often, these compositions may contain isotonic agents, e.g., sugars, polyalcohols, e.g., mannitol, sorbitol, or sodium chloride. Extended absorption of injectable compositions can be achieved by including absorption-delaying agents, e.g., monostearate and gelatin, in the composition.

[0156] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent, along with one or a combination of the components described above, and then performing sterilization microfiltration as necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those 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), from which a powder of the active component plus any further desired components is obtained from the solution that has been previously sterile-filtered. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the subject being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form is the amount of the composition that produces the therapeutic effect. Generally, out of 100 percent, this amount, combined with a pharmaceutically acceptable carrier, ranges from about 0.01 percent to about 99 percent of the active ingredient, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent of the active ingredient.

[0157] The drug regimen is adjusted to provide the optimal desired response (e.g., 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 urgent demands of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in drug unit forms. When used herein, drug unit form refers to a physically distinct unit appropriate as a unit dose for the subject being treated; each unit contains a calculated predetermined amount of the active compound to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of drug unit forms described herein are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the field of formulation for the treatment of susceptibility in an individual.

[0158] For example, for the administration of anti-TREM-1 antibodies as described herein, the dosage is in the range of approximately 0.0001 to 100 mg per kg of host body weight, more typically in the range of 0.01 to 5 or 10 mg. 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 in the range of 1 to 10 mg / kg. An exemplary treatment regimen requires 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 administered intravenously, the antibody being given using one of the following dosing schedules: (i) six doses every four weeks, then every three months; (ii) every three weeks; (iii) one dose of 3 mg / kg body weight, followed by 1 mg / kg body weight every three weeks. In some embodiments, this anti-TREM-1 antibody is administered in a flat dose (flat dose regimen). In other embodiments, this anti-TREM-1 antibody is administered in a fixed dose along with another antibody. In certain embodiments, this anti-TREM-1 antibody is administered in a body weight-based dose. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the indicated range. Antibodies are usually administered on multiple occasions. The interval between single doses may be, for example, weekly, monthly, every three months, or annually. The interval may also be irregular, if indicated by measuring the blood levels of antibodies against the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of approximately 1–1000 μg / ml, and in others, approximately 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 have the longest half-lives, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals are required occasionally until disease progression is reduced or terminated, or until the patient shows partial or complete remission of disease symptoms. Thereafter, the patient may be administered a prophylactic regime.

[0160] The actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient effective in achieving 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 various pharmacokinetic factors, including factors well known in the medical field, such as the activity of the particular composition described herein or its ester, salt, or amide used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, and the age, sex, weight, condition, overall health, and prior medical history of the patient being treated.

[0161] The compositions described herein may be administered via one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. Routes of administration for the anti-TREM-1 antibodies described herein may include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes by injection or infusion. The term “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, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the antibodies described herein may potentially be administered via non-parenteral routes, such as topical, dermal, or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0162] The active compound may be prepared using a carrier that protects the compound from rapid release, such as in controlled-release formulations including grafts, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

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

[0164] In some embodiments, the anti-TREM-1 antibodies described herein may 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), they may be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more portions that are selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PG 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); K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: See also 123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0165] VIII. Use and Method The anti-TREM-1 antibodies and compositions comprising such antibodies (e.g., pharmaceutical compositions, formulations, polynucleotides, vectors, and cells) of this disclosure may be used for the treatment of inflammatory diseases (e.g., by inhibiting TREM-1 activity).

[0166] Accordingly, in one embodiment, the present disclosure provides a method for treating an inflammatory disease in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose of an anti-TREM-1 antibody to the subject. Examples of inflammatory diseases that can be treated with the anti-TREM-1 antibody 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 1 diabetes mellitus, 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 syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, and other autoimmune diseases resulting from either acute or chronic inflammation.

[0167] In one embodiment, these anti-TREM-1 antibodies are suitable for use in the treatment of individuals with inflammatory bowel disease (IBD). Inflammatory bowel disease (IBD) is a disease that can affect any part of the gastrointestinal tract from mouth to anus and causes a wide range of symptoms. IBD primarily causes abdominal pain, diarrhea (which may be bloody), vomiting, or weight loss, but it can also cause complications outside the gastrointestinal tract, such as skin rashes, arthritis, eye inflammation, fatigue, and lack of concentration. Patients with IBD can be divided into two main classes: those with ulcerative colitis (UC) and those with 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, inflammation is transmural and causes abscesses, fistulas, and strictures, whereas in UC, inflammation is typically limited to the mucosa. For Crohn's disease, neither a medicinal nor surgical cure is known, although some patients with UC may be cured by surgical removal of the colon. Treatment options are limited to symptom control, maintenance of improvement, and prevention of relapse. Efficacy in inflammatory bowel disease in a clinical setting can be measured as a reduction in the Crohn's Disease Activity Index (CDAI) score for CD, a scoring scale based on laboratory tests and quality of life questionnaires. In animal models, efficacy is largely measured by weight gain and also by the Disease Activity Index (DAI), a combination of stool consistency, body weight, and bloody stools.

[0168] In one embodiment, the anti-TREM-1 antibody of this disclosure is suitable for use in the treatment of individuals with rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease affecting almost, though not all, of the body and is one of the most common forms of arthritis. It is characterized by inflammation of the joints, causing pain, stiffness, warmth, redness, and swelling. This inflammation is a result of inflammatory cells infiltrating the joints, which release enzymes that can digest bone and cartilage. As a result, this inflammation can lead to severe bone and cartilage damage, as well as joint deterioration and severe pain, among other physiological effects. The affected joints may 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 that of human rheumatoid arthritis. Since CIA shares similar immunological and pathological characteristics with RA, this makes the model a suitable model for screening potential human anti-inflammatory compounds. Efficacy in this model is measured by a reduction in joint swelling. Efficacy in RA in the clinical setting is measured by the ability to reduce symptoms in patients, measured as a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein levels, and serum factors, such as the level of anti-citrullinated protein antibodies.

[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. This is a disease for which there is currently no cure and it 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 often have 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). Potency in this model is measured by reduction in skin pathology using a scoring system. Similarly, potency 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 having psoriasis. In these patients, joint swelling similar to that seen in rheumatoid arthritis accompanies the skin pathology / symptoms. This is characterized by patchy raised red areas of skin inflammation with scaling. Psoriasis often affects the elbows and knees tips, scalp, umbilicus, and around the genital or anal areas. Approximately 10% of patients having psoriasis also develop associated joint inflammation.

[0171] With respect to this disclosure, prophylactic, palliative, symptomatic, and / or curative treatments may represent separate aspects of this disclosure. The antibodies of the present invention may be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously. Alternatively, the antibodies of the present invention may be administered via parenteral routes, for example, orally or topically. The antibodies of the present invention may be administered prophylactically. The antibodies of the present invention may be administered therapeutically (as required). The following examples are provided for illustrative purposes only and not for limiting purposes. The contents of all references cited throughout this application are expressly incorporated herein by reference. [Examples]

[0172] (Example 1) Analysis of interaction kinetics of 318 antibody variants against TREM-1 in both humans and cynomolgus monkeys using surface plasmon resonance. The binding kinetics of the mAb 0318 variant to human TREM-l-Fc (hTREM-1) and cynomolgus monkey TREM-l-Fc (cTREM-1) were determined. Binding studies were performed using a ProteOn Analyzer (BioRad) to measure molecular interactions in real time via surface plasmon resonance. Experiments were conducted at 25°C, and samples were stored at 15°C in the sample compartment. The signal (RU, response unit) reported by ProteOn directly correlated with the mass on the surface of the individual sensor chip in six parallel flow cells. Anti-human Fc monoclonal antibodies or anti-mouse Fc polyclonal antibodies from Biacore's human or mouse Fc capture kits were 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. Capture of purified monoclonal mouse or recombinantly expressed anti-hTREM-1 antibody was performed 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 it vertically at 30 μl / min over 60 seconds to create reference interspots adjacent to all flow cells, where only anti-Fc antibody was immobilized. This typically resulted in a final capture level of approximately 100–300 RU of test antibody and an analyte Rmax value of 30–90 RU. Binding of hTREM-1 or cTREM-1 protein was performed by injecting the analyte (antigen) horizontally into all flow cells, allowing for comparative analysis of binding to different captured anti-TREM-1 antibodies compared to binding to the reference interspots. hTREM-1 or cTREM-1 protein was serially diluted 1:3 in running buffer to concentrations of 1.2–100 nM and injected at 100 μl / min for 250 seconds, followed by dissociation for 600 seconds. The GLM surface was regenerated after each injection cycle of the analyte by 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 for subsequent binding of the next interacting sample pair. The regeneration procedure did not directly remove the immobilized anti-Fc capture antibody from the chip surface.

[0173] The binding affinity between the antibody and the antigen is determined by the equilibrium dissociation constant (K) measured by the kinetics of complex formation and dissociation. D Quantified by the determination of the rate constant corresponding to the association and dissociation of monovalent complexes, e.g., k a (Meeting velocity) and k d The dissociation rate was extracted by fitting the data to a 1:1 Langmuir model using ProteOn evaluation software for data analysis. D is, equation K D =k d / k a Through, k a and k d It can be associated with this. The binding curves were processed with a dual reference (subtraction of the reference surface signal and blank buffer injection for the captured anti-TREM-1 antibody) before data analysis. This allowed for correction of instrument noise, bulk shift, and drift during sample injection. As shown in Figures 1A and 1B, all 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-1 similar to that of mAb 0318-IgG4. The 0318-IgG1.3f variant also bound to cynomolgus monkey TREM-1, albeit with a slightly reduced affinity compared to human TREM-1. See Figure 1A.

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

[0175] Next, to determine the fate of the TREM-1 receptor after internalization, the TREM26 antibody (catalog number 314902, Biolegend; see paragraph

[0005] of U.S. Patent Application Publication No. 20150274825) was used for comparison. The TREM26 antibody does not compete with the 0318 antibody variant described herein. As shown in Figure 2B, a significant reduction (approximately 51%) in TREM26+ expression was observed at 20 hours compared to 0 hours. A significant reduction (one-quarter) in TREM26+ MFI (mean fluorescence index) was also observed at 20 hours (after mAb 0318-IgG1.3f treatment), suggesting that the TREM-1 receptor is degraded during 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 a BWZ / hTREM-1 reporter cell assay. The ability of the anti-TREM-1 mAb 0318 variant to inhibit human TREM-1 signaling was determined, for example, using the BWZ.36 / hTREM-1DAP12:NFAT-LacZ cell line (also referred to herein as "BWZ / hTREM-1 reporter cell") assay, as described in U.S. Patent No. 9,550,830 and International Patent Publication No. WO2016 / 009086. In short, approximately 40,000 hTREM-1 / BWZ.36 cells / well were plated in clear-bottomed black 96-well plates in the presence of 75 ng / ml PGLYRP1 (SEQ ID NO: 8) along with 2.5 μg / ml PGN-ECndi (catalog no. tlrl-kipgn, Invivogen San Diego, Calif., USA) to provide a submaximal positive signal, or in the presence of a submaximal level (1 μg / ml) of plastic-adsorbed anti-TREM-1 monoclonal antibody (catalog no. MAB1278, R&D Systems, Minneapolis, Minn., USA) to provide a positive signal.

[0177] mAb 0318 variants (i.e., 0318-IgG1.3f; 0318-IgG1.1f; 0318-IgG1-Aba; and 0318-IgG4-Aba) were titrated during the assay starting at 10 μg / ml in five sequential 2-fold dilutions. The assay was incubated overnight at 37°C and subsequently color-developed with Beta Glo (catalog no. E4740, Promega Madison, Wis., USA) according to the Beta Glo protocol, with luminescence recorded. Data were plotted to show Beta Glo relative luminescence units versus 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 20150274825), a known agonist of TREM-1 signaling, was also used as a positive control (see inserted boxed figure). As shown in Figure 3, all mAb 0318 variants were potent in inhibiting human TREM-1 signaling, as previously observed with the mAb 0318 IgG4 antibody in international patent publication number 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 anti-TREM-1 mAb 0318 variants, their potency in blocking the release of various inflammatory cytokines (e.g., TNF-α, IL-6, or IL-8) from activated human primary cells was assessed. 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 lacking TLR2 activity).

[0179] As shown in Figure 4, all mAb 0318 variants (i.e., IgG1.3f, IgG1.1f, IgG1-Aba, and IgG4-Aba) were potent in inhibiting the TREM-1-mediated release of TNF-α from PBMCs and monocytes (IC4 in the range of approximately 10–20 pM). 50 (Value). 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 (approximately 32 pM IC50). 50 (Value). For neutrophils, the mAb 0318-IgG1.3f variant appeared to be more effective than the mAb 0318-IgG4 antibody in blocking 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 variant, a monocyte-neutrophil co-culture assay was also used. When co-cultured with monocytes, neutrophil-associated PGRP1 can bind to the TREM-1 receptor, resulting in monocyte-derived TNF-α production. As shown in Figure 4, all mAb 0318 antibody variants effectively blocked this endogenous activation (IC₂ in the range of 19–44 pM). 50 (Value). Similar results were observed in whole blood with RBC sedimentation (see Figure 4).

[0181] (Example 5) In vitro analysis of the potency of mAb 0318-IgG1.3f, which blocks IL-8 production from stimulated whole blood. One of the major challenges in developing whole blood pharmacodynamic (PD) assays to measure the antagonist properties of anti-TREM-1 antibody variants is the high background resulting from PGN stimulation. To help address this problem, whole blood was stimulated with pre-complexed PGRP1+PGN in the presence of a NOD2 inhibitor (to block background cytokines resulting from 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 percentage inhibition ranging from approximately 60 to 90% (see Figure 5A). The observed IC50 values ​​(mean values ​​of 12 pM for HTRF and 19.6 pM for ICS) were similar to those observed in other functional assays (see, e.g., Example 4).

[0183] (Example 6) In vitro analysis of the potency of mAb 0318-IgG1.3f blocking 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 stimulated overnight with pre-complexed hPGRP1 (50 μg / ml) and PGN-ECndss (10 μg / ml) (Invivogen tlrl-ksspgn) in the presence of varying concentrations of mAb 0318-IgG1.3f (0-1 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. Next, the isolated mRNA was converted to cDNA using SuperScript VILO Master Mix (Thermo Fisher 11755250). Then, 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 standardized relative to HPRT1, and ΔΔCT values ​​were generated. The results were then plotted, and IC50 was calculated. 50 The value was determined.

[0184] As shown in Figures 6A-6C and consistent with the examples described above, 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 herein is antagonistic and can effectively block TREM-1-mediated production of inflammatory cytokines from various human cells.

[0185] [Table 1]

[0186] (Example 7) Viscosity of mAb 0318 variant The samples were exchanged for buffer, dialyzed in an 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 centrifugation molecular weight cutoff filter. The aggregation state of the samples was measured by size exclusion chromatography to control for potential changes in monomericity during concentration, but no such changes were observed. Concentration-dependent viscosity was determined for each measured concentration using a RheoSense m-VROC solution viscometer with a 3-point ascending shear sweep. The concentration was determined by measuring the absorbance at 280 nm using 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. This viscosity profile was very similar to that previously observed with mAb 0318-IgG4 (see International Patent 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 carried a low to intermediate risk of immunogenicity in human patients. Only about 22–30% of donors showed an immunogenic response to these antibodies (in vitro CD4 + The mAb had T cell proliferation (measured by T cell proliferation). 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 even more immunogenic (55%) in human patients. KLH (keyhole limpet hemocyanin) and VL6 (IL-21R mAb), used as positive controls, were highly immunogenic (100% and 40%, respectively) in human patients. [Table 2]

[0189] (Example 9) Coupling analysis of mAb 0318 variants to FcRn using surface plasmon resonance (SPR) To determine whether different mAb 0318 variants could bind to FcRn, FcRn receptors (mouse, human, and cynomolgus monkey) were immobilized on BIAcore CM5 biosensor chips (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 and 0.05% Tween-20™ pH 6.0 (GE Healthcare Bioscience) as running and dilution buffers. Different mAb 0318 variants (200 nM) were injected at a flow rate of 50 μL / min at pH 6.0 at room temperature. The association time was 180 seconds, and the dissociation phase (also at pH 6.0) took 360 seconds. Regeneration of the chip surface back to baseline was achieved by short-term injections of 50 mM Tris, pH 8.0, and 150 mM NaCl. SPR data were evaluated by comparing the height of the biological response signal at 180 seconds and 300 seconds after infusion. The corresponding parameters were RU max level (180 seconds after infusion) and late stability (300 seconds after the end of infusion).

[0190] As shown in Figures 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 against certain cell surface immunoreceptors has the potential to induce cytokine release, which can lead to the induction of a common toxic clinical complication known as cytokine release syndrome (CRS). Due to concerns that FcγR binding may result in potential TREM-1 agonist activity via cross-linking, mAb 0318-IgG4 was reengineered into one of the variant formats described herein (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 Figures 10A and 10B, the 318-IgG1.1f and 318-IgG1.3f variants showed minimal binding to all 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 FcγRs.

[0193] (Example 11) Analysis of inflammatory cytokine induction by mAb 0318 variant To further determine whether in vivo treatment with the mAb 0318 variant poses a risk of cytokine release syndrome, whole blood was collected from eight human donors and monocytes were isolated. Subsequently, monocytes (4 × 10⁻¹⁴) were analyzed. 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 - 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. Then, these cells were 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 the 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 antibody identity 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 identified at N301 on the heavy chain, with a glycan profile consistent with the glycan profiles (G0F, G1F, and G2F) of the monoclonal antibody expressed in CHO. The thermal stability of mAb 0318-IgG1.3f (T m 1 = 66.2℃; T m 2 = 78.4℃; T m The reversibility (3 = 83.2°C) and thermal reversibility (24% at 77°C) 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), no physical stability issues were observed at 150 mg / mL during freeze-thaw stress (3 cycles). Forced degradation studies of the 150 mg / mL study formulation were set at 4, 25, and 40°C (for a maximum of 3 months). Chemical modifications of the CDR remained low throughout all temperature conditions, as determined by SPR, and did not affect activity. VSNK deamide was below prediction (a 3% increase / month at 40°C storage) compared to other monoclonal antibodies in the IgG1.3f framework, and the changes therein were time and temperature dependent. All other chemical modifications (oxidation, deamide, isomerization) also remained low when monitored during stability studies. Notably, storage at 40°C exhibited the formation of both HMW 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 under storage at 4°C over the study period, while LMW remained unchanged. The low LMW formation under storage at 40°C was characterized by the cumulative loss of a single Fab, as well as, in estimation, a Fab arm in a conserved sequence within the upper hinge region, by 2D-LC / MS with high-resolution, accurate mass measurements.

[0199] (Example 13) PK / TK / PD study of the mAb 0318-IgG1.3f variant in cynomolgus monkeys Single-dose pharmacokinetic (PK), toxicokinetic (TK), and pharmacodynamic (PD) studies of the anti-TREM-1 0318-IgG1.3f antibody were conducted in cynomolgus monkeys. Some animals received 2 mg / kg of 0318-IgG1.3f antibody intravenously (n=3). Other animals received one of the following doses of 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). Pharmacokinetics, anti-drug antibody (ADA), TREM-1 receptor occupancy (RO), and ex vivo pharmacodynamic responses were examined at predetermined time points following antibody administration.

[0200] Pharmacokinetics (PK) To evaluate the pharmacokinetics, serum concentrations of the 0318-IgG1.3f antibody were assessed in animals using a ligand-binding assay that utilized biotinylated recombinant TREM-1 protein as the capture reagent and a commercially available polyclonal goat anti-TREM-1 antibody as the detection reagent. As shown in Figure 12, the pharmacokinetics of the 0318-IgG1.3f antibody were determined to be nonlinear 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 an IV dose of 2 mg / kg was 0.1 ± 0.02 mL / hour / kg in monkeys, 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 a 0.1 mg / kg dose to 10 days for 2 and 10 mg / kg (single subcutaneous dose). 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 antibodies (ADA) Serum ADA was detected in most monkeys (16 out of 18) after a single dose (regardless of the route of administration), but exposure to 0318-IgG1.3f antibody and TREM-1 receptor occupancy (RO) were not impaired in the majority of ADA-positive animals. See Tables 4 and 5. Accelerated decay of 0318-IgG1.3f antibody in terminal exposure accompanied by ADA formation was observed in only two monkeys (one in the 0.1 mg / kg dose group on day 7 and one in the 0.5 mg / kg dose group on day 21). Since ADA affected exposure in these two monkeys in the terminal 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 rate of TREM-1 receptors expressed on peripheral blood monocytes and granulocytes was evaluated. As shown in Figures 15A and 15B, for all doses tested, the percentage of TREM-1 receptors occupied (i.e., bound to anti-TREM-1 antibody) was similar between monocytes and granulocytes. Furthermore, the duration of receptor occupancy appeared to be dependent on the dose of 0318-IgG1.3f antibody administered to the animals. At 0.5 mg / kg, an RO of ≥85% was observed for up to 2 weeks after antibody administration. In contrast, at 2 and 10 mg / kg, ≥85% of TREM-1 receptors remained occupied for at least 1 month after administration. Following administration of the 0318-IgG1.3f antibody, total TREM-1 receptor levels were reduced on both monocytes and granulocytes. See Figures 14A and 14B. As previously discussed, this reduction is likely attributable 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] Soluble TREM-1 (sTREM-1) levels 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). It is unclear whether the increase in sTREM-1 levels is related to the administration of anti-TREM-1 antibody or to the overall handling of the animals during the course of the study. In either case, drug concentrations far exceeded sTREM-1 levels (>1000-fold), suggesting that the soluble target does not result in the binding of the mAb 0318 mAb-IgG1.3f variant to cell surface TREM-1.

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

[0208] [Table 7]

[0209] [Table 8] This PCT application, TIFF0007867999000009.tif90170, claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 651,605, filed 2 April 2018, which is incorporated herein by reference in its entirety.

Claims

1. An isolated antibody that specifically binds to trigger receptor-1 (TREM-1) expressed on myeloid cells, comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 52, SEQ ID NO: 87, SEQ ID NO: 53, or SEQ ID NO: 86, and the light chain comprises SEQ ID NO:

54.

2. A bispecific molecule comprising the antibody according to claim 1, linked to a molecule having a second binding specificity.

3. A nucleic acid encoding the antibody according to Claim 1.

4. A vector comprising the nucleic acid described in Claim 3.

5. A host cell comprising the vector according to claim 4.

6. An immunoconjugate comprising the antibody described in Claim 1, linked to a drug.

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.

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.

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: 86 and the light chain comprises SEQ ID NO:

54.

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: 53 and the light chain comprises SEQ ID NO:

54.

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: 87 and the light chain comprises SEQ ID NO:

54.

12. 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: 52 and the light chain comprises SEQ ID NO:

54.

13. A pharmaceutical composition for blocking TREM-1 activity in humans, comprising an antibody according to claim 1 or any one of claims 9 to 12, a bispecific molecule according to claim 2, or an immunoconjugate according to claim 6.

14. 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 12, the bispecific molecule according to claim 2, or the immunoconjugate according to claim 6.

15. The pharmaceutical composition according to claim 14, wherein 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 1 diabetes mellitus, 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 syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma, other autoimmune diseases resulting from any acute or chronic inflammation, and any combination thereof.

16. The pharmaceutical composition according to claim 13, further comprising one or more further therapeutic agents.

17. The pharmaceutical composition according to claim 15, wherein the inflammatory disease or autoimmune disease is Crohn's disease.

18. The pharmaceutical composition according to claim 15, wherein the inflammatory disease or autoimmune disease is ulcerative colitis.

19. 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: 86 and the light chain comprises SEQ ID NO:

54.

20. 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: 86 and the light chain comprises SEQ ID NO:

54.

21. 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: 53 and the light chain comprises SEQ ID NO:

54.

22. 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: 53 and the light chain comprises SEQ ID NO:

54.

23. 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: 87 and the light chain comprises SEQ ID NO:

54.

24. 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: 87 and the light chain comprises SEQ ID NO:

54.

25. 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: 52 and the light chain comprises SEQ ID NO:

54.

26. 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: 52 and the light chain comprises SEQ ID NO: 54.

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