EA046142B1 - ANTIBODIES TO TREM-1 AND THEIR APPLICATIONS - Google Patents

Abstract

The invention describes antibodies or antigen-binding fragments thereof that specifically bind to TREM-1 and inhibit TREM-1 signaling, wherein the antibodies do not bind to one or more FcyRs and do not induce the production of proinflammatory cytokines by myeloid cells. Therapeutic uses of such antibodies or their antigen-binding fragments are also described, for example, in the treatment of autoimmune diseases.

Description

Link to the sequence listing presented in electronic form on the EFS-WEB website

The contents of the electronically submitted sequence listing as an ASCII text file (name: 3338_092PC01_SeqListing.txt; size: 106162 bytes; and creation date: March 27, 2019), generated during the creation of the application, are fully incorporated into this description by reference.

Prerequisites for the creation of the invention

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 involved in inflammation. TREM-1 mRNA and protein expression are up-regulated in patients with rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and TREM-1-positive cells accumulate at sites of inflammation, which correlates with disease severity (see Bouchon et al., Nature, 410 (2001), pp. 1103–1107; Schenk et al., Clin Invest, 117 (2007), pp. 3097–3106; and Kuai et al., Rheumatology, 48 (2009), pp. 1352–1358). Peptidoglycan recognition protein 1 (PGLYRP1), expressed primarily by activated neutrophils, is a ligand for TREM-1, and upon binding, it mediates TREM-1 signaling.

In vitro, TREM-1 engagement triggers the secretion of proinflammatory cytokines, including TNF, IL-8, and monocyte chemotactic protein-1. Furthermore, TREM-1 signaling synergistically interacts with numerous Toll-like receptors (TLRs), further amplifying proinflammatory signals. In turn, this upregulates TREM-1 expression, leading to a vicious cycle that increases inflammation (see Bouchon et al., J. Immunol., 164 (2000), pp. 4991–4995). Growing evidence suggests that TLRs contribute to the onset and progression of chronic inflammatory diseases such as RA and IBD.

Humanized anti-TREM-1 mAbs that inhibit TREM-1 function in both humans and cynomolgus monkeys have been described in various documents (see WO 2013/120553 A1 and WO 2016/009086 A1). However, these antibodies either have a viscosity profile that complicates their manufacturing process or present other problems that may limit their therapeutic potential (such as cytokine storm and ADCC) (see Shire et al., J. Pharm. Sci., 93 (2004), pp. 1390–1402; and Warncke et al., J. Immunol., 188 (2012), pp. 4405–4411). Therefore, there is a need for anti-TREM-1 antibodies that can specifically bind to TREM-1 and inhibit its function, but do not cause the problems associated with previously developed anti-TREM-1 antibodies.

Brief summary of the invention

The present invention provides isolated antibodies, such as monoclonal antibodies, particularly human (e.g., monoclonal) antibodies, that specifically bind to the trigger receptor-1 expressed on myeloid cells (TREM-1) and exhibit the desired functional properties. 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 comprises 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 TREM-1, blocking it, 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 comprises 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 binds to the same TREM-1 epitope as mAb 0318. In some embodiments, the antibody specifically binds to a TREM-1 epitope comprising one or more amino acid residues selected from the group consisting of D38, V39, K40, C41, D42, Y43, T44, L45, E46, K47, F48, A49, S50, S51, Q52, K53, A54, W55, Q56, Y90, H91, D92, H93, G94, L95, and L96 of SEQ ID NO: 1. In some embodiments, the antibody specifically binds to a TREM-1 epitope comprising amino acids D38 to L45, E46 to Q56, and/or Y90 to L96 of SEQ ID NO: 1.

In some embodiments, the constant region of the IgG1 heavy chain of an antibody provided herein comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, and L358M (EU numbering). In some embodiments, the constant region of the IgG1 heavy chain comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M (EU numbering). In some embodiments, the constant region of the IgG1 heavy chain comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S (EU numbering). In some embodiments of the invention, the constant region of the IgG1 heavy chain 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 (EU numbering).

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In some embodiments, an antibody as provided herein comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR3 comprises DMGIRRQFAY (SEQ ID NO: 26) or DMGIRRQFAY (SEQ ID NO: 26) except for one or two substitutions. In some embodiments, the heavy chain CDR3 comprises DQGIRRQFAY (SEQ ID NO: 72).

In some embodiments, an antibody as provided herein comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR2 comprises RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) or RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) except for one or two substitutions.

In some embodiments, an antibody as provided herein comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1 comprises TYAMH (SEQ ID NO: 24) or TYAMH (SEQ ID NO: 24) except for one or two substitutions.

In some embodiments, an antibody as provided herein comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein the light chain CDR1 comprises RASQSVDTFDYSFLH (SEQ ID NO: 27) or RASQSVDTFDYSFLH (SEQ ID NO: 27) except for one or two substitutions. In some embodiments, the light chain CDR2 comprises RASNLES (SEQ ID NO: 28) or RASNLES (SEQ ID NO: 28) except for one or two substitutions. In some embodiments, the light chain CDR3 comprises QQSNQDPYT (SEQ ID NO: 29) or QQSNQDPYT (SEQ ID NO: 29) except for one or two substitutions.

In some embodiments, the VH of an antibody as provided herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the VL of an antibody as provided herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown in SEQ ID NO: 15. In some embodiments of the invention, VH and VL comprise SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

In some embodiments, an antibody provided herein comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. In some embodiments, the light chain comprises SEQ ID NO: 54.

The present invention provides an isolated antibody that specifically binds to TREM-1, comprising

CDR1, CDR2, CDR3 of the heavy chain;

CDR1, CDR2, CDR3 of the light chain; and the constant region of the heavy chain of IgG1, wherein CDR1, CDR2 and CDR3 of the heavy chain comprise TYAMH (SEQ ID NO: 24).

RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) _and DMGIRRQFAY (SEQ ID NO: 26), respectively, wherein CDR1, CDR2 and CDR3 of the light chain comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively, and wherein the constant region of the IgG1 heavy chain comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E and G237A (EU numbering).

The present invention provides an isolated antibody that specifically binds to TREM-1, comprising

CDR1, CDR2, CDR3 of the heavy chain;

CDR1, CDR2, CDR3 of the light chain; and a constant region of the heavy chain of IgG1, wherein the CDR1, CDR2 and CDR3 of the heavy chain comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively, wherein the CDR1, CDR2 and CDR3 of the light chain comprise RASQSVDTFDYSFLH (SEQ ID NO. 27),

RASNLES (SEQ ID NO: 28) _and QQSNQDPYT (SEQ ID NO: 29), respectively, and wherein the constant region of the IgG1 heavy chain comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S and P331S (EU numbering).

The present invention provides an isolated antibody that specifically binds to

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TREM-1 containing

CDR1, CDR2, CDR3 of the heavy chain;

CDR1, CDR2, CDR3 of the light chain; and the constant region of the heavy chain of IgG1, wherein CDR1, CDR2 and CDR3 of the heavy chain comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) with DMGIRRQFAY (SEQ ID NO: 26) _{correspondingly} ^v and and respectively, wherein CDR1, CDR2 and CDR3 of the light chain comprise RASQSVDTFDYSFLH (SEQ ID NO: 27),

RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively, and wherein the constant region of the IgG1 heavy chain comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S and P238S (EU numbering).

The present invention provides an isolated antibody that specifically binds to TREM-1, comprising

CDR1, CDR2, CDR3 of the heavy chain;

CDR1, CDR2, CDR3 of the light chain; and an IgG1 heavy chain constant region, wherein the heavy chain CDR1, CDR2 and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively, wherein the light chain CDR1, CDR2 and CDR3 comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively, and wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131G, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S (EU numbering).

In some embodiments, TREM-1 comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

In some embodiments, an antibody as provided herein has a lower binding affinity for FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof, compared to an antibody comprising a heavy chain that consists of the amino acid sequence set forth in SEQ ID NO: 76 and a light chain that consists of the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the antibody has a binding affinity for FcγRI (CD64) that is at least twofold, at least threefold, at least fourfold, at least fivefold, at least sixfold, at least sevenfold, at least eightfold, at least ninefold, or at least 10fold lower than an antibody comprising a heavy chain that consists of the amino acid sequence presented in SEQ ID NO: 76, and a light chain, which consists of the amino acid sequence presented in SEQ ID NO: 54.

In some embodiments, an antibody provided herein is less immunogenic than an antibody comprising a heavy chain consisting of the amino acid sequence presented in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence presented in SEQ ID NO: 54. In some embodiments, the antibody does not have an agonistic effect on TREM-1 signaling upon binding to TREM-1 and in the absence of a stimulator. In some embodiments, the antibody does not induce proinflammatory cytokine expression in immature dendritic cells (iDCs) when the cells are incubated in the presence of the antibody and in the absence of a stimulator, compared to an antibody comprising a heavy chain consisting of the amino acid sequence presented in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence presented in SEQ ID NO: 54.

In some embodiments, an antibody provided herein blocks the production of a proinflammatory cytokine in a cell upon cell activation in the presence of both the antibody and a stimulant. In some embodiments, the stimulant is a TREM-1 ligand. In some embodiments, the proinflammatory cytokine is selected from the group consisting of IL-6, TNF-α, IL-8, IE-1β, IL-12, chitinase-3-like protein 1 (CHI3L1), and combinations thereof.

In some embodiments, an antibody of the invention binds to human FcRn, cynomolgus monkey FcRn, and/or mouse FcRn in a pH-dependent manner. In some embodiments, an antibody as provided herein has higher thermal stability than a reference antibody comprising a heavy chain consisting of the amino acid sequence as set forth in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as set forth in SEQ ID NO: 54, according to

- 3 046142 measurements using a capillary differential scanning calorimeter (CAP-DSC). In some embodiments, upon heating to 77°C, the state of about 10 to 20%, about 20 to 30% (e.g., 24%), or about 30 to 40% of the antibodies is reversible. In some embodiments, the antibody has a higher melting temperature ( _Tm ) compared to an antibody comprising a heavy chain that consists of the amino acid sequence presented in SEQ ID NO: 76 and a light chain that consists of the amino acid sequence presented in SEQ ID NO: 54.

In some embodiments, an antibody as provided herein 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, the antibody has a viscosity of less than 10 cP (e.g., 9 cP) at a concentration of 130 mg/mL.

In some embodiments, antibody binding to human TREM-1 is characterized by a KD of less than 4 nM (e.g., 3.4 nM), as measured by Biacore. In some embodiments, antibody binding to cynomolgus monkey TREM-1 is characterized by a KD of less than 1 nM (e.g., 0.91 nM), as measured by Biacore.

In some embodiments, the antibody is monomeric as determined by gel filtration-high performance liquid chromatography (Gel Filtration-High Performance Liquid Chromatography) analysis. In some embodiments, the antibody exhibits minimal risk of fragmentation as determined by two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC/MS) analysis or by intact protein mass analysis using liquid chromatography-tandem mass spectrometry (LC/MS). In some embodiments, the antibody has an isoelectric point of 8 to 9 (e.g., 8.75).

In some embodiments, the antibody is stable in a composition comprising histidine, sucrose, arginine, and NaCl. In some embodiments, the antibody is stable for at least 2 months in a composition comprising 20 mM histidine, 150 mM sucrose, 25 mM arginine, and 50 mM NaCl. In some embodiments, the composition has a pH of 6.0 and/or the composition is stored at 4, 25, or 40°C.

The present disclosure also provides bispecific molecules comprising an anti-TREM-1 antibody as described herein linked to a molecule having a second binding specificity.

The present disclosure provides nucleic acids encoding the antibody described herein, vectors containing the nucleic acids, and cells transformed with the vectors.

The present disclosure provides immunoconjugates comprising the anti-TREM-1 antibodies described herein linked to an agent.

The present disclosure provides compositions comprising anti-TREM-1 antibodies or antigen-binding fragments thereof, bispecific molecules or immunoconjugates as described herein, and a carrier. The present disclosure also provides kits comprising anti-TREM-1 antibodies or antigen-binding fragments thereof, bispecific molecules or immunoconjugates as described herein, and instructions for their use.

The present disclosure provides a method for inhibiting TREM-1 activity in an individual in need thereof, comprising administering an anti-TREM-1 antibody, bispecific molecule, nucleic acid, vector, cell, or immunoconjugate as described herein.

The present disclosure provides a method for treating an inflammatory disease or an autoimmune disease in an individual in need thereof, comprising administering an anti-TREM-1 antibody, a bispecific molecule, a nucleic acid, a vector, a cell, or an immunoconjugate as described herein. In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, vasculitis, sepsis, systemic inflammatory response syndrome (SIRS), type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's disease, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, other autoimmune diseases that are the result of

- 4 046142 either acute or chronic inflammation, and any combination thereof. In some embodiments, the method also includes administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an anti-IP-10 antibody or an anti-TNF-α antibody.

Brief description of drawings/figures

The figures show the following.

Figures 1A and 1B show data demonstrating that all mAb 0318 variants bind to human and cynomolgus monkey TREM-1 with affinities similar to those of the parent mAb 0318 (IgG4). Figure 1A shows the binding affinity of the 318-IgG1.3f variant to human (top row) and cynomolgus monkey (bottom row) TREM-1. Figure 1B shows the binding affinity of several different mAb 0318 variants to human TREM-1. The different variants presented 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 mutants). The binding affinity data of mAb 0318 (IgG4 variants A and B) are shown in Fig. 1B for comparison purposes.

Figures 2A and 2B show internalization data for mAb 0318-IgG1.3f after binding to TREM-1 expressed on CD14 ⁺ monocytes at different time points after TREM-1 binding. Figure 2A shows TREM-1 receptor expression at 0, 6, and 24 h after binding. Figure 2B shows TREM-1 receptor expression at 0, 4, and 20 h after binding. Figure 2B also shows data obtained using the TREM26 antibody, which does not compete with 0318 antibody variants for TREM-1 binding. TREM26 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 shows that mAb 0318 variants do not exert agonistic effects on TREM-1 signaling in a BWZ/hTREM-1 reporter cell assay in which the reporter cell line was incubated with (both) or without CHO-CD32 (BWZ 36 only). The antibody variants shown included (i) 318-IgG1.1f (IgG1.1f), (ii) 318-IgG1.3f (gG1.3f'), (iii) 318-IgG4-Aba (FcAba-4), (iv) 318-IgG1-Aba (FcAba-1). MAB 1278, a known agonist of TREM-1 signaling, was used as a positive control antibody (see boxed inset). Antibody 5C8 (isotype control) was used as a negative control.

Figure 4 shows the efficacy of different mAb 0318 variants in blocking TREM-1-mediated proinflammatory cytokine production by various human cells. The mAb 0318 variants shown in Figure 4 inhibited TREM-1-mediated release of proinflammatory cytokines (e.g., TNF-α, IL-6, or IL-8) from various human cell types: PBMCs, monocytes, neutrophils, and whole blood after RBC sedimentation (specifically, the majority of red blood cells were removed according to the RBC sedimentation protocol using dextran according to the procedure described in the Examples). To produce proinflammatory cytokines, cells were stimulated with either plate-immobilized PGRP1 and soluble peptide devoid of TLR2 activity (PGRP+PGN-Ecndss) or phorbol-12-myristate-13acetate (PMA)-stimulated neutrophils expressing PGRP1 (PMA-Stim Neutrophil Endogenous PGRP). Antibody variants shown included (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 particular proinflammatory cytokine was not determined.

Figures 5A and 5B show the efficacy of mAb 0318-IgG1.3f in blocking IL-8 production in whole blood following PGN stimulation with or without PGRP1 in the presence of a NOD2 inhibitor. Figure 5A shows inhibition data obtained by pharmacodynamic analysis of whole blood according to the method described in the examples. Figure 5B shows inhibition data obtained by intracellular cytokine analysis in whole blood according to the method described in the examples.

Figures 6A–6B show the RNA expression levels of chitinase-3-like protein 1 (CHI3L1) (Figure 6A), GE-1β (Figure 6B), and IL-6 (Figure 6C) in whole blood stimulated with different concentrations of 0318-IgG1.3f mAb (0–1 nM). Whole blood was collected from three different donors (no. 126, 290, and 322) and stimulated with soluble PGRP1 and soluble peptide glycan lacking TLR2 activity (soluble PGRP+ soluble PGN-Ecndss). In each of Figures Figures 6A-6B show RNA expression levels (y-axis) as both % inhibition (right column) and AAC't (the difference between the reference gene level and the gene level in the test sample) (left column). The x-axis shows different concentrations of the 0318-IgG1.3f antibody.

Figure 7 shows the viscosity-concentration profile of two antibody variants, 318-IgG1.1f (squares) and 318-IgG1.3f (circles). The viscosity profile was determined based on the dilution scheme using a Rheosense m-VROC dynamic viscometer using a 3-point shear force sweep for each point at a constant temperature. The solid line represents the curve

- 5 046142 obtained by nonlinear data approximation using the best-fit method. The dotted line indicates the maximum viscosity level acceptable for efficiency.

Figure 8 presents data demonstrating that both antibody variants 318-IgG1.1f and 318-IgG1.3f had a low to moderate 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.

Figures 9A and 9B show data demonstrating that all mAb 0318 variants have the ability to bind to FcRn (human (black), cynomolgus monkey (white), and mouse (gray)) in a pH-dependent manner. Figure 9A shows FcRn binding data as % Rmax (maximum ability to bind to FcRn). Figure 9B shows FcRn binding data as sensorgrams. The 0318 antibody variants shown include (i) IgG1-Aba, (ii) IgG4-Aba, (iii) IgG1.1f, and (iv) IgG1.3f. Data for mAb 0318 (IgG4) are also shown for comparison.

Figures 10A and 10B show data demonstrating that all mAb 0318 variants have reduced binding affinity to one or more human FcyRs (namely, CD64, CD32a-H131 variant, CD32a-R131 variant, CD32b, CD16a-V158 variant, and CD16B-NA2 variant). Figure 10A shows binding affinity data as % Rmax (maximum FcyR binding affinity). Figure 10B shows sensorgrams. The 0318 variants shown include (i) IgG1-Aba, (ii) IgG4-Aba, (iii) IgG1.1f, and (iv) IgG1.3f. For comparison purposes, data for mAb 0318 (IgG4) are shown. The antibody lF4-hIgG1, which is known to bind to several FcyRs, was used as a control.

Figures 11A-11I show data demonstrating that antibody 0318 variants by themselves (i.e., in the absence of stimulus) are not agonistic, as evidenced by measurements of the release of various proinflammatory cytokines (e.g., IL-6, TNF-α, and IL-12) when cultured with immature dendritic cells. Figures 11A, 11B, and 11C show the amounts of IL-6 produced by immature dendritic cells isolated from donors D179, D276, and D341, respectively. Figures 11D, 11E, and 11E show the amounts of TNF-α produced by immature dendritic cells isolated from donors D179, D276, and D341, respectively. Figures 11G, 11H, and 11I show the amounts of IL-12 produced by 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 antibody doses (μg/ml) are given in parentheses. Immature dendritic cells were cultured with anti-TREM-1 antibody variants in the presence and absence of CHO cells expressing CD32a on the membrane (to stimulate Fc cross-linking). PGRP+PGN, CD40L trimer (Trimer), and Pfizer anti-CD40 agonist antibody (CP870) were used as positive controls. An isotype antibody (CHIL-6) was used as a negative control. The dotted line shows the lower limit of detection of the assay.

Figure 12 shows the pharmacokinetic characteristics of the mAb variant 0318-IgG1.3f in cynomolgus monkeys following a single subcutaneous dose 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 shows a target-mediated drug distribution (TMDD) diagram used to describe pharmacokinetic (PK), pharmacodynamic (PD), and receptor occupancy (RO) data in cynomolgus monkeys.

Figures 14A and 14B show the total TREM-1 receptor density on monocytes (Figure 14A) and granulocytes (Figure 14B) following administration of a single dose of the cynomolgus mAb variant 0318-IgG1.3f to monkeys (subcutaneously (sc) or intravenously (iv)) injected. TREM-1 receptor density is presented as a percentage of the pre-antibody TREM-1 receptor density. Each animal was administered one of the following doses: (a) 0.1 mg/kg (sc) (n=4) (A), (b) 0.5 0.1 mg/kg (n=4) (sc) (B), (c) 2 0.1 mg/kg (n=3) (sc) (C), (d) 2 0.1 mg/kg (n=3) (iv) (D), or (f) 10 0.1 mg/kg (n=4) (sc) (E). Data are presented as mean ± standard deviation.

Figures 15A and 15B show TREM-1 receptor occupancy data on monocytes (Fig. 15A) and granulocytes (Fig. 15B) following a single dose of mAb variant 0318-IgG1.3f in cynomolgus monkeys. Receptor occupancy data are presented as a percentage of the total number of 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) (D), or (e) 10 mg/kg (n=4) (sc) (E). Data are presented as mean ± standard deviation. The dotted line corresponds to 85% receptor occupancy.

Figures 16A and 16B show experimentally determined (open circles) and model-predicted (solid line) PK (upper left panel), PD (upper right panel), RO (lower left panel), and total surface expression level of TREM-1 receptor (lower right panel) after a single dose of mAb variant 0318-IgG1.3f in cynomolgus monkeys, using a 2-compartment PK model with TMDD for theoretical estimation.

- 6 046142 in the central compartment. Fig. 16A shows the data obtained after a single subcutaneous dose of 0.1 mg/kg antibody was administered to each monkey. Fig. 16B shows the data obtained after a single subcutaneous dose of 10 mg/kg antibody was administered to the animals.

Detailed description of the invention

To facilitate understanding of this description, definitions of some concepts are first provided. Additional definitions are provided in the detailed description.

It should be understood that the use of an element in the singular also implies its use in the plural; for example, the term "nucleotide sequence" should be understood to refer to one or more nucleotide sequences. Thus, the terms "one (or more), one or more, and at least one" may be used interchangeably in the context of the present description.

Furthermore, it should be understood that in the context of the present description and/or refers specifically to each of the two specified characteristics or components in combination with each other or separately.

Thus, it should be understood that the concept and/or, when used in such an expression as A and/or B in the context of the present description, includes A and B, A or B, A (individually) and B (individually). Similarly, it should be understood that the concept and/or, when used in such an expression as A, B and/or C, includes the following options: 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 (individually); B (individually) and C (individually).

It should be understood that when the expression “comprising” is used in relation to any aspects in this description, it also includes similar aspects described in another way using the concepts “consisting of” and/or “essentially consisting of”.

Unless otherwise defined, all technical and scientific terms used in this specification have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., CRC Press, 2002; The Dictionary of Cell and Molecular Biology, 3rd ed., Academic Press, 1999; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, Oxford University Press, 2000 are well-known dictionaries in which one skilled in the art can find many of the terms used in this specification.

Units, prefixes, and symbols are used in the International System of Units (SI). Numerical ranges include the numbers defining the range boundaries. Unless otherwise noted, nucleotide sequences are written left to right in a 5'^3' orientation. Amino acid sequences are written left to right in an amino-to-carboxyl-terminal orientation. The headings in this specification do not limit the scope of the various aspects of the invention, which is defined by the application as a whole. Accordingly, concepts defined immediately below in the text are more fully defined by the application as a whole.

The term "about," as used herein, means approximately, roughly, around, or within the range of this. When the term "about" is used in relation to a numerical range, it modifies the stated range by extending the boundaries of the numerical values above and below the stated values. In general, the term "about" can modify a numerical value upward or downward (making it higher or lower) by a difference of, for example, 10%.

Myeloid cell-derived triggering receptor 1 (also known as TREM1, TREM-1, and CD354) refers to a receptor expressed on monocytes, macrophages, and neutrophils. The primary ligand for TREM-1 is peptide glycan recognition protein 1 (PGLYRP1), a member of the peptide (PGN)-binding protein (PGRP) family. Upon activation, TREM-1 associates with the ITAM-containing signaling adaptor protein, DAP12. Downstream signaling may involve activation of the transcription factor NFAT, resulting in upregulation of proinflammatory cytokine production. TREM-1 encompasses any variants or isoforms of TREM-1 naturally expressed by cells. Therefore, in some embodiments, the antibodies provided herein may cross-react with TREM-1 from non-human species (e.g., TREM-1 from cynomolgus monkeys).

Three TREM-1 isoforms 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 the transmembrane domain, the cytoplasmic domain, and part of the extracellular domain. Amino acid residues 138-150 also differ from the canonical sequence described above.

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The amino acid sequences of the three known isoforms of human TREM-1 are shown below.

(A) Human TREM-1 isoform 1 (accession number NP_061113.1; SEQ ID NO: 1; encoded by the nucleotide sequence having the accession number NM_018643; SEQ ID NO: 4): MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGOTLDVKCDYTLEKFASSQ

KAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVE DSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKA LCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKS LVFSVLFAVTLRSFVP (signal sequence underlined);

(b) Human TREM-1 isoform 2 (accession number NP_001229518.1; SEQ ID NO: 2; encoded by the nucleotide sequence having the accession number NM_001242589; SEQ ID NO: 5):

MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGOTLDVKCDYTLEKFASSO

KAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVE DSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKA LCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRYSFQVPGPLVWTLSPLF PSLCAERM (signal sequence underlined);

(B) Human TREM-1 isoform 3 (accession number NP_001229519; SEQ ID NO: 3; encoded by the nucleotide sequence having the accession number NM_001242590; SEQ ID NO: 6):

MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGOTLDVKCDYTLEKFASSO

KAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVE

D SGLYQC VIYQPPKEPHMLFDRIRL VVTKGFRC STLSF S WLVD S (signal sequence underlined).

The TREM-1 protein from cynomolgus monkeys (accession number XP_001082517; SEQ ID NO: 7) is predicted to have the following amino acid sequence:

MRKTRLWGLLWMLFVSELRATTELTEEKYEYKEGOTLEVKCDYALEKYANSR

KAWQKMEGKMPKILAKTERPSENSHPVQVGRITLEDYPDHGLLQVQMTNLQVE DSGLYQCVIYQHPKESHVLFNPICLVVTKGSSGTPGSSENSTQNVYRTPSTTAKA LGPRYTSPRTVTQAPPESTVVVSTPGSEINLTNVTDIIRVPVFNIVIIVAGGFLSKS LVFSVLFAVTLRSFGP (signal sequence underlined).

The present invention relates to antibodies that specifically bind to and block TREM-1 function. The antibodies block TREM-1 function by reducing/blocking TREM-1 activation and downstream signaling.

The anti-TREM-1 antibodies provided herein block TREM-1 signaling through one or a combination of different mechanisms, blocking TREM-1 directly or indirectly. In one embodiment, the antibodies prevent the natural ligand for TREM-1, peptide glycan recognition protein 1 (PGLYRP1), from forming a functional complex with TREM-1. In another embodiment, the antibodies block TREM-1 by preventing individual TREM-1 molecules from forming dimers or multimers. In some embodiments, the reduction or prevention of TREM-1 dimerization or multimerization is achieved by anti-TREM-1 antibodies that are capable of binding to a portion of TREM-1 that would otherwise be at the interface in a TREM-1 dimer, thereby preventing the association of individual TREM-1 molecules with one another. In other embodiments of the invention, the reduction or prevention of TREM-1 dimerization or multimerization is caused by antibodies to TREM-1 that interfere with the interaction of TREM-1 with its ligand.

In some embodiments, anti-TREM-1 antibodies can block PGLYRP1-induced TREM-1 activation. PGLYRP1, a highly conserved 196-amino acid protein consisting of a signal peptide and a peptide-binding domain, is expressed on neutrophils and released upon their activation. The amino acid sequence of PGLYRP1 (Accession No. NP_005082.1; SEQ ID NO: 8) is shown below:

- 8 046142

MSRRSMLLAWALPSLLRLGAAQETEDPACCSPIVPRNEWKALASECAOHLSLPL RYVVVSHTAGSSCNTPASCQQQARNVQHYHMKTLGWCDVGYNFLIGEDGLVY EGRGWNFTGAHSGHLWNPMSIGISFMGNYMDRVPTPQAIRAAQGLLACGVAQ GALRSNYVLKGHRDVQRTLSPGNQLYHLIQNWPHYRSP (signal sequence underlined).

Thus, in some embodiments, the anti-TREM-1 antibodies of the present invention down-regulate or block the release of pro-inflammatory cytokines from myeloid cells (e.g., dendritic cells and monocytes). In some embodiments, the 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 cells, and/or a reporter cell, as demonstrated herein.

Although the controlled release of proinflammatory cytokines in response to foreign antigens can be beneficial (e.g., by eliciting an effective adaptive immune response), excessive release of proinflammatory cytokines can have serious consequences. For example, one common and toxic complication observed with the in vivo administration of certain antibodies against immune cell surface receptors (e.g., anti-human CD3 antibodies, such as OKT3) is cytokine release syndrome (CRS), which is associated with the excessive release of various cytokines (e.g., TNF-alpha, IFN-gamma, and IL-2) into the bloodstream. CRS can result from the simultaneous binding of antibodies to their cognate antigen (e.g., CD3 on T cells) (via the antibody variable region) and Fc receptors (e.g., FcyR) and/or complement receptors (via the antibody constant region) on accessory cells (e.g., antigen-presenting cells). This interaction leads to cell activation (e.g., T cells and/or accessory cells) and the release of various cytokines, which induce a systemic inflammatory response characterized by hypotension, hypothermia, and chills. Other symptoms of CRS include fever, chills, nausea, vomiting, and shortness of breath.

In one embodiment, the anti-TREM-1 antibodies described herein, in addition to blocking PGLYRP1-induced proinflammatory cytokine production, result in a reduction in the incidence of, or the elimination of, cytokine release syndrome after administration to an individual in need thereof. In some embodiments, the anti-TREM-1 antibodies do not induce the expression of proinflammatory cytokines by cells (e.g., dendritic cells) when the cells are incubated in the presence of the antibody alone, compared to an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the anti-TREM-1 antibodies have reduced binding to one or more FcyRs, which contributes to a reduction in the incidence of CSR.

In some embodiments, the anti-TREM-1 antibodies of the present invention bind to both human TREM-1 and TREM-1 from other species. Thus, as used herein, the term TREM-1 includes any naturally occurring form of TREM-1, which may be from any suitable organism. For example, a TREM-1 suitable for use herein may be a vertebrate TREM-1, such as a mammalian TREM-1, such as a primate (such as a human, chimpanzee, cynomolgus monkey, or rhesus macaque); a rodent (such as a mouse or rat), a lagomorph (such as a rabbit), or an artiodactyl (such as a cow, sheep, pig, or camel). In some embodiments, the TREM-1 has the sequence of SEQ ID NO: 1 (human TREM-1, isoform 1). TREM-1 may represent a mature form of TREM-1, such as a TREM-1 protein that has undergone post-translational processing in the respective cell. Such a mature TREM-1 protein may, for example, be glycosylated. TREM-1 may represent the full-length TREM-1 protein.

In some embodiments, the anti-TREM-1 antibodies of the present invention are monoclonal antibodies in that they are obtained directly or indirectly from a single B lymphocyte clone. In some embodiments, the anti-TREM-1 antibodies are obtained, screened, and purified using methods such as those described in the Examples of International Patent Application Publication WO 2013/120553. In general, the method consists of immunizing a suitable mouse, such as a TREM-1 knock-out (KO) or TREM-1/TREM-3 mouse, with TREM-1, a TREM-1-expressing cell, or a combination thereof. In another embodiment, the anti-TREM-1 antibodies are polyclonal antibodies in that they are a mixture of the monoclonal antibodies described herein.

In some embodiments, the anti-TREM-1 antibodies provided herein

- 9 046142 further described, are recombinantly expressed in prokaryotic or eukaryotic cells. In some embodiments, the prokaryotic cell is E. coli. In particular embodiments, the eukaryotic cell is a yeast, insect, or mammalian cell, such as a cell obtained from an organism that is a primate (such as a human, chimpanzee, cynomolgus monkey, or rhesus macaque); a rodent (such as a mouse or rat), a lagomorph (such as a rabbit), or an artiodactyl (such as a cow, sheep, pig, or camel). Suitable mammalian cell lines include, but are not limited to, HEK293 cells, CHO cells, and HELA cells. The anti-TREM-1 antibodies described herein can also be produced by other methods known to one of skill in the art, such as phage display or yeast display. Once produced, the antibodies can be screened for binding to, for example, full-length TREM-1 or its mutants using the methods described in the Examples section of International Patent Application Publication WO 2013/120553.

The term "antibody" as used herein refers to a protein derived from the immunoglobulin germline sequence that has the ability to specifically bind to an antigen (TREM-1) or a portion thereof. This includes full-length antibodies of a class or subclass (namely, IgA, IgE, IgG, IgM, and/or IgY) and any single-chain antibodies or fragments. An antibody that specifically binds to an antigen or a portion thereof may bind only to that antigen or a portion thereof, or it may bind to a limited number of homologous antigens or portions thereof. Full-length antibodies typically contain at least four polypeptide chains: two heavy (H) chains and two light (L) chains, which are linked to each other by disulfide bonds.

One of the immunoglobulin subclasses of pharmaceutical interest is the IgG family. In humans, the IgG class can be divided into four subclasses: IgG1, IgG2, IgG3, and IgG4, based on the sequences of the constant regions of their heavy chains. Light chains can be subdivided into kappa and lambda types based on differences in their sequence composition. IgG molecules consist of two heavy chains linked to each other by two or more disulfide bonds and two light chains, each attached to the heavy chain by a disulfide bond. A heavy chain may contain a heavy chain variable region (VH) and up to three heavy chain constant regions (CH): CH1, CH2, and CH3. A light chain may contain a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), flanked by more conserved regions called framework regions (FRs). The VH and VL regions typically consist of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy and light chains form the binding domain, which has the ability to interact with antigen, while the constant region of the antibody can mediate the binding of immunoglobulin to tissues or host factors, including (but not limited to) various cells of the immune system (effector cells), Fc receptors, and the first component (C1q) of the classical complement system. The antibodies provided in the present invention may be in isolated form. The term "isolated antibody" refers to an antibody that has been separated and/or separated from other component(s) of the environment in which it was produced and/or purified from a mixture of components present in the environment in which it was produced. Specific antigen-binding fragments of antibodies may be considered in the context of the present invention if it has been demonstrated that fragments of a full-length antibody can perform the antigen-binding function of the antibody.

The term "antigen-binding fragment of an antibody" refers to one or more fragments of an antibody that retain 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 one arm of an antibody), single-chain Fv (scFv; see, e.g., Bird et al., Science, 1988, 242:42S-426; Huston et al., 1988, PNAS, 85:5879-5883), dsFv, Fd (typically the VH and CH1 domains), and dAb (typically the VH domain) fragments; VH, VL, VhH, and VNAR domains; monovalent molecules containing one VH and one VL chain; minibodies, diabodi, triabodi, tetrabodies, and kappa-all-antibodies (see, e.g., I11 et al., Protein Eng., 10:949-957 (1997)); camel IgG; IgNAR; and one or more isolated CDRs or a functional paratope, wherein the isolated CDRs or antigen-binding residues or polypeptides may be associated or linked to each other to form a functional antibody fragment. Various types of antibody fragments are described or reviewed in, e.g., Holliger and Hudson, Nat. Biotechnol., 2S:1126-1136 (2005); in International Patent Application Publication No. WO 2005/040219 and in US Patent Publications Nos. 2005/0238646 and 2002/0161201. These antibody fragments can be produced using conventional methods known to those skilled in the art, and the fragments can be screened for their suitability in the same manner as

- 10 046142 and intact antibodies.

The term "human antibody" (huMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The anti-TREM-1 antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, are grafted onto human framework sequences. The terms "human antibodies" and "fully human antibodies" are used synonymously.

The term "humanized antibody" refers to a human/non-human chimeric antibody that contains one or more sequences (CDR regions or portions thereof) derived from immunoglobulin of a non-human species. A humanized antibody is a human immunoglobulin (recipient antibody) in which at least residues from the hypervariable region of the recipient antibody are replaced with residues from the hypervariable region of an antibody of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, sequence composition, and functionality. In some cases, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. An example of such a modification is the introduction of one or more so-called back mutations, which typically represent amino acid residues derived from the donor antibody. Humanization of an antibody can be accomplished using recombinant techniques known to those skilled in the art (see, e.g., Antibody Engineering, Methods in Molecular Biology, Vol. 248, ed. Benny K. C. Lo). A suitable human recipient framework for the variable domain of either the light or heavy chain can be identified, for example, based on sequence homology or structural homology. Alternatively, fixed recipient frameworks can be used, for example, based on structural information, biophysical, and biochemical properties. Recipient frameworks can be of germline or mature antibody origin. CDR regions from a donor antibody can be transferred by CDR grafting. A humanized antibody with a grafted CDR can be further optimized, for example, for affinity, functionality, and biophysical properties, by identifying critical framework positions where reintroduction (backmutation) of an amino acid residue from the donor antibody favorably impacts the properties of the humanized antibody. In addition to using backmutations obtained using the donor antibody, a humanized antibody can be created by introducing germline residues into CDRs or framework regions, eliminating immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

Furthermore, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. Such modifications are made to further improve the antibody's characteristics. In general, a humanized antibody should contain at least one, typically two, variable domains in which all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin and in which all or substantially all of the FR residues represent residues from a human immunoglobulin sequence. A humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The term "derivative of a humanized antibody" refers to any modified form of a humanized antibody, such as a conjugate comprising the antibody and another agent or another antibody.

The term recombinant human antibody as used herein includes all human antibodies that are produced, expressed, created, or isolated using recombinant techniques, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchomosomal for human immunoglobulin genes or derived from a hybridoma;

(b) antibodies isolated from a host cell transformed to express the antibody, such as from a transfectoma;

(c) antibodies isolated from a recombinant combinatorial library of human antibodies; and (d) antibodies produced, expressed, generated, or isolated by any other method that involves splicing of human immunoglobulin gene sequences with other DNA sequences.

- 11 046142

Such recombinant human antibodies contain variable and constant regions based on specific human germline immunoglobulin sequences but incorporate subsequent transformations and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg, Nature Biotech., 23(9), 2005, pp. 1117–1125), variable regions contain an antigen-binding domain encoded by various genes that is transformed to produce an antibody specific for a foreign antigen. In addition to transformation, the variable region can be further modified by multiple single amino acid substitutions (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region can be altered to provide an additional response to the antigen (i.e., by isotype switching). Thus, nucleic acid molecules that encode immunoglobulin light chain and heavy chain polypeptides that have been reshaped and somatically mutated to respond to an antigen may not have sequence identity with the original nucleic acid molecules, but they must be substantially identical or similar (i.e., at least 80% identical).

The term chimeric antibody refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

In one embodiment, the anti-TREM-1 antibodies described herein are IgG antibodies. As used herein, an IgG antibody, such as human IgG1, in some 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, an IgG1 anti-TREM-1 antibody consists of two heavy chains (HC) and two light chains (LC), where the two heavy chains and the light chains are linked by the same number and arrangement of disulfide bridges as in a naturally occurring IgG1 antibody (unless the antibody is mutated to modify the disulfide bridges).

As used herein, the term "isotype" refers to the class of antibodies (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) that are encoded by the heavy chain constant region genes.

The term "allotype" refers to naturally occurring variants within a particular isotypic group, where the variants differ by multiple amino acids (see, e.g., Jefferis et al., mAbs 1, 2009, p. 1). The anti-TREM-1 antibodies provided herein can be of any allotype. In some embodiments, the anti-TREM-1 antibodies have an IgG1.3f' allotype that comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, and G237A (EU numbering), compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In other embodiments, the anti-TREM-1 antibodies have an IgG1.1f allotype that comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S (EU numbering), compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In specific embodiments, the anti-TREM-1 antibodies have an IgG1-Aba allotype that comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S (EU numbering), compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In other embodiments, the anti-TREM-1 antibodies have an IgG4-Aba allotype that comprises 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, the 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 (EU numbering), compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 9).

The expressions "antibody recognizing an antigen" and "antibody having specificity for an antigen" are used herein interchangeably with the term "antibody that specifically binds to an antigen."

In the context of the present description, the term "isolated antibody" is intended to refer to an antibody that is separated and/or separated from other component(s) in the environment in which it is produced and/or purified from a mixture of components present in the environment in which it is produced.

The term effector function refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the biochemical event that results from it. Examples of effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcYR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and cell surface receptor downregulation.

- 12 046142 function (e.g., B cell receptor; BCR). Such effector functions typically require the combination of an Fc region with a binding domain (e.g., the variable domain of an antibody). In one embodiment, the anti-TREM-1 antibodies provided herein comprise Fc regions that do not bind one or more FcγRs and therefore do not have effector function (i.e., are not effectors).

An Fc receptor, or FcR, is a receptor that binds to the Fc region of immunoglobulin. FcRs that bind to antibody in the form of IgG include receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating receptors (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory receptor (FcyRIIB). Diverse properties of human FcyRs are known in the art. Most naturally occurring effector cell types are characterized by coexpression of one or more activating FcyRs and inhibitory FcyRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not inhibitory FcyRIIB in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the types of activating Fc receptors to which it binds.

The term Fc region (the region representing the crystallizable fragment), or Fc domain, or Fc refers to the C-terminal region of the antibody heavy chain that mediates binding of immunoglobulin to tissues or host 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. Thus, the Fc region comprises the constant region of the antibody minus the first domain of the immunoglobulin constant region (e.g., CH1 or CL).

In IgG, the Fc region contains the CH2 and CH3 immunoglobulin domains and a hinge between the CH1 and CH2 domains. Although the definition of the boundaries of the Fc region of the immunoglobulin heavy chain may vary, for the purposes of the present disclosure, the Fc region of the human IgG heavy chain is defined as the segment extending from amino acid residues D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxyl terminus of the heavy chain (numbering according to the EU Cabot index). The CH2 domain of the Fc region of human IgG extends from amino acid 237 to amino acid 340, and the CH3 domain is located at the C-terminal side of the CH2 domain of the Fc region, i.e., it 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, including any allotypic variants, or an Fc variant (e.g., a non-naturally occurring Fc). The term Fc may also refer to the region considered individually or in the context of an Fc-containing protein polypeptide, such as a binding protein containing an Fc region, which is also referred to as an Fc fusion protein (e.g., an antibody or an immunoadhesive agent).

A native-sequence Fc region or native-sequence Fc region comprises an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Native-sequence human Fc regions include the native-sequence human IgG1 Fc region;

having a native sequence human IgG2 Fc region;

having a native sequence human IgG3 Fc region; and having a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

The native Fc sequence includes various Fc allotypes (see, e.g., Jefferis et al., mAbs 1, 2009, p. 1).

A variant Fc region sequence or non-naturally occurring Fc region comprises a modification that typically serves to alter one or more functional properties, such as, but not limited to, serum half-life, complement fixation, Fc receptor binding, protein stability, and/or antigen-dependent cellular cytotoxicity or the absence thereof. In some embodiments, the anti-TREM-1 antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or may be modified to alter its glycosylation or to alter one or more functional properties of the antibody. In one embodiment of the invention, the anti-TREM-1 antibody is an IgG1 antibody and carries a modified Fc domain comprising one or more, and possibly all, of the following mutations that result in reduced affinity for certain Fc receptors (L234A, L235E, and G237A) and reduced C1q-mediated complement fixation (A330S and P331S), respectively.

- 13 046142 (residues are numbered according to the EU index).

The terms hinge, hinge domain, hinge region, and hinge region of an antibody refer to the domain of the heavy chain constant region that connects the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (Roux et al., J. Immunol., 1998, 161, p. 4083). The hinge allows varying degrees of flexibility between the binding and effector regions of the antibody and also provides sites for intermolecular disulfide bonding between the constant regions of the two heavy chains. For the purposes of this description, the hinge begins at Glu216 and ends at Gly237 in all IgG isotypes (Roux et al., J. Immunol., 1998, 161, p. 4083). The hinge sequences of wild-type IgG1, IgG2, IgG3, and IgG4 are known in the art (see, for example, International Patent Application Publication No. WO 2017/087678). In one embodiment, the CH1 hinge region of anti-TREM-1 antibodies is modified to alter, for example, increase or decrease, the number of cysteine residues in the hinge region. This approach is described in more detail, for example, in U.S. Patent No. 5,677,425.

The constant region can be modified to stabilize the antibody, for example, to reduce the risk of partitioning a divalent antibody into two monovalent VH-VL fragments. For example, in the constant region of IgG4, residue S228 (residues are numbered according to the EU index) can be mutated to a proline (P) residue to stabilize disulfide bridge formation at the hinge between the heavy chains (see, for example, Angal et al., Mol. Immunol., 30 (1995), pp. 105-108). Antibodies or fragments thereof can also be assessed in terms of their complementarity-determining regions (CDRs). The term complementarity-determining region or hypervariable region, as used herein, refers to the regions of an antibody that contain the amino acid residues involved in antigen binding. A hypervariable region, or CDR, can be identified as the regions characterized by the greatest variability by comparative analysis of the amino acids of the variable domains of an antibody. Databases such as the Kabat database can be used to identify CDRs; for example, CDRs are defined as regions containing amino acid residues 24-34 (CDR1), 50-59 (CDR2), and 89-97 (CDR3) of the variable light chain domain and 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) of the variable heavy chain domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91, 1991, p. 3242). Alternatively, the CDRs may be defined as regions containing residues from the hypervariable loop (residues 26-33 (L1), 50-52 (L2), and 91-96 (L3) in the variable light chain domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the variable heavy chain domain (Chothia and Lesk, J. Mol. Biol., 1987, 196:901-917). Typically, the amino acid residues in this region are numbered using the method described by Kabat et al., supra. Expressions such as Kabot position, Kabot residue, and Kabot as used herein refer to this numbering system for the variable heavy chain and variable light chain domains. When using the Kabot numbering system, the actual linear The amino acid sequence of a peptide may contain fewer or additional amino acids, corresponding to a shortening or the presence of an insertion in the framework region (FR) or CDR of the variable domain. For example, the variable domain of a heavy chain may include amino acid insertions (residues 52a, 52b, and 52c according to Kabot) after residue 52 of CDR H2 and insertional residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabot) after residue 82 of the heavy chain FR. Kabot residue numbers can be determined for a given antibody by comparing the regions of homology of the antibody sequence with a standard Kabot-numbered sequence.

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 determined, for example, by a specific assay designed to identify it. Epitopes can be formed by either contiguous amino acids (usually a linear epitope) or non-contiguous amino acids brought into proximity by tertiary protein folding (usually a conformational epitope). Epitopes formed by contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a subject antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays that test overlapping or continuous peptides (e.g., from TREM-1) to assess reactivity with the subject antibody (e.g., an antibody to TREM-1). Methods for determining the spatial conformation of epitopes include methods known in the art and methods described herein, such as X-ray crystallography, antigen mutation analysis, 2-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols

- 14 046142 in Methods in Molecular Biology, vol. 66, ed. G. E. Morris, 1996).

The term "binds to the same epitope" when applied to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by the method in question. Methods for determining whether antibodies bind to the same epitope on TREM-1 as the antibodies described herein include, for example, epitope mapping methods such as X-ray crystal analysis of antigen:antibody complexes, which provides atomic-level epitope resolution, and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor antibody binding to antigen fragments or mutant variants of the antigen, where loss of binding capacity due to modification of an amino acid residue in the antigen sequence is often considered indicative of the presence of an epitope component. Computational combinatorial methods can also be used for epitope mapping. These methods rely on the ability of the antibody of interest to affinity-based isolate specific short peptides from combinatorial phage display peptide libraries. It is assumed that antibodies with the same VH and VL sequences or the same CDR1, 2, and 3 sequences should bind to the same epitope.

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

As used herein, the terms "specific binding,""selectivebinding,""selectivelybinds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, antibody binding is characterized by (i) a dissociation equilibrium constant (K _D ) of less than about 10 ^-7 M, such as less than about 10 ^-8 , 10 ^-9 , or 10 ^-10 M, or even less, when determined, for example, using surface plasmon resonance (SPR) technology with a BIACORE® 2000 device using a predetermined antigen, such as recombinant human TREM-1, as the analyte and the antibody as the ligand, or a Scatchard assay of antibody binding to antigen-positive cells; and (II) a binding affinity for a predetermined antigen that is at least two times greater than its binding affinity for a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen.

Thus, an antibody that specifically binds to human TREM-1 is an antibody whose binding to soluble or cell-bound human TREM-1 is characterized by a KD of ^10-7 M or less, such as less than about ^10-8 , ^10-9 , or ^10-10 M, or even less. An antibody that cross-reacts with cynomolgus monkey TREM-1 refers to an antibody whose binding to cynomolgus monkey TREM-1 is characterized by a KD of ^10-7 M or less, such as less than about ^10-8 , ^10-9 , or ^10-10 M, or even less. In some embodiments, such antibodies that do not cross-react with TREM-1 from non-human species are characterized by a substantially undetectable level of binding to these proteins in a standard binding assay.

As used herein, the term binding specificity refers to the interaction of a molecule, such as an antibody or antibody fragment, with a single antigen or a limited number of highly homologous antigens (or epitopes). Conversely, antibodies that specifically bind to TREM-1 lack the ability to bind to dissimilar molecules. The antibodies of the invention may lack the ability to bind to Nkp44, a protein related to p44 on natural killer cells.

The specificity of the interaction and the equilibrium constant of the binding reaction can be determined directly using well-known methods. Standard assays for assessing

- 15 046142 The ability of ligands (such as antibodies) to bind to their targets is known in the art and includes, for example, ELISA, Western blotting, RIA, and flow cytometry analysis. The binding kinetics and affinity of an antibody can also be assessed using standard assays known in the art, such as SPR.

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

In the context of this description, the term "group" is defined in relation to the reference antibody. If a second antibody cannot bind to an antigen simultaneously with the reference antibody, the second antibody is considered to belong to the same group as the reference antibody. In this case, the reference antibody and the second antibody bind to the same portion of the antigen and are referred to as competing antibodies. If the second antibody is capable of binding to the antigen simultaneously with the reference antibody, the second antibody is considered to belong to a different group. In this case, the reference antibody and the second antibody do not compete for binding to the same portion of the antigen and are referred to as non-competing antibodies.

Antibody grouping does not provide direct information about the epitope. For competing antibodies, i.e., antibodies within the same group, the epitopes may be identical, overlapping, or even different. The latter occurs when a reference antibody, bound to its epitope on the antigen, occupies the space required for the second antibody to interact with its epitope on the antigen (steric hindrance). Non-competing antibodies typically have different epitopes.

In the context of this description, the term binding affinity refers to the measurement of the strength of a non-covalent interaction between two molecules, such as an antibody or its fragment and an antigen. Binding affinity is used to describe univalent interactions (the activity inherent to molecules).

The binding affinity of two molecules, for example, an antibody or its fragment and an antigen, due to a monovalent interaction, can be quantitatively assessed by determining the equilibrium constant of the dissociation reaction (KD). In turn, KD can be determined by measuring the kinetic characteristics of the process of complex formation and dissociation, for example, using the SPR method. The rate constants corresponding to the association and dissociation of a monovalent complex are designated as the association reaction rate constant _ka (or kon) and the dissociation reaction rate constant k _d (or k _on ), respectively. K _D is related to ka and kd by the equation K _D = k _d /ka. In accordance with the above definition, when comparing the binding affinities of different antibodies to a given antigen, the binding affinities associated with different molecular interactions can be compared, for example, by comparing the KD values for individual antibody/antigen complexes.

In the context of the present description, the term "high affinity" for an IgG antibody for a target antigen refers to an antibody having a _KD value of ^10-8 M or less, ^10-9 M or less, or ^10-10 M or less. However, high binding affinity may differ from the specified level for other antibody isotypes. For example, high binding affinity for the IgM isotype refers to an antibody having a _KD value of ^10-10 M or less or ^10-8 M or less.

The term EC ₅₀ in the context of an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof refers to the concentration of the antibody or antigen-binding fragment thereof that induces a response that is 50% of the maximal response, i.e., is midway between the maximal response and the baseline level.

In the context of this description, the term "naturally occurring" applies to an object, reflecting the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses), which can be isolated from a natural source and which has not been intentionally modified by humans in a laboratory, is a naturally occurring sequence.

- 16 046142

The term "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 contain modifications such as (but not limited to) glycosylation, phosphorylation, or disulfide bond formation. A protein may contain one or more polypeptides.

In the context of this description, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and it may be cDNA.

Conservative amino acid substitutions refer to the replacement 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 this field. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predetermined nonessential 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 conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem., 32 (1993), pp. 1180-1187; Kobayashi et al., Protein Eng., 12(10), 1999, pp. 879-884; and Burks et al., Proc. Natl. Acad. Sci., USA, 94 (1997), pp. 412-417).

As applied to nucleic acids, the term "substantial homology" means that, in two nucleic acids or the sequences denoting them, when optimally aligned and compared, taking into account appropriate nucleotide insertions or deletions, at least about 80% of the nucleotides, at least about 90 to 95%, or about 98 to 99.5% of the nucleotides are identical. Alternatively, substantial homology occurs when segments hybridize under selective hybridization conditions with the complement of the strand.

With respect to polypeptides, the term substantial homology means that in two polypeptides or the sequences denoting them, when optimally aligned and compared taking into account appropriate amino acid insertions or deletions, at least about 80% of the nucleotides, at least about 90 to 95%, or about 98 to 99.5% of the amino acids are identical.

The percentage identity of two sequences is a function of the number of identical positions present in the sequences (i.e., % homology = number of identical positions/total number of positions/100), taking into account the number of gaps and the length of each gap that must be introduced to achieve an optimal alignment of the two sequences. Sequence comparison and determination of the percentage identity of two sequences can be performed using a mathematical algorithm, as described below in the non-limiting examples.

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

The nucleotide and protein sequences provided herein may also be used as a query sequence to search public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST (version 2.0) programs described in Altschul et al., J. Mol. Biol., 215 (1990), pp. 403-410. BLAST searches for nucleotide sequences can be performed using NBLAST, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules provided herein. BLAST searches for protein sequences can be performed using XBLAST, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules provided herein. Gap-guided alignments for comparison purposes can be performed using

- 17 046142 Run the Gapped BLAST program according to the procedure described in Altschul et al., Nucleic Acids Res., 25(17), 1997, pp. 3389–3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see worldwideweb.ncbi.nlm.nih.gov).

Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially purified form. Nucleic acid is isolated or considered substantially pure if it is purified from other cellular components or other contaminants, such as other nucleic acids present in the cell (e.g., other parts of the chromosome) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other well-known techniques (see Current Protocols in Molecular Biology, F. Ausubel et al., eds., Greene Publishing and Wiley Interscience, New York, 1987).

Nucleic acids, such as cDNA, can be mutated using standard techniques to produce gene sequences. For coding sequences, these mutations may affect the amino acid sequence, if desired. Specifically, DNA sequences that are substantially homologous to or derived from native V, D, and J gene segments, constant sequences, switches, and other similar sequences described herein (where "derived" means that a sequence is identical to another sequence or is derived from one by modification) can be considered.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a plasmid, which refers to a loop of circular double-stranded DNA into which additional DNA segments can be inserted by ligation. Another type of vector is a viral vector, in which case additional DNA segments can be inserted by ligation into the viral genome. Some vectors are capable of autonomously replicating within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal vectors for mammalian cells). Other vectors (e.g., non-episomal vectors for mammalian cells) can be integrated into the host cell genome by introduction into the host cell, where they replicate alongside the host genome. Additionally, some vectors have the ability to direct the expression of genes to which they are functionally linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein, as the plasmid is the most widely used form of vector. However, the term also includes other forms of expression vectors, such as viral vectors (e.g., retroviruses, adenoviruses, and replication-deficient adeno-associated viruses), which perform equivalent functions.

As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the specific cell in question but also to the progeny of such a cell. Because certain modifications may arise in subsequent generations, either through mutations or environmental influences, such progeny may not actually be identical to the parent cell, but they still fall within the scope of the term "host cell" as used herein.

In the context of this description, the term "linked" refers to the association of two or more molecules. The linkage may be covalent or non-covalent. The linkage may also be genetically determined (i.e., obtained by recombinant fusion). Such linkages can be created using a wide variety of methods accepted in the field, such as chemical conjugation and protein production by recombination.

As used herein, the term "administration" refers to the physical introduction of a composition containing a therapeutic agent into an individual's body using any of the various delivery methods and systems known to those skilled in the art. Various routes of administration of the anti-TREM-1 antibody described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as administration by injection or infusion. The term "parenteral administration" as used herein refers to routes of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous,

- 18 046142 intravascular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibody provided herein can be administered by a non-parenteral route, such as topical administration, epidermal administration or mucosal administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administration can also be carried out, for example, once, several times and/or over one or more extended periods of time.

As used herein, the terms "inhibits" or "blocks" (e.g., when referring to inhibiting/blocking the binding of a TREM-1 ligand to TREM-1 on cells) are used interchangeably and include both partial and complete inhibition/blocking. In some embodiments, an anti-TREM-1 antibody inhibits the binding of a TREM-1 ligand to TREM-1 by at least about 50%, such as about 60, 70, 80, 90, 95, 99, or 100%, as assessed by the methods provided later herein. In some embodiments, an anti-TREM-1 antibody inhibits the binding of a TREM-1 ligand to TREM-1 by no more than 50%, such as about 40, 30, 20, 10, 5, or 1%, as assessed by the methods provided later herein.

The terms "treat," "treatment," and "treatment" as used herein refer to any type of intervention or process performed on an individual, or the administration of an active substance to an individual, with the purpose of reversing, alleviating, reducing, inhibiting, or slowing, or preventing the progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical marker associated with a disease, or improving overall survival. Treatment may be performed on an individual with a disease or an individual without a disease (e.g., for prophylaxis).

The term "effective dose" or "effective dosage" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. A therapeutically effective amount or therapeutically effective dose of a drug or therapeutic agent is an amount of the drug that, when administered alone or in combination with another therapeutic agent, enhances disease regression, which is manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or the prevention of deterioration or disability due to disease. A therapeutically effective amount or dose of a drug includes a prophylactically effective amount or a prophylactically effective dose, which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to an individual at risk of developing a disease or suffering a relapse of the disease, inhibits the development or relapse of the disease. The ability of a therapeutic agent to enhance disease regression or inhibit the progression or recurrence of a disease may be assessed by a variety of methods known to the practitioner, such as through human clinical trials, through animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

The term patient includes humans and other mammals undergoing prophylactic or therapeutic treatment.

As used herein, the term "individual" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat an individual with cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

In the context of this description, the terms microgram and micromole are used interchangeably with the terms µg and µM, respectively.

In the following subsections, various aspects of the invention presented in this description are described in more detail.

I. Antibodies to TREM-1.

Disclosed herein are antibodies, e.g., fully human antibodies, that have particular functional characteristics or properties. For example, the antibodies provided herein specifically bind to human TREM-1, and more particularly to a particular domain (e.g., a functional domain) within the extracellular domain of human TREM-1. In one embodiment, the antibodies specifically bind to a site on TREM-1 to which a ligand for TREM-1 (e.g., PGLYRP1) binds. In some embodiments, the antibodies are antagonist antibodies, i.e., they inhibit or suppress TREM-1 activity (i.e., do not have agonist activity when bound) on cells, such as monocytes, macrophages, and neutrophils. In some embodiments, the anti-TREM-1 antibodies cross-react with TREM-1 from one

- 19 046142 or several non-human primates, such as TREM-1 from cynomolgus monkeys. In some embodiments, anti-TREM-1 antibodies block the production of pro-inflammatory cytokines (e.g., IL-6, TNF-α, IL-8, IL-1e, IL-12, and combinations thereof) by cells (e.g., macrophages, dendritic cells, neutrophils) after activation. In other embodiments, anti-TREM-1 antibodies comprise Fc regions that do not bind one or more FcyRs. In further embodiments, 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 onset of a cytokine storm following administration of an anti-TREM-1 antibody to an individual in need thereof.

In some embodiments, the specific anti-TREM-1 antibodies described herein are antibodies, such as monoclonal, recombinant, and/or human antibodies, that cross-compete with mAb 0318 for binding to human TREM-1. In some embodiments, the 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 described herein are, in certain embodiments, included in the same group as mAb 0318.

The mAb 0318 antibody has a heavy chain variable region (VH) comprising SEQ ID NO: 14 and a light chain variable region (VL) comprising SEQ ID NO: 15 (see International Patent Application Publication No. 2016/009086). mAb 0318 also has heavy chain CDR1, CDR2, and CDR3, which correspond 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.

Therefore, in some embodiments, the anti-TREM-1 antibody provided herein comprises a VH and VL having SEQ ID NO: 14 and SEQ ID NO: 15, respectively. In another embodiment, the anti-TREM-1 antibody VH comprises a CDR1 sequence consisting of amino acids 31-35 (TYAMH) of SEQ ID NO: 14, in which one of the amino acids may be replaced with another amino acid. In specific embodiments, the anti-TREM-1 antibody VH comprises a CDR2 sequence consisting of amino acids 50-68 (RIRTKSSNYATYYAASVKG) of SEQ ID NO: 14, in which one, two, or three amino acids may be replaced with another amino acid. In some embodiments, the VH of an anti-TREM-1 antibody comprises a CDR3 sequence consisting of amino acids 101-110 (DMGIRRQFAY) of SEQ ID NO: 14, in which one, two or three amino acids may be replaced with another amino acid.

In some embodiments, the anti-TREM-1 antibody VL comprises a CDR1 sequence consisting of amino acids 24-38 (QQSNQDPYT) of SEQ ID NO: 15, in which one, two, or three amino acids may be replaced with another amino acid. In other embodiments, the anti-TREM-1 antibody VL comprises a CDR2 sequence consisting of amino acids 54-60 (RASNLES) of SEQ ID NO: 15, in which one or two amino acids may be replaced with another amino acid. In some embodiments, the anti-TREM-1 antibody VL comprises a CDR3 sequence consisting of amino acids 93-101 (QQSNQDPYT) of SEQ ID NO: 15, in which one or two amino acids may be replaced with another amino acid.

Methionine residues in the CDRs of antibodies can be oxidized, leading to possible chemical cleavage and subsequent reduction in antibody potency. Therefore, in the anti-TREM-1 antibodies provided herein, one or more methionine residues in the CDRs of the heavy and/or light chain can be replaced with amino acid residues that are not subject to oxidative cleavage. In some embodiments, methionine residues in CDR1 and CDR3 of the heavy chain are replaced with amino acid residues that are not subject to oxidative cleavage (e.g., glutamine or leucine). Thus, in one embodiment, the VH of an anti-TREM-1 antibody comprises a CDR3 sequence consisting of amino acids 101-110 (DQGIRRQFAY) of SEQ ID NO: 81 or amino acids 101-110 (DLGTRRQFAY) of SEQ ID NO: 82

In other embodiments of the invention, the VH of the anti-TREM-1 antibody comprises a CDR1 sequence consisting of amino acids 31-35 (TYAQH) of SEQ ID NO: 83 or amino acids 31-35 (TYALH) of SEQ ID NO: 84. Likewise, in some embodiments of the invention, deamination sites may be removed from the anti-TREM-1 antibody, particularly from the CDRs.

In some embodiments, the anti-TREM-1 antibody VH and VL comprise the anti-TREM-1 antibody VH and VL sequences described in International Patent Application Publication No. WO 2017/152102 A2, the entire contents of which are incorporated herein by reference. In some embodiments, the anti-TREM-1 antibody VL comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 9-27 as set forth in WO 2017/152102, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 28-40 as set forth in WO 2017/152102, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 41-119 as set forth in WO 2017/152102. In one embodiment, the anti-TREM antibody VH

- 20 046142 comprises a CDR1 sequence selected from the group consisting of SEQ ID NO: 120-143 presented in WO 2017/152102, a CDR2 sequence selected from the group consisting of SEQ ID NO: 144-172 presented in WO 2017/152102, and/or a CDR3 sequence selected from the group consisting of SEQ ID NO: 173-247 presented in WO 2017/152102.

In some embodiments, the anti-TREM-1 antibodies provided herein comprise CDR and/or variable region sequences that are at least 80% identical (e.g., at least 85% identical, at least 95% identical, at least 95% identical, or at least 99%) to the CDR and/or variable region sequences of mAb 0318.

In some embodiments, anti-TREM-1 antibodies comprise a heavy chain variable region (VH) and a light chain variable region (VL) comprising SEQ ID NO: 14 and SEQ ID NO: 15, respectively. In some embodiments, anti-TREM-1 antibodies comprise a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. In some embodiments, the LC comprises SEQ ID NO: 54.

In some embodiments, the anti-TREM-1 antibodies provided herein comprise a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 396-475 as provided in WO 2017/152102 and/or a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 316-395 as provided in WO 2017/152102.

In some embodiments, an anti-TREM-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain and light chain comprise the amino acid sequences shown in Table 7. In some embodiments, an anti-TREM-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 50 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, an anti-TREM-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 51 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, an anti-TREM-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 52 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, an anti-TREM-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 53, and the light chain comprises the amino acid sequence presented in SEQ ID NO: 54.

Heavy and light chains comprising an amino acid sequence that is at least 99, 98, 97, 96, 95, 90, 85, or 80% identical to any of the heavy and light chains described herein, such as in SEQ ID NOs: 50-54, can be used to generate anti-TREM-1 antibodies having desired characteristics, such as those described below in the present disclosure.

In some embodiments, an anti-TREM-1 antibody provided herein comprises a heavy chain and a light chain, wherein the heavy chain comprises 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 set forth in SEQ ID NO: 50, 51, 52, or 53, and wherein the light chain comprises 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 the sequence presented in SEQ ID NO: 54.

In some embodiments, an anti-TREM1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, L358M, and any combination thereof (EU numbering). In some embodiments, an anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, L358M, and any combination thereof (EU numbering). In some embodiments, an anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, P238S, and any combination thereof (EU numbering). In some embodiments, an anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D,

- 21 046142

K214R, C226S, C229S, P238S and any combination thereof (EU numbering).

In one embodiment, an anti-TREM-1 antibody has the ability to bind to human TREM-1 variants (e.g., TREM-1 isoforms 2 and 3, SEQ ID NO: 2 and SEQ ID NO: 3, respectively) as assessed, for example, by surface plasmon resonance. In another embodiment, an anti-TREM-1 antibody has the ability to bind to cynomolgus monkey TREM-1 (SEQ ID NO: 7) as assessed, for example, by surface plasmon resonance.

In some embodiments, the anti-TREM-1 antibodies provided herein bind to human TREM-1 with high affinity, e.g., having a KD of 10 ^-7 M or less, 10 -8 M or less, 10 ^-9 M (1 nM) or less, 10 -10 M or less, 10 ^-11 M or less, 10 ^-12 M or less, 10 -12 to 10 ^-7 M, 10 ^-11 to 10 ^-7 M, 10 ^-10 to 10 ^-7 M, or ^{10 -9 to 10 -7 M} , as assessed by ^BIACORE ™ (e.g., using the assay described in the Examples). In some embodiments, the anti-TREM-1 antibodies provided herein bind to cynomolgus monkey (supo) TREM-1, e.g., with a KD of as assessed by BIACORE™ (e.g., according to the method described in the Examples section) 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 to 10 ^-7 M, 10 ^-11 to 10 ^-7 M, 10 ^-10 to 10 ^-7 M, or 10 ^-9 to 10 ^-7 M.

In one embodiment, the antibodies provided herein bind to the same one or more of the same epitopes on TREM-1 as the mAb 0318 antibody. In some embodiments, the anti-TREM-1 antibody is capable of specifically binding to (i) at least one amino acid residue selected from the group consisting of A21, T22, K23, L24, T25, E26, and any combination thereof;

(II) at least one amino acid residue selected from the group consisting of A49, S50, S51, Q52, K53, A54, W55, Q56, 157, 158, R59, D60, G61, E62, M63, P64, K65, T66, L67, A68, C69, T70, E71, R72, P73, S74, K75, N76, S77, H78, P79, V80, Q81, V82, G83, R84, 185 and any combination thereof; and (III) at least one amino acid residue selected from the group consisting of C113, V114, I115, Y116, Q117, P118, P119 and any combination thereof, of human TREM-1 (e.g., isoform 1, SEQ ID NO: 1) (see WO 2016/009086).

In one embodiment, an anti-TREM-1 antibody has the ability to specifically bind to amino acids D38 through F48 of SEQ ID NO: 1 (human TREM-1), as analyzed by, for example, NH-MS or X-ray diffraction. In some embodiments, an epitope for an anti-TREM-1 antibody comprises one, two, three, four, five, six, seven, or all of the amino acid residues D38, V39, K40, C41, D42, Y43, T44, and L45 of SEQ ID NO: 1 (human TREM-1) and one, two, or all of the amino acid residues selected from the group consisting of E46, K47, and F48 of SEQ ID NO: 1 (human TREM-1), as analyzed by, for example, NH-MS or X-ray diffraction. In specific embodiments of the invention, the epitope for an antibody to TREM-1 comprises one, two, three or all of the amino acid residues selected from the group consisting of D42, E46, D92 H93 of SEQ ID NO: 1 (human TREM-1), as determined by analysis using TREM-1 variants and surface plasmon resonance.

In one embodiment, an epitope for an anti-TREM-1 antibody as provided herein comprises at least amino acid residues E46 and/or D92 of SEQ ID NO: 1 (human TREM-1), as determined by analysis using TREM-1 variants and surface plasmon resonance. In another embodiment, an epitope for an anti-TREM-1 antibody comprises one, two, or all amino acid residues selected from the group consisting of L31, 186, and V101 of SEQ ID NO: 1 (human TREM-1). In particular embodiments, an anti-TREM-1 antibody is capable of specifically binding to a polypeptide comprising amino acid residues E19 through L26 of cynomolgus monkey TREM-1 (SEQ ID NO: 7), as determined by analysis, for example, NH-MS or X-ray diffraction.

In one embodiment of the invention, an anti-TREM-1 antibody has the ability to specifically bind to human TREM-1, wherein the epitope for the antibody comprises one, two, three, four, five, six, seven, eight, nine, or all 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.

In one embodiment, an anti-TREM-1 antibody has the ability to specifically bind to human TREM-1, wherein the epitope for the antibody comprises D42 of SEQ ID NO: 1. In other embodiments, an anti-TREM-1 antibody has the ability to specifically bind to human TREM-1, wherein the epitope for the antibody comprises E46 of SEQ ID NO: 1. In some embodiments, the epitope for the antibody may comprise

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V39, C41, D42, Y43, L45 of SEQ ID NO: 1. In further embodiments of the invention, the epitope for the antibody may comprise E46, K47 and A49 of SEQ ID NO: 1. In a specific embodiment of the invention, the epitope for the antibody to TREM-1 may further comprise F48 of SEQ ID NO: 1.

In some embodiments, the anti-TREM-1 antibody has a viscosity profile similar to that of mAb 0318. In some embodiments, the anti-TREM-1 antibody provided herein 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, the anti-TREM-1 antibody has a viscosity of less than 10 cP (e.g., 9 cP) at a concentration of 130 mg/mL.

In some embodiments, an anti-TREM-1 antibody provided herein comprises mutations that replace one or more negatively charged residues in the CDR1 and CDR3 regions of the light chain of the antibody with uncharged residues. In some embodiments, an anti-TREM-1 antibody comprises a replacement of 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-hiding mutations.

In some embodiments, the anti-TREM-1 antibody described herein contains mutations in the Fab-Fab interaction region of SEQ ID NO: 14 to reduce Fab-Fab dimerization. It was previously determined for mAb 0318 that, because the antibody contains two Fabs, multimerization can affect viscosity. Such mutations are referred to as Fab-Fab interaction mutations. In specific embodiments, an anti-TREM-1 antibody comprises a mutation replacing any one of residues Y32, R52, S55, S56, N57, A59, M102, 1104, 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.

In one embodiment, the anti-TREM-1 antibody described herein comprises a mutation at position 32 of SEQ ID NO: 15, whereby phenylalanine is replaced with an amino acid selected from the following amino acid residues: glycine, serine, threonine, cysteine, alanine, valine, leucine, isoleucine, and methionine. The use of such a mutation is based on the discovery that an Ala substitution at position Y90 of SEQ ID NO: 1 increases the affinity of SEQ ID NO: 3 for TREM-1. Y90 was found to interact with the phenylalanine residue of SEQ ID NO: 15. The mutation of SEQ ID NO: 15, carried out to enhance the Fab-TREM-1 interaction, is referred to as the Fab-TREM-1 interaction mutation. Disclosed herein are anti-TREM-1 antibodies whose variable regions are linked (e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc, which may be of any allotype or isoallotype, e.g., for IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Km1, Km2, Km3 (see, e.g., Jeffries et al., mAbs 1, 2009, p. 1). In some embodiments, the variable regions of the anti-TREM-1 antibodies provided herein are linked to an Fc that has no or substantially no effector function, such as IgG1. In some embodiments of the invention, the variable regions of the anti-TREM-1 antibodies are linked to an Fc that has reduced binding affinity or lacks binding affinity to one or more FcγRs.

In one embodiment, the VH domain of an anti-TREM-1 antibody as described herein may be fused to a human IgG constant domain (i.e., Fc), such as IgG1, IgG2, IgG3, or IgG4, which may be naturally occurring or modified, such as further described herein. For example, the VH domain may comprise the amino acid sequence of any VH domain as described herein fused to a human IgG constant domain, such as IgG1, having the amino acid sequence of a wild-type human IgG1 constant domain:

- 23 046142

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP , AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

SLSPGK (SEQ ID NO: 9) or the sequence of an allotypic variant of SEQ ID NO: 9, and having the following amino acid sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 77;

Allotype-specific amino acid residues are shown in bold and underlined).

In one embodiment, the VH domain of an anti-TREM-1 antibody as described herein may comprise the amino acid sequence of any VH domain as described herein fused to a non-effector-function constant region, such as the following amino acid sequences of a non-effector-function constant domain of human IgG1:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 78;

IgG1.1f containing the substitutions L234A, L235E, G237A, A330S and P331S (EU numbering, the indicated substitutions are underlined) or

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO: 79;

IgG1.1f containing the substitutions L234A, L235E and G237A (EU numbering, the indicated substitutions are underlined).

For example, an allotypic variant of IgG1 comprises K97R, D239E and/or L241M (underlined and highlighted in bold, see above), and their numbering corresponds to the numbering of SEQ ID NO: 77-79. In the full-length region of the heavy chain and according to EU numbering, these amino acid substitutions have the following numbers: K214R, D356E L358M. In some embodiments, the constant region of the anti-TREM-1 antibody further comprises one or more mutations or substitutions of amino acids L117, A118, G120, A213 and P214 (underlined above), if they are numbered as in SEQ ID NO: 77-79, or L234, A235, G237, A330 and P331 according to EU numbering. In further embodiments of the invention, the constant region of the anti-TREM-1 antibody comprises one or more mutations or substitutions of the amino acids L117A, A118E, G120A, A213S and P214S in SEQ ID NOs: 77-79 or L234A, L235E, G237A, A330S and P331S according to EU numbering. The constant region of the anti-TREM-1 antibody may also comprise one or more mutations or substitutions L117A, A118E and G120A in SEQ ID NO: 9 or L234A, L235E and G237A according to EU numbering.

In some embodiments of the invention, the VH domain of the anti-TREM-1 antibodies represented by

- 24 046142 in the present description, contains the amino acid sequence of any VH domain presented in the present description, fused with an IgG1 constant domain containing the following amino acid sequences:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTS

PPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: AND;

IgG1-Aba containing the substitutions K214R, C226S, C229S and P238S (EU numbering, which are underlined); or

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQ S S GL YSL S S V VT VP S S SLGTKT YTCN VDHKP SNTK VDKRVEPKS CDKTHT S

PPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 12;

IgG4-Aba containing the substitutions S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S (EU numbering, which are underlined).

The VL domain 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 comprise the amino acid sequence of any VL domain described herein fused to the following amino acid sequence of a human IgG1 kappa light chain:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13)

In specific embodiments, the heavy chain constant region comprises a lysine or other amino acid at the C-terminus, such as the following amino acids: LSPGK (SEQ ID NO: 48) in the heavy chain. In specific embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus and has, for example, the C-terminal sequence LSPG (SEQ ID NO: 49) or LSP.

In one embodiment of the invention, the variable region of an anti-TREM-1 antibody is linked to an Fc that has no effector function or substantially no effector function. In specific embodiments, the variable region of an anti-TREM-1 antibody is linked to an Fc selected from the group consisting of IgG1.1f, IgG1.3f, IgG1-Aba, and IgG4-Aba, as described herein.

Typically, the variable regions provided herein may be linked to an Fc containing one or more modifications typically designed to alter one or more functional properties of the antibody, such as Fc receptor binding, proinflammatory cytokine release, serum half-life, complement fixation, and/or antibody-dependent cellular cytotoxicity. Additionally, an antibody provided herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation to alter one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of residues in the Fc region corresponds to the EU Kabot index.

The Fc region includes domains derived from the constant region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM), including a fragment, analog, variant, mutant, or derivative of the constant region. The constant region of an immunoglobulin is a naturally occurring or synthetically produced polypeptide homologous to the C-terminal region of an immunoglobulin and may include a CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain, alone or in combination.

Ig molecules interact with several classes of cellular receptors. For example, IgG molecules interact with three classes of antibody-specific Fcy receptors (FcyRs).

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IgG class, namely FcγRI, FcγRII, and FcγRIII. Data have been published indicating that sequences crucial for IgG binding to FcγR receptors are located in the CH2 and CH3 domains. The ability of this antibody to bind to the Fc receptor (FcR) influences its serum half-life.

In one embodiment of the invention, the Fc region of the anti-TREM-1 antibody is a variant Fc region, e.g., has an Fc sequence that is modified (e.g., by amino acid substitution, deletion, and/or insertion) compared to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is then modified to create a variant) to provide desired structural characteristics and/or biological activity.

For example, modifications can be made to the Fc region to create an Fc variant that has (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC);

(b) increased or decreased complement-mediated cytotoxicity (CDC);

(c) increased or decreased affinity for C1q; and/or (d) increased or decreased affinity for the Fc receptor compared to the parental Fc.

Such Fc region variants typically should contain at least one amino acid modification within the Fc region. A combination of amino acid modifications is considered particularly preferred. For example, an Fc region variant may include two, three, four, five, etc., substitutions, such as those at the specific positions within the Fc region described herein.

A variant Fc region may also have a sequence alteration whereby amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid interaction with other cysteine-containing proteins present in the host cell used to produce the anti-TREM-1 antibody described herein. Even when cysteine residues are removed, the single-chain Fc domains can still form a dimeric Fc domain in which they are present together through non-covalent interaction. In other embodiments, the Fc region can be modified to make it more compatible with the selected host cell. For example, the PA sequence present near the N-terminus of a typical native Fc region, which can be recognized by a digestive enzyme in E. coli, such as proline-specific immunopeptidase, can be removed. In other embodiments, one or more glycosylation sites in the Fc domain can be deleted. Residues that are typically glycosylated (e.g., asparagine) can mediate a cytolytic response. Such residues can be deleted or replaced with non-glycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement, such as the C1q binding site, can be deleted from the Fc region. For example, the human IgG1 NK sequence can be deleted or replaced. In particular embodiments, sites that affect binding to Fc receptors, preferably sites other than the salvage receptor binding sites, can be deleted. In other embodiments, the Fc region can be modified to remove an ADCC site. ADCC sites are known in the art; see, for example, Sarmay et al., Molec. Immunol., 29 (5), 1992, p. 633-639 regarding ADCC sites in IgG1. Specific examples of Fc domain variants are described, for example, in WO 97/34631 and WO 96/32478.

In one embodiment of the invention, the Fc hinge region is modified to alter, for example, increase or decrease, the number of cysteine residues in the hinge region. This approach is described in detail in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the Fc hinge region is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment of the invention, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 interface of the Fc hinge region such that the antibody has reduced ability to bind Staphylococcyl protein A (SpA) compared to the ability to bind SpA of an antibody having a native Fc hinge region. This approach is described in more detail in U.S. Patent No. 6,165,745 to Ward et al.

In further embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330 and/or 331 can be replaced with a different amino acid residue such that the antibody has an altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered can be, for example, an Fc receptor or the C1 component of the complement system. This approach is described in more detail in U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260,

- 26 046142 both in the name of Winter and others.

In another example, one or more amino acids at amino acid positions 329, 331, and 322 can be substituted with a different amino acid residue in a manner that alters the antibody's ability to bind C1q and/or reduces or eliminates complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 to Idusogie et al.

In another example, one or more amino acid residues at amino acid positions 231 and 239 can be substituted to alter the antibody's ability to fix complement. This approach is described in more detail in PCT Publication WO 94/29351 to Bodmer et al.

In yet another example, the Fc region can be modified to decrease antibody-dependent cell-mediated cytotoxicity (ADCC) and/or to decrease affinity for the FcY receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270,

272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312,

313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,

382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439. Examples of substitutions include

236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Examples of variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T. Other modifications to enhance interactions 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. A review of these and other modifications is provided in Strohl, Current Opinion in Biotechnology 20, 2009, pp. 685–691.

Other Fc modifications that can be made to Fc include those designed to reduce or eliminate the ability to bind to FcyR and/or complement proteins, resulting in a reduction or elimination of Fc-mediated effector functions such as ADCC, ADCP, and CDC. Examples of 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) (numbering corresponds to the EU index). Examples of substitutions include (but are not limited to) 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S) (numbering corresponds to the EU index). The Fc variant may contain 236R/328R. Other modifications intended to reduce interactions with FcγR and complement include substitutions at 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as abolition of glycosylation at position 297 by mutation, enzymatically, or through production in organisms such as bacteria that do not glycosylate proteins. A review of these and other modifications is provided in Strohl, Current Opinion in Biotechnology, 20 (2009), pp. 685–691.

Optionally, the Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one of skill in the art (see, e.g., U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; International Patent Application Publications WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/0201 14).

The affinities and binding affinities of an Fc region for its ligand can be determined using a variety of in vitro assays (biochemical and immunoassays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., BIACORE assay), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may employ a label on one or more components to be analyzed and/or employ a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed discussion of issues related to binding affinity and kinetics is presented in Fundamental Immunology, 4th ed., edited by Paul W.E., Lippincott-Raven, Philadelphia, 1999, where the focus is on antibody-immunogen interactions.

In particular embodiments, the anti-TREM-1 antibodies provided herein comprise an Fc that has reduced or no binding affinity to FcγR. In some embodiments, an anti-TREM-1 antibody has reduced binding affinity to FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD 6b), or any combination thereof, compared to an antibody comprising a heavy chain that consists of the amino acid sequence set forth in SEQ ID NO: 76 and a light chain that consists of the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, an anti-TREM-1 antibody has a binding affinity to

- 27 046142 affinity with FcyRI (CD64) reduced by at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times or at least 10 times compared to an antibody comprising a heavy chain consisting of the amino acid sequence presented in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence presented in SEQ ID NO: 54.

In some embodiments, the anti-TREM-1 antibodies comprise an IgG1 Fc variant comprising (a) one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, and any combination thereof (EU numbering);

(b) one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, P331S and any combination thereof (EU numbering);

(c) one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, P238S and any combination thereof (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 (EU numbering).

In some embodiments, an anti-TREM-1 antibody provided herein is (a) an IgG1 isotype and comprises one or more amino acid substitutions of an amino acid residue in the Fc region selected from the group consisting of

N297A, N297Q, D270A, D265A,

L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G,

P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E,

L328E, P238D, S267E, L328F, E233D, G237D, H268D, P271G, A330R and any combination thereof, where the residue numbering corresponds to EU numbering or Cabot numbering, or has an amino acid deletion in the Fc region at the position corresponding to glycine 236;

(b) is an IgG2 isotype and comprises one or more amino acid substitutions of an amino acid residue in the Fc region selected from the group consisting of

P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q,

A330S, P331S, C232S, C233S, M252Y, S254T, T256E and any combination thereof, wherein the residue numbering corresponds to EU numbering or Cabot numbering; or (c) the IgG4 isotype and comprises one or more amino acid substitutions of an amino acid residue in the Fc region 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, where the residue numbering corresponds to EU numbering or Cabot numbering.

In some embodiments of the invention (a) the Fc region further comprises one or more amino acid substitutions of an amino acid residue selected from the group consisting of A330L, L234F; L235E, P331S, and any combination thereof, wherein the residue numbering corresponds to EU numbering or Kabot numbering;

(b) the Fc region also comprises one or more additional amino acid substitutions of an amino acid residue selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein the numbering of the residues corresponds to EU numbering or Kabot numbering; or (c) the Fc region further comprises an amino acid substitution S228P, wherein the numbering corresponds to EU numbering or Kabot numbering (see WO 2017/152102).

In specific embodiments of the invention, an Fc characterized by reduced complement fixation capacity is selected. An example of an Fc, such as an IgG1 Fc, with reduced complement fixation capacity is an Fc that has the following two amino acid substitutions: A330S and P331S.

In specific embodiments of the invention, an Fc is selected that exhibits virtually no effector function, i.e., is characterized by reduced binding to FcγR and reduced complement fixation. An example of an Fc, such as an IgG1 Fc, that exhibits no effector function is an Fc containing the following five mutations: L234A, L235E, G237A, A330S, and P331S.

II. Physical properties of antibodies.

Anti-TREM-1 antibodies, such as those described herein, have some or

- 28 046142 all physical characteristics of the specific antibodies to TREM-1 described herein, such as those described in the Examples section.

Glycosylation sites, particularly those present in variable regions, may confer increased immunogenicity of an antibody or alter the pKa of an antibody due to altered binding to antigen (Marshall et al., Annu. Rev. Biochem., 41 (1972), pp. 673–702; Gala and Morrison, J. Immunol. 172 (2004), pp. 5489–5494; Wallick et al., J. Exp. Med., 168 (1988), pp. 1099–1109; Spiro, Glycobiology, 12 (2002), pp. 43R–56R; Parekh et al., Nature, 316 (1985), pp. 452–457; Mimura et al., Mol. Immunol. 37 (2000), pp. 697-706). Glycosylation is known to occur at motifs containing the sequence N-X-S/T. In some embodiments, the anti-TREM-1 antibodies provided herein lack or have a reduced level of glycosylation of the variable region. This can be achieved by selecting antibodies that do not contain a glycosylation motif in the variable region or by mutating residues in the glycosylation region. Thus, in some embodiments, the anti-TREM-1 antibody provided herein is less immunogenic than an antibody comprising a heavy chain that consists of the amino acid sequence presented in SEQ ID NO: 76 and a light chain that consists of the amino acid sequence presented in SEQ ID NO: 54.

In some embodiments, anti-TREM-1 antibodies lack asparagine isomerization sites. Asparagine deamination can occur at the N-G or D-G sequences, resulting in the formation of an isoaspartic acid residue, which leads to bending of the polypeptide chain and decreased stability (the isoaspartic acid effect).

Each antibody must have a unique isoelectric point (pi), which is typically in the pH range of 6 to 9.5. Pi for an IgG1 subclass antibody is typically in the pH range of 7 to 9.5, and Pi for an IgG4 subclass antibody is typically in the pH range of 6 to 8. It is expected that antibodies with a Pi outside the normal range may exhibit some degree of protein unfolding and instability in vivo. Thus, the anti-TREM-1 antibodies described herein may have a Pi within the normal range (e.g., 8 to 9). This can be achieved either by selecting antibodies with a Pi within the normal range or by mutating charged surface residues.

Each antibody must have a characteristic melting temperature, and the higher the melting temperature, the greater the overall stability in vivo (Krishnamurthy R. and Manning MC, Curr. Pharm. Biotechnol., 3, 2002, pp. 361-371). Typically, the T _M i (temperature at which the antibody begins to unfold) can be above 60°C, above 65°C, or above 70°C. In some embodiments, the anti-TREM-1 antibodies provided herein have a higher melting temperature compared to an antibody comprising a heavy chain that consists of the amino acid sequence presented in SEQ ID NO: 76 and a light chain that consists of the amino acid sequence presented in SEQ ID NO: 54. Thus, in some embodiments, the anti-TREM-1 antibodies provided herein have a higher thermal stability compared to a reference antibody comprising a heavy chain that consists of the amino acid sequence presented in SEQ ID NO: 76 and a light chain that consists of the amino acid sequence presented in SEQ ID NO: 54, as measured, for example, by a capillary differential scanning calorimeter (CAP-DSC). The melting point of an antibody can be measured by differential scanning calorimetry (Chen et al., Pharm. Res. 20 (2003): 1952-1960; Ghirlando et al., Immunol. Lett., 68 (1999): 47-52) or circular dichroism (Murray et al., J. Chromatogr. Sci., 40 (2002): 343-349). In some embodiments, about 10% to 20%, about 20% to 30% (e.g., 24%), or about 30% to 40% of the antibody is reversible upon heating to 77°C.

In some embodiments, the anti-TREM-1 antibodies described herein do not undergo rapid degradation. Antibody degradation can be assessed using capillary electrophoresis (CE) and MALDI-MS (Alexander A.J., Hughes D.E., Anal. Chem., 67, 1995, pp. 3626-3632).

In some embodiments, the anti-TREM-1 antibodies provided herein exhibit minimal aggregation that may lead to the initiation of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable at a level of aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be assessed using several methods, including size exclusion column assay (SEC), high performance liquid chromatography (HPLC), and dynamic light scattering (DLS). In some embodiments, the anti-TREM-1 antibodies provided herein are monomeric assays by size exclusion column assay (SEC-HPLC). In some embodiments, the anti-TREM-1 antibodies exhibit minimal risk of fragmentation as assessed by two-dimensional liquid

- 29 046142 chromatography-tandem mass spectrometry (2O-LC/MS) or intact mass spectrometric analysis by liquid chromatography-tandem mass spectrometry (LC/MS).

III. Nucleic acids, vectors and cells.

Another object of the invention are nucleic acid molecules encoding the anti-TREM-1 antibodies described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. A nucleic acid is considered isolated or considered substantially pure if it is purified from other cellular components or other contaminants, such as other nucleic acids present in the cell (e.g., other chromosomal DNA, such as chromosomal DNA naturally linked to the isolated DNA) or proteins, using standard techniques, including alkaline/SDS digestion, CsCl banding, column chromatography, restriction enzyme digestion, agarose gel electrophoresis, and other techniques well known in the art (see Current Protocols in Molecular Biology, F. Ausubel et al., eds., Greene Publishing and Wiley Interscience, New York, 1987). The nucleic acid 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.

The nucleic acids described herein can be prepared using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas generated using transgenic mice carrying human immunoglobulin genes, as described in more detail below), cDNAs encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained using standard PCR amplification or cDNA cloning methods. For antibodies generated from an immunoglobulin gene library (e.g., using phage display methods), the nucleic acid encoding the antibody can be isolated from the library.

In some embodiments, the nucleic acids provided herein encode the VH and VL sequences of the anti-TREM-1 antibodies provided herein. Exemplary DNA sequences encoding the VH and VL sequences are provided in SEQ ID NOs: 58-61 and SEQ ID NOs: 62-65, respectively.

The method for producing the anti-TREM-1 antibody described herein may comprise expressing the heavy chains and light chains in a cell line containing nucleotide sequences encoding the heavy and light chains with a signal peptide, such as SEQ ID NOs: 58-61 and SEQ ID NOs: 62-65, respectively. Host cells containing such nucleotide sequences fall within the scope of the present invention.

Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody genes, Fab fragment genes, or an scFv gene. During these manipulations, the VL or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (hinge, CH1, CH2, and/or CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242, 1991), and DNA fragments including these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, such as an IgG2 and/or IgG4 constant region. In the case of the Fab fragment heavy chain gene, the VH encoding DNA can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.

Isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by functionally linking the VL encoding DNA to another DNA molecule encoding the light chain constant region, CL. Sequences of human light chain constant region genes are known in the art (see, e.g., Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242, 1991), and DNA fragments including these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

Another object of the present invention are cells (e.g., host cells) expressing (e.g., recombinantly) the anti-TREM-1 antibodies described herein and correspondingly

- 30 046142 relevant polynucleotides and expression vectors. The present disclosure also provides vectors containing polynucleotides that contain nucleotide sequences encoding anti-TREM-1 antibodies or a fragment thereof. In some embodiments, the vectors can be used to recombinantly express the anti-TREM-1 antibodies described herein in host cells, such as mammalian cells. In some embodiments, the vectors can be used for gene therapy.

Vectors useful herein include expression vectors, viral vectors, and plasmid vectors. In some embodiments, the vector is a viral vector.

As used herein, the term "expression vector" refers to any nucleic acid construct that contains elements necessary for the transcription and translation of an inserted coding sequence, or, in the case of a viral RNA vector, elements necessary for replication and translation after introduction into an appropriate host cell. Expression vectors may include plasmids, phagemids, viruses, and their derivatives.

The expression vectors described herein may include polynucleotides encoding an antibody or antigen-binding fragment thereof described herein. In some embodiments, the coding sequences of the antibody or antigen-binding fragment thereof are operably linked to an expression control sequence. In the context of the present disclosure, two nucleotide sequences are considered to be operably linked if they are covalently linked in such a way that each component of the nucleotide sequence maintains its functionality. A coding sequence and a gene expression control sequence are considered to be operably linked if they are covalently linked in such a way that expression or transcription and/or translation of the coding sequence is influenced or controlled by the gene expression control sequence. Two DNA sequences are said to be functionally linked if induction of a promoter in the 5' region of the expressed sequence of a gene results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation;

(2) does not impair the ability of the promoter region to control transcription of the coding sequence; or (3) does not impair the ability of the corresponding RNA transcript to be translated into protein.

Thus, an expressed gene sequence may be operably linked to a coding nucleotide sequence if the expressed gene sequence is capable of transcribing said coding nucleotide sequence, resulting in the resulting transcript being translated into the desired antibody or antigen-binding fragment thereof.

Viral vectors include, but are not limited to, nucleotide sequences from the following viruses: a retrovirus such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; poliovirus; and an RNA virus such as a retrovirus. The use of other vectors well known in the art is acceptable. Particular viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes are replaced with the gene of interest. Non-cytopathic viruses include retroviruses whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by proviral integration into the host cell DNA. Retroviruses are approved for experiments in therapeutics using human genes. The most suitable are replication-deficient retroviruses (i.e., they are able to direct the synthesis of the required proteins, but are unable to produce an infectious particle). Such genetically modified retroviral expression vectors are widely used for highly efficient gene transduction in vivo. Standard protocols for the production of replication-deficient retroviruses (including the steps of inserting exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retroviruses by the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with viral particles) are presented in Kriegler M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York, 1990; and Murry E.J., Methods in Molecular Biology, vol. 7, Humana Press, Inc., Cliffton, N.J., 1991.

In some embodiments, the virus is an adeno-associated virus, a virus containing double-stranded DNA. The adeno-associated virus can be engineered to be replication-deficient and capable of infecting a wide range of cell types and species. It has additional advantages such as thermal stability and stability in lipid solvents; high transduction frequencies in cells of various lineages, including hematopoietic cells; and lack of inhibition of superinfection, which

- 31 046142 allows for multiple transduction series. Data have been published demonstrating that adeno-associated virus can integrate into human cell DNA in a site-specific manner, minimizing the potential for insertional mutagenesis and variability in the expression characteristics of the integrated gene during retroviral infection. Furthermore, wild-type adeno-associated virus infections in tissue culture were monitored for more than 100 passages in the absence of selective pressure, indicating that adeno-associated virus integration into the genome is a relatively stable event. Adeno-associated virus can also function extrachromosomally.

IV. Immunoconjugates.

The present disclosure also provides immunoconjugates comprising any of the anti-TREM-1 antibodies disclosed herein. In some embodiments, an immunoconjugate comprises an antibody or antigen-binding fragment thereof linked to an agent. In some embodiments, an immunoconjugate comprises a bispecific molecule disclosed herein linked to an agent (e.g., a therapeutic agent or a diagnostic agent).

For diagnostic purposes, suitable agents are detectable labels, which include radioisotopes for whole-body imaging and radioisotopes, enzymes, fluorescent labels, and other antibody-compatible labels used for sample analysis. Detectable labels that can be linked to any of the TREM-1 antibodies described herein may be any of the various types of labels currently used in the field of in vitro diagnostics, including particulate labels, including metal sols such as colloidal gold, isotopes such as I or Tc ⁹⁹ used, for example, with a peptide chelating agent such as N2S2, N3S or _N4 , chromophores including fluorescent markers, luminescent markers, phosphorescent markers, and the like, as well as enzymatic labels that convert the substrate in question into a detectable marker, and polynucleotide labels that are detected after amplification, such as by the polymerase chain reaction. Suitable enzymatic labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label may be an alkaline phosphatase enzyme that is detected by assessing the presence of or generating chemiluminescence after conversion of 1,2-dioxetane substrates such as adamantyl methoxyphosphoryloxyphenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl)phenyl phosphate) (CSPD), as well as CDP and CDP-STAR® or other luminescent substrates well known in the art, such as chelates of suitable lanthanides such as terbium(III) and europium(III). The means of detection are determined by the selected label. Detection of the label or its reaction products can be carried out by the naked eye in the case where the label consists of particles and accumulates in appropriate quantities, or by using instruments such as a spectrophotometer, luminometer, fluorimeter, etc., in all cases in accordance with standard practice.

In some embodiments, conjugation techniques are used to produce linkages that are substantially (or nearly) non-immunogenic, such as peptide (i.e., amide), sulfide (sterically hindered), disulfide, hydrazone, and ether linkages. These linkages are substantially non-immunogenic and have acceptable stability in serum (see, e.g., Senter P.D., Curr. Opin. Chem. Biol., 13, 2009, pp. 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the fragment and the antibody, various conjugation strategies can be used. In the case where the fragment is a naturally occurring or recombinant fragment consisting of 50-500 amino acids, standard procedures are described in textbooks on the chemistry of protein conjugate synthesis that can be easily performed by a person skilled in the art (see, for example, Hackenberger C.P.R., Schwarzer D., Angew. Chem. Int. Ed. Engl., 47, 2008, pp. 10030-10074). In some embodiments, the maleimide moiety is reacted with a cysteine residue present in the antibody or fragment thereof. Such a reaction is the most suitable chemical coupling reaction in the case of using, for example, the Fab or Fab' fragment of the antibody. Alternatively, in some embodiments, the coupling is carried out with the C-terminal region of the antibody or fragment. C-terminal modification of a protein, for example, a Fab fragment, can be carried out according to the method described by Sunbul M. and Yin J., Org. Biomol. Chem., 7, 2009, pp. 3361–3371).

In general, site-specific interaction and covalent coupling is based on the transformation of a naturally occurring amino acid into an amino acid whose reactivity is orthogonal to the reactivity of other functional groups present. For example, a specific cysteine in the context of a rare sequence can be enzymatically converted to an aldehyde (see Frese M.A., Dierks T., ChemBioChem., 10, 2009, pp. 425-427). The desired amino acid modification can also be created by exploiting the specific enzymatic reactivity of certain enzymes towards a naturally occurring amino acid in the context of the sequence in question (see, e.g., Taki M. et al., Prot. Eng. Des. Sel., 17, 2004, pp. 119-126; Gautier A. et al., Chern.

- 32 046142

Biol., 15, 2008, pp. 128-136; and protease-catalyzed formation of C-N bonds is described in Bordusa F., Highlights in Bioorganic Chemistry, 2004, pp. 389-403). Site-specific interaction and covalent coupling can also be achieved through selective interaction of terminal amino acids with appropriate modifying reagents.

To achieve site-specific covalent coupling, the reactivity of the N-terminal cysteine towards benzonitriles can be exploited (see Ren H. et al., Angew. Chem. Int. Ed. Engl., 48, 2009, pp. 9658–9662).

Native chemical ligation can also be based on the use of C-terminal cysteine residues (Taylor E. Vogel, Imperiali B., Nucleic Acids and Molecular Biology, 22 (Protein Engineering), 2009, pp. 65-96).

US Patent No. 6,437,095 B1 describes a conjugation method based on the rapid interaction of a cysteine present in a segment of negatively charged amino acids with a cysteine located in a segment of positively charged amino acids.

The fragment may also be a synthetic peptide or peptide mimetic. When the polypeptide is synthesized chemically, amino acids with orthogonal chemical reactivity can be incorporated during the synthesis process (see, e.g., de Graaf A.J. et al., Bioconjug. Chem., 20, 2009, pp. 1281–1295). Since a wide variety of orthogonal functional groups are available and can be introduced into a synthetic peptide, conjugation of such a peptide to a linker is a standard chemical procedure.

To obtain a polypeptide bearing a single label, the conjugate with a 1:1 stoichiometry can be separated from other conjugation byproducts using chromatography. This procedure can be facilitated by using a dye-labeled pairing partner and a charged linker. Using such a labeled pairing partner bearing a highly negative charge allows for the easy separation of monoconjugated polypeptides from unlabeled polypeptides and polypeptides bearing more than one linker, as the difference in charge and molecular weight can be used for separation. A fluorescent dye, such as a labeled monovalent coupling agent, can be used to purify the complex from unbound components.

In some embodiments of the invention, the moiety attached to the antibody to TREM-1 is selected from the group consisting of a binding moiety used to label the moiety and a biologically active moiety.

The anti-TREM-1 antibodies described herein can also be conjugated to a therapeutic agent to produce an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binding agents, DNA intercalators, DNA crosslinkers, histone deacylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. To produce an ADC, the antibody and therapeutic agent are preferably conjugated using 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. The ADC can be prepared according to the method described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO 02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO 08/103693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295.

Anti-TREM-1 antibodies, such as those described herein, can also be used to detect TREM-1, such as human TREM-1, such as human TREM-1, in tissues or tissue samples. The antibodies can be used, for example, in an ELISA assay or a flow cytometric assay. In some embodiments, an anti-TREM-1 antibody is contacted with cells, such as cells in tissue, for a period of time sufficient to achieve specific binding, and then a reagent, such as an antibody that detects the anti-TREM-1 antibody, is added. Examples of assays are provided in the Examples section. The anti-TREM-1 antibody can be a fully human antibody or it can be a chimeric antibody, such as an antibody having human variable regions and murine constant regions, or fragments thereof. Examples of methods for detecting TREM-1, such as human TREM-1, in a sample (a cell or tissue sample) include (i) contacting the sample with an anti-TREM-1 antibody for a period of time sufficient to allow specific binding of the anti-TREM-1 antibody to TREM-1 in the sample, and (ii) contacting the sample with a detection reagent, such as an antibody that specifically binds to the anti-TREM-1 antibody, such as the Fc region of an anti-TREM-1 antibody, to detect TREM-1 bound to the anti-TREM-1 antibody. Washing steps may be applied after incubation with the antibody and/or detection reagent. The anti-TREM-1 antibodies used in these methods do not require coupling to a label or detection agents, as the detection agent can be applied separately.

- 33 046142

Other uses of TREM-1 antibodies, such as monotherapy or combination therapy, are presented below, for example in the section on combination treatment.

V. Bispecific molecules.

The anti-TREM-1 antibodies described herein can be used to create bispecific molecules. An anti-TREM-1 antibody or its antigen-binding fragments can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to create a bispecific molecule that binds to at least two different binding sites or target molecules. For example, an anti-TREM-1 antibody can be linked to an antibody or scFv that specifically binds to any protein that can be used as potential targets for combination treatments, such as the proteins described herein (e.g., antibodies to IP-10 or TNF-α). In fact, the antibody described herein can be derivatized or linked to more than one other functional molecule to create multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to fall within the term bispecific molecule as used herein. To create the bispecific molecule described herein, the antibody described herein can be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, to produce a bispecific molecule.

Thus, the present disclosure provides bispecific molecules comprising at least one first binding specificity for TREM-1 and a second binding specificity for a second target epitope. In some embodiments of the invention presented herein, in which the bispecific molecule is multispecific, the molecule may additionally have a third binding specificity.

In some embodiments, the bispecific molecules described herein comprise at least one antibody or antibody fragment as the binding specificity, including, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv). The antibody may also be a dimer of a light chain or a heavy chain, or any minimal fragment, such as Fv or the single-chain construct described in U.S. Pat. No. 4,946,778 to Ladner et al.

Although monoclonal antibodies are preferred, other antibodies, such as murine, chimeric, and humanized monoclonal antibodies, can be used in the bispecific molecules described herein.

The bispecific molecules described herein can be prepared by conjugating their constituent binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be created separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-N-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridylthio)propionate (SPDP), and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., J. Exp. Med., 1984, 160:1686; Liu, M.A., et al., Proc. Nail. Acad. Sci., USA, 1985, 82:8648). Other methods include those described by Paulus, Behring Ins. Mitt., 78 (1985), pp. 118–132; Brennan et al., Science, 229 (1985), pp. 81–83; and Glennie et al., J. Immunol., 139 (1987), pp. 2367–2375. Examples of conjugating agents include SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated via sulfhydryl linkage between the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region is modified to contain an odd number (preferably one) of sulfhydryl residues prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is most suitable when the bispecific molecule is a fusion protein such as MAtxMAb, MAtxFab, MAtx(scFv) ₂ , FabxF(ab') ₂ or LigandxFab. The bispecific antibody may comprise an antibody comprising an scFv at the C-terminus of each heavy chain. The bispecific molecule provided herein may be a single-chain molecule comprising a single-chain antibody and a binding determinant or a single-chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single-chain molecules. Methods for producing bispecific molecules are described, for example,

- 34 046142 measures, in US No. 5260203; US No. 5455030; US No. 4881175; US No. 5132405; US No. 5091513; US No. 5476786; US No. 5013653; US No. 5258498; and US No. 5482858.

Binding of bispecific molecules to their specific targets can be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition assay), or Western blot analysis. Each of these assays detects the presence of protein-antibody complexes of particular interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest.

VI. Sets.

The present disclosure provides kits comprising one or more anti-TREM-1 antibodies as provided herein, or antigen-binding fragments thereof, bispecific molecules, or immunoconjugates thereof. In some embodiments, the invention provides a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions as provided herein, such as one or more antibodies as provided herein, or antigen-binding fragments thereof, optionally instructions for use. In some embodiments, the kits comprise a pharmaceutical composition as provided herein and any prophylactic or therapeutic agent, for example, as provided herein.

VII. Compositions and formulations.

The present disclosure also provides compositions (e.g., pharmaceutical compositions) and formulations comprising one or more anti-TREM-1 antibodies (including polynucleotides, vectors, and cells that encode and/or express anti-TREM-1 antibodies) as described herein. For example, in one embodiment, the invention provides a pharmaceutical composition comprising one or more anti-TREM-1 antibodies as described herein, formulated together with a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody, immunoconjugate, or bispecific molecule, may be coated with a material that protects the compound from the action of acids or other environmental conditions that inactivate the compound.

Therefore, one of the objectives of the present invention is to provide a pharmaceutical formulation that increases the stability of an antibody to TREM-1 and thereby ensures its long-term storage. In some embodiments, the pharmaceutical formulation provided herein comprises (a) an antibody to TREM-1;

(b) buffering agent;

(c) stabilizer;

(g) salt;

(d) an excipient; and/or (e) a surfactant. In some embodiments, the pharmaceutical formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or more. In some embodiments, the formulation is stable when stored at 4, 25, or 40°C.

Buffering agent.

Buffering agents that can be used according to the present invention can be a weak acid or base used to maintain the acidity (pH) of a solution close to a selected value after the addition of another acid or base. Suitable buffering agents can maximize the stability of pharmaceutical formulations by maintaining the control pH of the formulation. Suitable buffering agents can also help ensure physiological compatibility or optimize solubility. Rheological characteristics, viscosity and other properties can also be affected by the pH of the formulation. Common buffering agents include, but are not limited to, histidine, citrate, succinate, acetate and phosphate. In some embodiments, the buffering agent comprises histidine (e.g., L-histidine) with isotonicity agents and can adjust the pH with an acid or

- 35 046142 base known in the art. In specific embodiments, the buffering agent is L-histidine. In specific embodiments, the pH of the composition is maintained at a level of from about 2 to about 10 or from about 4 to about 8.

Stabilizer.

Stabilizers are added to a pharmaceutical product to stabilize said product. Such agents can provide protein stabilization in a variety of 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, raffinose, or maltose; polyols such as glycerol, mannitol, sorbitol; cyclodextrins or dextrans of any type and molecular weight; or PEG. In one aspect of the invention, the stabilizer is selected to maximize the stability of the FIX polypeptide in lyophilized formulations. In specific embodiments, the stabilizer is sucrose and/or arginine.

Filler

Excipients can be added to a pharmaceutical product to increase its volume and weight, thereby facilitating precise dosing and handling. Common excipients include (but are not limited to) lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

Surfactant.

Surfactants are amphipathic substances having lyophilic and lyophobic groups. A surfactant can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethyl amine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

In some embodiments, the pharmaceutical formulation provided herein comprises (a) an anti-TREM-1 antibody at a concentration of about 0.25 to 250 mg/mL (e.g., 10 to 200 mg/mL);

(b) histidine at a concentration of approximately 20 mM;

(c) sucrose at a concentration of approximately 150 mM;

(d) arginine at a concentration of approximately 25 mM; and (e) NaCl at a concentration of approximately 50 mM.

The formulation may additionally contain one or more buffering systems, a preservative, a tonicity agent, a chelating agent, a stabilizer, and a surfactant, as well as various combinations thereof. The use of preservatives, isotonicity agents, chelating agents, stabilizers, and/or surfactants in pharmaceutical formulations is well known to those skilled in the art (see Remington: The Science and Practice of Pharmacy, 19th ed., 1995).

In some embodiments, the pharmaceutical formulation is an aqueous formulation. Such a formulation is typically a solution or suspension, but may also include colloids, dispersions, emulsions, and multiphase products. The term "aqueous formulation" refers to a formulation containing at least 50% by weight of water. Similarly, the term "aqueous solution" refers to a solution containing at least 50% by weight of water, and the term "aqueous suspension" refers to a suspension containing at least 50% by weight of water.

In some embodiments of the invention, the pharmaceutical formulation is a freeze-dried formulation to which the treating physician or patient adds solvents and/or diluents prior to use.

The pharmaceutical compositions provided herein can be administered as part of combination therapy, i.e., in combination with other agents. For example, combination therapy can include the use of an anti-TREM-1 antibody provided herein in combination with at least one other therapeutic agent. Examples of therapeutic agents that can be used in combination therapy can include other compounds, drugs, and/or agents used to treat a disease or disorder (e.g., an inflammatory disorder). Such compounds, drugs, and/or agents can include, for example, anti-inflammatory drugs or antibodies that block or reduce the production of proinflammatory cytokines. In some embodiments, therapeutic agents 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-W (e.g., BETASERON®, EXTAVIA®), glati

- 36 046142 amino acids acetate (e.g. COPAXONE®, GLATOPA®), mitoxantrone (e.g. NOVANTRONE®), nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids and their combinations.

The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not exhibit any undesirable toxicological effects (see, e.g., Berge S.M. et al., J. Pharm. Sci., 66, 1977, pp. 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts formed from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acid, etc., as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include salts formed from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as from non-toxic organic amines such as N.N-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

The pharmaceutical composition provided herein may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;

(2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of suitable coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by sterilization procedures (see above) and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may be advisable to include isotonicity agents such as sugars, sodium chloride, etc. in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be ensured by the use of agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of extemporaneous sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in pharmaceutical compositions is contemplated, except in cases where any medium or agent is incompatible with the active substance. The pharmaceutical composition may or may not contain a preservative. Additional active substances may be included in the composition.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition may be prepared in the form of a solution, microemulsion, liposome, or other ordered structure suitable for incorporating a drug at a high concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of suitable coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, the compositions may include agents for imparting isotonicity, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearates and gelatin.

Sterile injectable solutions may be prepared by incorporating the active substance in the required amount in an appropriate solvent, if necessary, with one or a combination of ingredients enumerated above, and then sterilizing by

- 37 046142 by microfiltration. Typically, dispersions are prepared by incorporating the active substance into a sterile vehicle containing the basic dispersion medium and the required other ingredients listed herein. For sterile powders intended for the preparation of sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying (lyophilization), which yield a powder of the active substance plus any additional required ingredient from a previously sterile-filtered solution containing them.

The amount of active ingredient that can be combined with the carrier material to produce a single-dose dosage form will vary depending on the individual being treated and the specific route of administration. The amount of active ingredient that can be combined with the carrier material to produce a single-dose dosage form will generally represent the amount of the composition that produces a therapeutic effect. Typically, this amount, unless it is 100%, can range from about 0.01% to about 99% of the active ingredient, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus dose may be administered, several divided doses may be administered over a predetermined period of time, or the dose may be proportionately decreased or increased as needed by the acute therapeutic situation. Parenteral compositions are most advantageously prepared in dosage unit form for ease of administration and uniformity of dosage. As used herein, the term dosage unit form refers to physically discrete units suitable as unitary dosages for the individuals to be treated; each unit containing the active substance in a predetermined quantity calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification of the dosage unit forms described herein is dictated by and directly related to (a) the unique characteristics of the active substance and the particular therapeutic effect desired to be achieved; and (b) the limitations that exist in the field of formulations using the active substance for the treatment of susceptible individuals.

For administration of an anti-TREM-1 antibody, such as that provided herein, doses in the range of from about 0.0001 to 100 mg/kg or more, typically from 0.01 to 5 or 10 mg/kg of host body weight, are used. For example, doses can 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 can be in the range of 1-10 mg/kg. Examples of treatment regimens include administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. Examples of dosing regimens for the anti-TREM-1 antibody provided herein include intravenous administration at a dose of 1 mg/kg body weight or 3 mg/kg body weight, where the antibody is administered using one of the following dosing regimens:

(I) every four weeks for six doses, then every three months;

(II) every three weeks;

(III) a single dose of 3 mg/kg body weight, then 1 mg/kg body weight every three weeks.

In some embodiments, the anti-TREM-1 antibody is administered at a constant dose (constant dosing regimen). In other embodiments, the anti-TREM-1 antibody is administered at a fixed dose with another antibody. In specific embodiments, the anti-TREM-1 antibody is administered at a dose calculated based on body weight.

Some methods involve the simultaneous administration of two or more monoclonal antibodies with different binding specificities, in which case the dose of each antibody administered falls within the specified ranges. The antibody is typically administered multiple times. The intervals between individual doses may be, for example, one week, one month, three months, or one year. Intervals may also be irregular and may be determined based on measured levels of antibody to the target antigen in the patient's blood. In some methods, the dose is adjusted to achieve plasma antibody concentrations of approximately 1-1000 μg/mL, and in others, approximately 25-300 μg/mL.

The antibody can be administered in an extended-release formulation, which requires less frequent administration. The dose and frequency of administration vary depending on the half-life of the antibody in the patient's body. In general, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In the case of prophylactic use

- 38 046142 [Therapeutic applications] involve administering a relatively low dose at relatively infrequent intervals over a long period of time. Some patients continue treatment for the rest of their lives. In therapeutic applications, a relatively high dose may be administered at relatively short intervals until disease progression is reduced or stopped and the patient experiences partial or complete improvement in symptoms. After this, the patient can be switched to a prophylactic regimen.

The actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient when using the specified composition and route of administration, but is not toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular compositions provided herein or their ester, salt or amide being used, the route of administration, the time of administration, the excretion rate of the particular compound being used, the duration of treatment, other drugs, compounds and/or products used in combination with the particular compositions being used, the age, sex, body weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical art.

The composition described herein can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be apparent to those skilled in the art, the route and/or form of administration will vary depending on the desired results. Routes of administration of the anti-TREM-1 antibodies described herein can include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. The term parenteral administration as used herein refers to routes of administration other than enteral and local administration, typically administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

Alternatively, the antibody described herein can be administered by a non-parenteral route, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

Active ingredients can be formulated with carriers that protect the compound from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, ed. J.R. Robinson, Marcel Dekker, Inc., New York, 1978).

Therapeutic compositions can be administered using medical devices known in the art. For example, in a particular embodiment, a therapeutic composition described herein can be administered using a needleless hypodermal injection device, such as the devices described in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use using the anti-TREM-1 antibodies described herein include those described in U.S. Patent No. 4,487,603, which describes an implantable microinfusion pump for controlled-rate drug release; U.S. Patent No. 4,486,194, which describes a therapeutic device for delivering medications through the skin; No. 4,447,233, which describes a drug infusion pump for delivering a drug by infusion at a precise rate; U.S. Patent No. 4,447,224, which describes an implantable infusion device with a variable flow for continuous delivery of a drug; U.S. Patent No. 4,439,196, which describes an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which describes an osmotic drug delivery system. The contents of these patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In some embodiments, the anti-TREM-1 antibodies described herein can be formulated to provide the desired in vivo distribution. For example, the blood-brain barrier (BBB) prevents the passage of many highly hydrophilic compounds. To allow the therapeutic compounds described herein to pass through the BBB (if desired, such as for brain cancers), they can be incorporated into, for example, liposomes. Methods for producing liposomes are described, for example, in U.S. Patent No. 4,522,811;

- 39 046142

5,374,548; and 5,399,331. Liposomes may contain one or more moieties that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, e.g., V.V. Ranade, J. Clin. Pharmacol., 29 (1989), p. 685). Examples of targeting moieties include folate or biotin (see, e.g., US 5,416,016 to Low et al.); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun., 153 (1988), p. 1038); antibodies (P.G. Bloeman et al., FEBS Lett., 357, 1995, p. 140; M. Owais et al., Antimicrob. Agents Chemother., 39, 1995, p. 180); surfactant protein A receptor (Briscoe et al., Am. J. Physiol., 1233, 1995, p. 134); p120 (Schreier et al., J. Biol. Chem., 269, 1994, p. 9090); see also K. Keinanen, M.L. Laukkanen, FEBS Lett., 346, 1994, p. 123; J.J. Killion,

I.J. Fidler, Immunomethods, 4, 1994, p. 273.

VIII. Applications and methods.

The TREM-1 antibodies described herein and formulations containing such antibodies (e.g., pharmaceutical composition, formulations, polynucleotides, vectors, and cells) can be used to treat an inflammatory disease (e.g., by inhibiting TREM-1 activity).

Thus, one of the objects of the present invention relates to methods of treating an inflammatory disease in an individual in need thereof, which comprises administering to the individual a therapeutically effective dose of an antibody to TREM-1. Examples of inflammatory diseases that can be treated with the TREM-1 antibodies provided herein include, but are not limited to, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma and other autoimmune diseases resulting from acute or chronic inflammation.

In one embodiment, anti-TREM-1 antibodies can be used to treat individuals with inflammatory bowel disease. Inflammatory bowel disease (IBD) is a condition that can affect any part of the gastrointestinal tract from the mouth to the anus, causing a wide variety of symptoms. IBD primarily causes abdominal pain, diarrhea (which may be bloody), vomiting, or weight loss, but can also cause complications outside the gastrointestinal tract, such as skin rashes, arthritis, eye inflammation, fatigue, and loss 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 typically affects the ileum and colon; it can affect any area of the intestine, but the disease is often focal (areas affected by the disease spread in patches throughout the intestine). UC always involves the rectum (i.e., refers to the large intestine (colon)) and the lesion is more continuous. In CD, inflammation is transmural, leading to abscesses, fistulas, and strictures, while in UC, inflammation is typically limited to the mucosa. There is no pharmaceutical or surgical cure for Crohn's disease, while some patients with UC can be cured by surgical removal of the colon. Treatment options are limited to symptom control, maintenance of remission, and prevention of relapse. The effectiveness of inflammatory bowel disease treatment in a clinical setting can be assessed by a reduction in the CD score, calculated using the Crohn's Disease Activity Index (CDAI) score based on laboratory tests and a quality-of-life questionnaire. In animal models, efficacy is assessed primarily by weight gain and also by the disease activity index (DAI), which is calculated based on a combination of stool consistency, body weight, and the presence of blood in the stool.

In one embodiment, the anti-TREM-1 antibodies described herein can be used to treat individuals with rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease that affects most, if not all, of the body and is one of the most common forms of arthritis. It is characterized by joint inflammation, causing pain, stiffness, warmth, redness, and swelling. This inflammation is caused by inflammatory cells that invade the joints, and these inflammatory cells release enzymes that can break down bone and cartilage. As a result, this inflammation can lead to, among other physiological effects, severe bone and cartilage damage, joint destruction, and severe pain. The affected joint can lose its shape and proper alignment, leading to pain and loss of mobility. Several animal models of rheumatoid arthritis are known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop inflammatory arthritis that resembles human rheumatoid arthritis.

- 40 046142 rit. Since CIA has immunological and pathological features similar to RA, this makes it a suitable model for screening potential anti-inflammatory compounds for humans. Efficacy using this model is assessed by reducing joint swelling.

Efficacy in RA in a clinical setting is assessed by the ability to reduce symptoms in patients, which is assessed by a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein levels, and plasma levels of factors such as anti-citrullinated protein antibodies.

In one embodiment, the anti-TREM-1 antibodies described herein can be used to treat individuals with psoriasis. Psoriasis is a T-cell-mediated inflammatory skin disorder that can cause significant discomfort. It is a disease that currently has no cure and affects people of all ages. Although individuals with mild psoriasis can often control their disease with topical agents, more than one million people worldwide require ultraviolet light treatment or systemic immunosuppressant therapy. Unfortunately, the inconvenience and risks associated with ultraviolet radiation and the toxicity inherent in many therapies limit their long-term use. Furthermore, patients typically experience relapses of psoriasis, which in some cases occur shortly after discontinuing immunosuppressant therapy. A psoriasis model developed in recent years is based on CD4 ⁺ T-cell transfer; it mimics many aspects of human psoriasis and can therefore be used to identify compounds suitable for the treatment of psoriasis (Davenport et al., Internal Immunopharmacol., 2, 2002, pp. 653-672). Efficacy in this model is assessed using a scoring system based on reduction in skin lesions. Similarly, efficacy in patients is assessed based on reduction in skin lesions.

In one embodiment, anti-TREM-1 antibodies can be used to treat individuals with psoriatic arthritis. Psoriatic arthritis (RA) is a type of inflammatory arthritis that occurs in a subset of patients with psoriasis. In these patients, skin pathology/symptoms include joint swelling similar to that seen in rheumatoid arthritis. It is characterized by the presence of patchy, raised, reddened areas of skin inflammation characterized by scaling. Psoriasis often affects the folds of the elbows and knees, the scalp, the navel, and the areas around the genitals and anus. Approximately 10% of patients with psoriasis also develop associated joint inflammation.

For the purposes of the present description, prophylactic, palliative, symptomatic, and/or curative treatments may be considered as separate aspects of the invention. The antibody according to the invention may be administered parenterally, for example, intravenously, for example, intramuscularly, for example, subcutaneously. Alternatively, the antibody according to the invention may be administered non-parenterally, for example orally or topically. The antibody according to the invention may be administered prophylactically. The antibody according to the invention may be administered therapeutically (on-demand).

The following examples are provided to illustrate the invention and are not intended to limit its scope. The contents of all documents cited in this application are expressly incorporated herein by reference.

Examples

Example 1. Analysis of the kinetic characteristics of the interaction of antibody 318 variants with TREM-1 of humans and cynomolgus monkeys using the surface plasmon resonance method.

The kinetic characteristics of binding of mAb 0318 variants to human TREM-1-Fc (hTREM-1) and cynomolgus monkey TREM-1-Fc (cTREM-1) were determined. Binding studies were performed using a ProleOn analyzer (BioRad), which measures molecular interactions in real time using surface plasmon resonance. Experiments were performed at 25°C, and samples were stored at 15°C in the sample compartment. The signal (RU, response units) measured by ProleOn is directly correlated with the mass on the surfaces of individual sensor chips in six parallel flow cells. Monoclonal antibody to human Fc or polyclonal antibody to mouse Fc from Biacore 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 capture antibody capture level corresponded to approximately 2600-6000 RU in each experiment. Capture of purified monoclonal mouse or recombinantly expressed anti-hTREM-1 antibodies was achieved by diluting the antibodies to a concentration of 5-10 nM in running buffer (10 mM Hepes, 0.15 M NaCl, 5 mM EDTA, 0.05% surfactant P20, pH 7.4) and injecting vertically at 30 μl/min for 60 s, creating intermediate reference spots adjacent to all flow cells in which only anti-Fc antibody was immobilized. This typically resulted in final capture levels of the test antibodies corresponding to approximately

- 41 046142

100-300 RU and analyte Rmax values of 30-90 RU. Binding of hTREM-1 or cTREM-1 proteins was accomplished by injecting the analyte (antigen) horizontally into all flow cells to allow comparative analysis of the binding of different captured antibodies to TREM1 relative to binding to an intermediate reference spot. hTREM-1 or cTREM-1 proteins were serially diluted 1:3 in running buffer over a concentration range of 1.2-100 nM, injected at 100 μL/min for 250 s, and allowed to dissociate for 600 s. The GLM surface was regenerated after each analyte injection cycle by two 18-s 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 surface with the immobilized capture antibody, allowing subsequent binding of the next pair of samples intended for interaction analysis. The regeneration procedure did not directly remove the immobilized anti-Fc capture antibody from the chip surface.

Antibody-antigen binding affinities were quantified by determining the equilibrium dissociation constant (KD), which was determined by measuring the kinetic characteristics of complex formation and dissociation. Rate constants corresponding to the association and dissociation of the monovalent complex, such as ka (association rate) and kd (dissociation rate), were obtained by fitting the data to a 1:1 Langmuir binding model using ProteOn data analysis software. The KD coefficient is related to k _a and kd by the equation KD = kd/k _a .

Before data analysis, binding curves were processed using a double control (subtraction of signals from the reference surface and levels obtained by injecting pure buffer onto captured TREM-1 antibodies). This allowed for correction for instrumental noise, array shift, and drift during sample injection.

As shown in Figs. 1A and 1B, all mAb 0318 variants (namely, 318-IgG1.1f, 318-IgG1.3f, 318-IgG4-Aba, and 318-IgG1-Aba) were found to have affinity for human TREM-1-Fc similar to that of mAb 0318-IgG4. Variant 0318-IgG1.3f also bound to cynomolgus monkey TREM-1, although with a slightly lower affinity compared to its affinity for human TREM-1 (see Fig. 1A).

Example 2. Analysis of internalization of mAb 0318-IgG1.3f after binding to the TREM-1 receptor.

The mAb 0318-IgG1.3f antibody variant was tested for its internalization in primary human monocytes using both laser scanning confocal microscopy (data not shown) and flow cytometric imaging analysis using the Amnis ImageStream® system. As shown in Fig. 2A, at 0 h, TREM-1 was expressed primarily on the monocyte surface. However, by 24 h after antibody binding, a significant percentage (~36%) of the TREM-1 receptor (as assessed by 0318-IgG1.3f staining) was internalized, suggesting that the entire antibody-receptor complex was internalized into the cell following mAb 0318-IgG1.3f binding.

Next, the fate of the TREM-1 receptor after internalization was determined using the TREM26 antibody (catalog #314902, Biolegend; see US 20150274825, paragraph [0005]) for comparison. The TREM26 antibody does not compete with the 0318 antibody variants described herein. As shown in Fig. 2B, a significant decrease in TREM26+ expression (~51%) was observed at the 20 h time point compared to the 0 h time point. A significant decrease (4-fold) in the MFI (mean fluorescence index) of TREM26+ was also observed at the 20 h time point (following treatment with mAb 0318-IgG1.3f), suggesting that TREM-1 was degraded after internalization. The observed decrease in TREM-1 receptor expression following antibody treatment was reversible, but this occurred after antibody removal (data not shown).

Example 3. Analysis of the efficiency of blocking TREM-1 activation by mAb 0318 variants using a reporter assay using BWZ/hTREM-1 cells.

The ability of the mAb 0318 anti-TREM-1 antibody variants to inhibit human TREM-1 signaling was determined using the BWZ.36/hTREM-1DAP12:NFATLacZ cell line assay (also referred to herein as the BWZ/hTREM-1 reporter cell), as described, for example, in US Patent Publication No. 9,550,830 B2 and International Patent Application Publication No. WO 2016/009086 A1. In general, the method was as follows: approximately 40,000 hTREM-1/BWZ.36 cells/well were plated in a black 96-well plate with a clear flat bottom in the presence of 75 ng/mL PGLYRP1 (SEQ ID NO: 8) with 2.5 μg/mL PGN-ECndi (catalog no. tlrl-kipgn, Invivogen, San Diego, CA, USA) to provide a positive signal at half the maximum intensity, or alternatively in the presence of a submaximal level (1 μg/mL) of a plastic-adsorbed monoclonal antibody to TREM-1 (catalog no. MAB1278, R&D Systems, Minneapolis, MN, USA) to provide a positive signal.

In the assay, mAb 0318 variants (i.e., 0318-IgG1.3f; 0318-IgG1.1f; 0318-IgG1-Aba; and 0318-IgG4-Aba) were titrated starting at 10 μg/mL using 5 serial 2-fold dilutions.

- 42 046142 n. The sample was incubated overnight at 37°C, then developed using Beta Glo (catalog #E4740, Promega, Madison, WI, USA) according to the Beta Glo protocol, and luminescence was recorded. The results were plotted as a function of Beta Glo luminescence intensity (in relative units) versus antibody concentration. Each assay plate was tested with non-neutralizing mIgG1 (catalog #MAB002, R&D Systems, Minneapolis, MN, USA) as a negative control and neutralizing polyclonal goat antibody to hPGLYRP1 (catalog #AF2590, R&D Systems, Minneapolis, MN, USA) as a positive control. The antibody MAB1278 (catalog number MAB1278, R&D Systems; see US 20150274825, paragraph [0005]), a known agonist of TREM-1 signaling (see rectangular inset), was also used as a positive control.

As shown in Fig. 3, all mAb 0318 variants were potent inhibitors of human TREM-1 signaling, as previously described for the mAb 0318 IgG4 antibody in International Patent Application Publication WO 2016/009086 A1.

Example 4. In vitro analysis of the efficacy of mAb 0318 antibody variants in inhibiting TREM-1-mediated proinflammatory cytokine production by various primary human cells.

To further evaluate the antagonist properties of the mAb 0318 anti-TREM-1 antibody variants, we analyzed their efficacy in blocking the release of various proinflammatory cytokines (e.g., TNF-α, IL-6, or IL-8) by activated human primary cells. Primary monocytes, neutrophils, and peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood and stimulated with plate-immobilized PGRP1 and soluble peptide glycan (PGN-ECndss; a form of peptide glycan lacking TLR2 activity).

As shown in Fig. 4, all mAb 0318 variants (i.e., IgG1.3f, IgG1.1f, IgG1-Aba, and IgG4-Aba) were effective in inhibiting ( _IC50 values in the range of ~10-20 pM) TREM-1-mediated TNF-α release from PBMCs and monocytes. The potency of these mAb 0318 variants was similar to that of mAb 0318-IgG4. mAb 0318-IgG1.3f was also effective in inhibiting IL-6 production ( _IC50 value ~32 pM). In the case of neutrophils, the mAb 0318-IgG1.3f variant was superior to the mAb 0318-IgG4 antibody in blocking TREM-1-mediated IL-8 production by neutrophils (see Fig. 4).

To further demonstrate the antagonistic properties of the mAb 0318 anti-TREM-1 variants, a monocyte-neutrophil coculture assay was also used. Neutrophil-associated PGRP1, when cocultured with monocytes, can bind to TREM-1 receptors, leading to monocyte production of TNF-α. As shown in Fig. 4, all mAb 0318 variants effectively blocked this endogenous activation (IC ₅₀ values ranged from 19 to 44 pM). Similar results were obtained for whole blood with pelleted RBCs (see Fig. 4).

Example 5. In vitro analysis of the efficacy of mAb 0318-IgG1.3f in blocking IL-8 production in stimulated whole blood.

One of the major challenges in developing a whole blood pharmacodynamic (PD) assay to evaluate the antagonist properties of anti-TREM-1 antibody variants is the high baseline levels associated with PGN stimulation. To alleviate this challenge, whole blood was stimulated with a preformed PGRP1+PGN complex in the presence of a NOD2 inhibitor (to block baseline cytokines produced by PGN stimulation of NOD2). IL-8 levels were measured using a standard pharmacodynamic ligand binding assay (HTRF®) (Fig. 5A) or an intracellular cytokine staining (ICS) assay (Fig. 5B).

As shown in Fig. 5A and 5B, the 0318-IgG1.3f antibody effectively blocked TREM-1-mediated IL-8 production, with an inhibition percentage of 60-90% (see Fig. 5A). The measured IC ₅₀ values (mean 12 pM using HTRF and 19.6 pM using ICS) were similar to those obtained using other functional assays (see, e.g., Example 4).

Example 6. In vitro analysis of the efficacy of mAb 0318-IgG1.3f in blocking the mRNA expression of various inflammatory mediators in stimulated whole blood.

To further demonstrate the antagonist properties of mAb 0318-IgG1.3f, the expression levels of several inflammatory mediators (i.e., chitinase-3-like protein 1 (CHI3L1), IL1e, and IL6) were measured using real-time PCR (qPCR). In general, human whole blood from three healthy volunteers (donors #126, 290, and 322) collected in EDTA tubes was stimulated with pre-complexed hPGRP1 (50 μg/mL) and PGN-ECndss (10 μg/mL) (Invivogen, catalog #tlrl-ksspgn) overnight in the presence of mAb 0318-IgG1.3f in various

- 43 046142 concentrations (0-1 nM). After stimulation, plasma was removed and frozen for cytokine measurement. mRNA was isolated from the samples using the MagMax-96 Blood Isolation kit (ThermoFisher, catalog number AMI837) according to the manufacturer's protocol. The isolated mRNA was then converted to cDNA using the Superscript VILO master mix (Thermo Fisher, 11755250). qPCR was then performed using the following probes: HPRT1 (Hs99999909_ml) (Thermo Fisher, 4351370), CHI3L1 (Hs01072228_ml) (Thermo Fisher, 4331182), IL1p (Hs00174097_ml) (Thermo Fisher, 4331182), and IL6 (Hs00985639_ml) (Thermo Fisher, 4331182) and TaqMan Fast Universal Master Mix (2x) (Thermo Fisher, 4366072). Gene expression levels were normalized to the HPRT1 probe level to obtain AACT values. The results were then plotted and IC ₅₀ values were determined.

As demonstrated in Figs. 6A-6B and consistent with the results presented in the above examples, mAb 0318-IgG1.3f was able to inhibit TREM-1-mediated expression of various inflammatory mediators in human whole blood. Inhibition was found to be dose-dependent. IC50 values are presented below in Table 1.

Taken together, the above results demonstrate that the mAb 0318 antibody variants described herein exhibit antagonist activity and can effectively block TREM-1-mediated proinflammatory cytokine production by various human cells.

Table 1

Cell type Proinflammatory mediator Incentive mAb 0318-IgG1.3f 1C ₅₀ (pM) whole blood CHI3L1 RNA Soluble PGRP + Soluble PGN-Ecndss 4.58±5.60 (N=3) ILlb RNA ll.15±16.01 (N=3) IL6 RNA 97.66±125.63 (N=3)

Example 7. Viscosity of mAb 0318 variants.

The sample was buffer-exchanged and dialyzed against the selected solution (20 mM histidine, 150 mM sucrose, 25 mM arginine, 50 mM sodium chloride, pH 6.0) and then concentrated using Amicon Ultra centrifugal filters with a molecular weight cutoff. The aggregation state of the samples was assessed using gel filtration to monitor for any changes in monomerism that occurred after concentration. The viscosity–concentration relationship was determined using a RheoSense m-VROC solution viscometer, measuring incremental shear forces at three points for each analyzed concentration. Concentration was determined based on absorbance measurements at 280 nm using a nanoDrop instrument using serial dilutions and extrapolation.

As shown in Fig. 7, both variants 318-IgG1.1f and 318-IgG1.3f had similar viscosity profiles. At a concentration of approximately 130 mg/mL, variant 0318-IgG1.3f had a viscosity of approximately 9 cP. This viscosity profile is similar to that previously determined for mAb 0318-IgG4 (see International Patent Application Publication No. WO 2016/009086).

Example 8. Immunogenic potential of mAb 0318 variants.

As shown in Fig. 8 and Table 2 (below), the 318-IgG1.1f and 318-IgG1.3f variants had a low to moderate risk of immunogenicity in human patients. Only approximately 22-30% of donors produced immunogenic responses (as assessed by in vitro CD4+ T cell proliferation) to these antibodies. The immunogenicity of the 0318-IgG1-Aba and 0318-IgG4-Aba variants was 10% and 42.5%, respectively (see Table 2 (below)). In contrast, the 0318-IgG4 mAb variant was much more immunogenic (55%) in human patients. KLH (keyhole limpet hemocyanin) and VL6 (MAb to IL-21R), which were used as positive controls, had high immunogenicity for sick people (100 and 40%, respectively).

Table 2

Analysis Cohort Option 0318 END (mg/ml) Host cell EU/mg %MOHOMqxi Assay Conc (µg/ml) % immunogenicity PBMC 40 IgG4 161 UCOE CHO <0.001 97.9 150 55.0 PBMC 40 IgGl-Aba 145 UCOE CHO <0.001 97.3 150 10.0 PBMC 40 IgGl.lf 170 UCOE CHO <0.006 98.6 150 22.5 PBMC 40 IgG4-Aba 5 HEK <0.02 98.9 150 42.5 PBMC 40 IgG1.3f 4.6 HEK <0.02 99.5 150 30.0

Example 9. Analysis of binding of mAb 0318 variants to FcRn using surface plasmon resonance (SPR).

To determine whether different variants of mAb 0318 have the ability to bind to

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FcRn, FcRn receptors (mouse, human, and cynomolgus monkey) were immobilized on a CM5 BIAcore biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) by amine coupling to a level corresponding to 400 response units (RU). The assay was performed at room temperature using PBS, 0.05% Tween-20™, pH 6.0 (GE Healthcare Bioscience) as running and dilution buffer. Variants of mAb 0318 (200 nM) were injected at a flow rate of 50 μl/min at room temperature and pH 6.0. The association time was 180 s, and the dissociation phase (also at pH 6.0) took 360 s. The chip surface was regenerated to its baseline level by briefly injecting 50 mM Tris, pH 8.0, and 150 mM NaCl. SPR data were analyzed by comparing the biological response signal height at 180 s and 300 s post-injection. The corresponding parameters were the maximum RU level (RU max) (at 180 s post-injection) and the late stability level (at 300 s post-injection).

As shown in Fig. 9A and 9B, all variants of mAb 0318 (IgG1-Aba mod, IgG4-Aba mod, IgG1.1f, and IgG1.3f) were able to bind to FcRn from humans, mice, and cynomolgus monkeys in a pH-dependent manner. This was also the case for mAb 0318 (IgG4).

Example 10. Analysis of binding of mAb 0318 variants to one or more FcyRs.

As noted above, in vivo administration of antibodies to certain immune cell surface receptors can induce cytokine release, which can lead to the induction of a common and toxic clinical complication known as cytokine release syndrome (CRS). Due to concerns that FcyR cross-linking may contribute to TREM-1 agonist activity, mAb 0318-IgG4 was converted to one of the format variants described herein (i.e., IgG1.1f, IgG1.3f, IgG1-Aba, or IgG4-Aba). The binding ability of mAb 0318 to various FcyRs was then assessed.

As shown in Fig. 10A and 10B, the 318-IgG1.1f and 318-IgG1.3f variants showed minimal binding to all FcyRs (i.e., FcyRI (CD64), FcyRIIA (CD32a-H131 and CD32a-R131 variants), FcyRIIB (CD32b), FcyRIIIA (CD16a-V158 variant), and FcyRIIIB (CD16b-NA2 variant)). The 318-IgG1-Aba and 318-IgG4-Aba variants did not bind to FcyRIIA, FcyRIIB, FcyRIIIA, and FcyRIIIB, but bound to FcyRI. In contrast, mAb 0318 (IgG4) showed significant binding to all FcyRs.

Example 11. Analysis of the induction of proinflammatory cytokines by mAb 0318 variants.

To further determine whether in vivo treatment with mAb 0318 variants results in the risk of cytokine release syndrome, whole blood was collected from 8 donors and monocytes were isolated. Monocytes were then allowed to differentiate into immature dendritic cells by seeding monocytes (4 x 106 cells/well) and culturing them in differentiation medium containing IL-4 and GM-CSF (100 ng/mL). Approximately 2–3 d after seeding, approximately half of the differentiation medium was replaced with fresh medium. On day 7, cells were harvested and differentiation efficiency was assessed by analyzing CD14 expression on the cells using a flow cytometer. Immature dendritic cells were then seeded on a flat-bottom plate (0.8 x 105 cells/well), and mAb 0318 variants (with or without CHO-CD32a) were added to the appropriate wells. Immature dendritic cells stimulated with PGRP+PGN were used as a positive control. The cells were then incubated overnight at 37°C. The following day, supernatants from the wells were collected, and the amounts of TNF-α, IL-6, and IL-12 produced were assessed using ELISA.

Results obtained for representative donors are shown in Figs. 11A-11I. Addition of different variants of mAb 0318 (0318-IgG1.1f, 0318-IgG1.3f, and 0318-IgG1.1 Aba) resulted in minimal production of IL-6 (Figs. 11A, 11B, and 11C), TNF-α (Figs. 11D, 11E, and 11E), and IL-12 (Figs. 11G, 11B, and 11J) by immature dendritic cells. These results, along with those obtained in Example 9, indicate that the anti-TREM-1 antibodies presented herein have a low risk of inducing cytokine release syndrome when administered to patients in vivo.

Example 12. Additional characteristics of mAb 0318 variants.

The biophysical characteristics of the 0318-IgG1.3f variant are shown below in Table 3.

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Table 3

Biophysical characteristics of the antibody to TREM-1 0318-IgG1.3f

Property Method Result Identity zhkh-ms Intact mass confirmed Peptide mapping by LC-MS/MS The sequence was confirmed by peptide mapping using Lys-C (yield 95.4%; glycosylation degree 99.7%). Purity/homogeneity SEC monomer 96.3%, HMW 3.6% SEC-MALS The main peak is 95% monomer and is homogeneous (145 kDa) LC-MS G0F (23.3%), GIF (51.3%), G2F (17.3%), SG2F (2.1%), S2G2F (1.0%) Capillary Isoelectric Focusing (cIEF) Main peak p! = 8.75 (55%); acidic variants = 29.5% and basic variants = 15.5% SDS-PAGE Under reducing conditions: 2 major bands—50 kDa and 25 kDa Under non-reducing conditions: major band—150 kDa with minor fragments—98 kDa, 75 kDa, 50 kDa, 25 kDa (artifacts caused by SDS distillation conditions) Affinity SPR (Biacore) huTREMl: K _D = 0.91 nM; k _on = 1.4 E+06 (1/Ms); k _off = 1.3 E-3 (1/s) CvnoTREMl: K _D = 3.4 nM; k _on = 4.0 E+05 (1/Ms); k _off = 1.4 E-3 (1/s) Thermostability DSC T _t 1 = 66.2°C; T _t 2 = 78.4°C; T _t 3 = 83.2°C Reversibility at 77°C is 24%. FcyR/FcRn interaction SPR (Biacore) Decreased binding to all FcyRs was demonstrated. The expected pH-dependent FcRn binding is comparable to that of wild-type IgG1 mAbs.

The biophysical characteristics of the mAb 0318-IgG1.3f variant are favorable for clinical development. The identity of the antibody was confirmed by mass spectrometric analysis (intact mass analysis and peptide mapping). Gel filtration analysis showed that >96% of the antibody was in the monomeric form. A single N-glycosylation site was confirmed at N301 of the heavy chain, and the glycan profile was consistent with that of CHO-expressed mAbs (G0F, G1F, and G2F). Thermal stability (T _sh 1 = 66.2°C; T _sh 2 = 78.4°C; T _sh 3 = 83.2°C) and heat reversibility (24% at 77°C) of mAb 0318-IgG1.3f were within the range characteristic of a typical human IgG1.3 mAb.

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

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Table 4

Stability of the antibody to TREM-1 0318-IgG1.3f

Property Method(s) Results Freeze/thaw (2 hours at -80°C, 4 hours at RT x 3) UV, SEC, DLS, iCIEF No risk to freeze/thaw stability was identified. Solubility/concentration profile UF, SEC >150 mg/ml in formulation buffer (20mM histidine, 150mM sucrose, 50mM sodium chloride, 25mM arginine, pH 6.0) Choosing a pH value that ensures conformational and colloidal stability Optim2 (melting and aggregation onset temperatures Tsh and Tagg) Optimal conformational stability at pH 6-8 Optimal colloidal stability at pH 5-6 Selecting buffer and excipients Optim2 (melting and aggregation onset temperatures Tsh and Tagg) Stabilizers: sucrose, sorbitol, arginine (high concentration), glycerin Accelerated stability test: 150 mg/mL for 12 weeks at 4°C, 25°C, and 40°C in formulation buffer (20 mM histidine, 150 mM sucrose, 50 mM sodium chloride, 25 mM arginine, pH 6.0) SEC, cIEF, HIC, LC-MS/MS (peptide mapping), LC/MS (intact mass), 2D LC/MS (fragments), Biacore, bioassay, UV-Vis, DLS 12 weeks at 40°C: HMW increase by 1.2%/month LMW increase by 2.4%/month 12 weeks at 40°C: Rh and Pd increase 12 weeks at 40°C: Acid variant increase by 11%/month 12 weeks at 40°C: VSNK deamination by 3%/month Viscosity assessment Determining the concentration-viscosity profile The solution viscosity reaches 9 cP at -130 mg/ml, the predicted viscosity is 12 cP at 150 mg/ml.

No physical stability issues were observed in a freeze-thaw stress assay (3 cycles) at 150 mg/mL in the buffer of the described composition (20 mM histidine, 150 mM sucrose, 50 mM sodium chloride, 25 mM arginine, pH 6.0). Accelerated degradation tests of the composition at a concentration of 150 mg/mL were performed at 4, 25, and 40°C (for periods of up to 3 months). Chemical modifications of the CDRs remained low at all temperature conditions and did not affect activity as determined by the SPR assay. Deamination of VSNK was lower than expected compared to other mAbs with the IgG1.3f backbone (increase of 3%/month when stored at 40°C), and changes were time- and temperature-dependent. All other chemical modifications (oxidation, deamination, isomerization) also remained at low levels during stability testing. It should be noted that under storage conditions at 40°C, the formation of both HMW and LMW variants was detected (at a rate of 1.2%/month and 2.4%/month, respectively). The detected changes were time- and temperature-dependent, with the amount of HMW increasing by 0.25%/month and the amount of LMW remaining unchanged under storage conditions at 4°C throughout the study period. Low LMW formation under storage conditions at 40°C was classified based on 2D-LC/1(t) measurements and accurate high-resolution mass measurements as a result of the accumulation of species with the loss of one Fab, as well as a Fab arm, presumably in the conserved sequence in the upper hinge region.

Example 13. Study of the PK/TK/PD variant of mAb 0318-IgG1.3f in cynomolgus monkeys.

The pharmacokinetic (PK), toxicokinetic (TK), and pharmacodynamic (PD) properties of the anti-TREM-1 antibody 0318-IgG1.3f were studied following single-dose administration to cynomolgus monkeys. Some animals (n=3) received 2 mg/kg of 0318-IgG1.3f intravenously. Other animals received 1 of the following doses of 0318-IgG1.3f 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). Following antibody administration, pharmacokinetic characteristics, anti-drug antibody (ADA) presence, TREM-1 receptor occupancy (RO), and ex vivo pharmacodynamic responses were analyzed at predetermined time points.

Pharmacokinetic characteristics (PK).

To evaluate the pharmacokinetic characteristics, the concentration of the 0318-IgG1.3f antibody was determined.

- 47 046142 in animal serum by a ligand binding assay using biotinylated recombinant TREM-1 protein as a capture reagent and a commercially available polyclonal goat antibody to TREM-1 as a detection reagent.

As shown in Fig. 12, the pharmacokinetic characteristics of the 0318-IgG1.3f antibody were found to be nonlinear over the concentration range of 0.1 to 10 mg/kg, primarily due to target-mediated clearance (i.e., internalization and degradation of the antibody after binding to the TREM-1 receptor; see Example 2).

The PK parameters obtained using non-compartmental analysis are presented in Tables 5 (i.v. administration) and 6 (subcutaneous administration). Overall, the results were as follows: a 100-fold increase in dose resulted in an 830-fold increase in serum exposure (AUC) (see Table 6). Clearance in monkeys after an IV dose of 2 mg/kg was 0.1 ± 0.02 mL/h/kg and was similar to the clearance of other IgG1 mAbs (see Table 4). The Vss value at 36 ± 5 mL/kg was similar to the plasma volume, indicating limited extravascular distribution. The half-life of the 0318-IgG1.3f antibody increased from 2 days at a dose of 0.1 mg/kg to 10 days at doses of 2 and 10 mg/kg (single-dose subcutaneous administration) (see Table 6). The bioavailability of the 0318-IgG1.3f antibody after subcutaneous administration (2 mg/kg) was high and amounted to 84%.

Table 5

PK parameters of mAb 0318-IgG1.3f after single-dose IV administration to cynomolgus monkeys

PK parameters of mAb 0318-IgG1.3f after IV administration to cynomolgus monkeys Dose (mg/kg) 2 N 3 Vss (ml/kg) 36 ± 5 CL (ml/h/kg) 0.1 ± 0.02 T1/2 (days) 10 ± 2 AUCiast (μMh) 116 ± 20 ADA-positive/all analyzed 3/3 (1/3)*

*Number of monkeys found to have persistent ADA among all those analyzed.

Table 6

PK parameters of mAb 0318-IgG1.3f after single-dose SC administration to cynomolgus monkeys

PK parameters of mAb 0318-IgG1.3f after SC administration to cynomolgus monkeys Dose (mg/kg) OD 0.5 2 10 N 4 4 3 4 Stakh (nM) 13 ± 4 50 ± 3 189 ± 22 1419 ± 93 Ttakh (h) 24 48 168 ± 96 84 ± 24 AUCiast (μMh) 1 ± 0.2 15 ± 1 94 ± 13 649 ± 64 T1/2 (days) 2 ± 0.4 4 ± 2 10 ± 5 10 ± 3 Bioavailability after SC administration 84% ADA-positive/all analyzed 3/4 (2/4)* 4/4 (2/4)* 3/3 (2/3)* 3/4 (1/4)*

*Number of monkeys found to have persistent ADA among all those analyzed.

Anti-drug antibodies (ADA).

While ADAs were detected in the serum of most monkeys (16 of 18) following a single dose (regardless of the route of administration), most ADA-positive animals did not exhibit a negative effect on 0318-IgG1.3f antibody exposure and TREM-1 receptor occupancy (RO) (see Tables 4 and 5). An accelerated decline in terminal 0318-IgG1.3f antibody exposure, accompanied by ADA formation, was detected in only two monkeys (one in the 0.1 mg/kg group on day 7 and one in the 0.5 mg/kg group on day 21). Because ADAs affected exposure in these two monkeys during the terminal phase, the corresponding data were excluded from the PK analysis.

TREM-1 receptor occupancy (RO) and total TREM-1 receptor levels.

TREM-1 receptor occupancy expressed on peripheral blood monocytes and granulocytes was then assessed. As shown in Figs. 15A and 15B, the percentages of occupied TREM-1 receptors (i.e., bound by anti-TREM-1 antibody) on monocytes and granulocytes were similar for all doses tested. Furthermore, the duration of receptor occupancy appeared to be dependent on the dose of 0318-IgG1.3f antibody administered to the animals. At the 0.5 mg/kg dose, TREM-1 receptor occupancy levels >85% were observed for up to 2 weeks after antibody administration. In contrast, at the 2 and 10 mg/kg doses, TREM-1 receptor occupancy levels >85% were maintained for at least 1 month after administration.

Following administration of doses of the 0318-IgG1.3f antibody, total TREM-1 receptor levels were reduced by both

- 48 046142 on both nodocytes and granulocytes (see Figs. 14A and 14B). As described previously, this reduction is primarily due to increased receptor turnover following antibody binding. The reduction in TREM-1 receptor surface expression was reversible, and the duration of receptor surface loss correlated with the duration of receptor occupancy, at least at the dose levels studied.

Soluble TREM-1 (sTREM-1) levels were found to increase 10- to 50-fold following a single dose of the mAb variant 0318 mAb-IgG1.3f in all dose-treated groups tested (data not shown). It remains unclear whether the increase in sTREM-1 levels was related to the administration of the anti-TREM-1 antibody or to the general animal care conditions during the study. In any case, because drug concentrations were much higher (>1000-fold) than sTREM-1 levels, it is unlikely that a soluble target influenced the binding of the mAb variant 0318 mAb-IgG1.3f to cell-surface TREM-1.

Example 14. Study of the PK/TK/PD of the mAb 0318-IgG1.3f variant in the body of cynomolgus monkeys based on a 2-compartment PK model with TMDD in the central compartment.

PK, RO, total receptor levels, and PD data in monkeys were analyzed using a 2-compartment PK model with saturable target-mediated drug distribution (TMDD) in the central compartment to accommodate the nonlinear PK observed in the experiment (see Example 12 and Fig. 13). A feed-forward inhibition model was used to describe the PD response. This model effectively described the time-dependent PK/RO/PD observed in the monkeys. The observed (open circles) and model-predicted endpoints (solid line) are shown in Figs. 16A (0.1 mg/kg, subcutaneous dosing) and 16B (10 mg/kg, subcutaneous dosing). The determined PK/PD parameters are presented in Table 7. When serum exposure data for the mAb variant 0318-IgG1.3f and TREM-1 RO were pooled for all monkeys, a concentration-dependent increase in RO was detected (see Figs. 16A and 16B). Based on the E _max model used to describe the concentration-RO relationship, the estimated EC ₅₀ of RO in vivo was 1.6 ± 0.2 nM.

Table 7

Estimates of the parameters of the PK/PD model used to describe PK/TC/PD data in monkeys and estimate predicted parameters in humans

FC parameters Description CV-based assessment (CV%) Predicted parameter estimate for a person Ka (h-1) Absorption rate constant 0.049 (15) 0.049 VI (ml/kg/kg) Volume of distribution in the central compartment 22 (5) 22 Ke (h-1) Elimination rate constant from the central compartment 0.003 (10) 0.0019 K12 (h-1) Distribution rate constants 0.024 (14) 0.015 K21 (h-1) 0.022 (7) 0.014 F (%) Bioavailability with SC administration 80 (5) 80 Kdeg (h-1) Velocity constant 0.09 (20) 0.09 Kint (h-1) The rate constant of internalization of the target-MAb complex 0.39 (18) 0.39 R0 (nmol/kg) Initial target level in the central compartment 0.024 (22) 0.024 KD (hM) Affinity of MAbs to TREM-1 0.64 (12) 0.24 Koff (h-1) Dissociation rate of MAbs from TREM-1 0.37 (7) 0.30 IC50 (hM) Concentration required to inhibit PGN+PGRP-mediated ex-vivo TREM-1 stimulation by 50% 1.9 (92) 0.1 E0 (%) PGN+PGRP-mediated ex vivo stimulation of TREM-1 (pre-dose) 100 (fixed value) 100

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Claims (31)

Table 8 SEQ ID Description Sequences 50 318-IgG1.3f, heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMHWVRQASGKGLEWVGRIRTKSSNYATYYAAS VKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRDMGIRRQFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 51 318-IgGl.lf heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMHWVRQASGKGLEWVGRIRTKSSNYATYYAAS VKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRDMGIRRQFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 52 318-IgGl-Aba, heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMHWVRQASGKGLEWVGRIRTKSSNYATYYAAS VKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRDMGIRRQFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 53 318-IgG4-Aba, heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMHWVRQASGKGLEWVGRIRTKSSNYATYYAAS VKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRDMGIRRQFAYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTK P ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 54 318-IgG1.3f, IgGl.lf, IgGlAba, IgG4-Aba, light chain DIVLTQSPDSLAVSLGERATINCRASQSVDTFDYSFLHWYQQKPGQPPKLLIYRASNLESGVPDR FSGSGSGTDFTLTISSLQAEDVAVYYCQQSNQDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLK SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKV YACEVTHQGLSSPVTKSFNRGEC This PCT application claims the benefit of US Provisional Patent Application 62/651605, filed April 2, 2018, the entire contents of which are incorporated herein by reference. CLAIM 1. An isolated antibody that specifically binds to trigger receptor-1 expressed on myeloid cells (TREM-1), containing CDR1, CDR2, CDR3 heavy chain; CDR1, CDR2, CDR3 light chain; and an IgG1 heavy chain constant region, in which the heavy chain CDR1, CDR2 and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively, in which CDR1, CDR2 and the light chain CDR3 comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively, and wherein the IgG1 heavy chain constant region contains three or more amino acid substitutions, where the substitutions include L234A, L235E and G237A (EU numbering), or in which the IgG1 heavy chain constant region contains three or more amino acid substitutions and one or more deletions, where the substitutions include L234A, L235E and G237A (EU numbering). 2. The antibody of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein VH comprises SEQ ID NO: 14 and VL comprises VL SEQ ID NO: 15. 3. The antibody of claim 2, comprising a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 85 and wherein the light chain contains SEQ ID NO: 54. 4. The antibody of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NO: 85 and wherein the light chain contains SEQ ID NO: 54. 5. The antibody according to claim 1, containing a heavy chain, wherein the heavy chain lacks a C-terminal lysine residue. 6. The antibody according to claim 1, wherein the IgG1 heavy chain constant region contains three amino acid substitutions, where the substitutions are L234A, L235E and G237A (EU numbering). 7. The antibody of claim 1, wherein the IgG1 heavy chain constant region contains three amino acid substitutions, wherein the substitutions are L234A, L235E and G237A (EU numbering), and wherein the C-terminal residue in the IgG1 heavy chain constant region is present. 8. The antibody of claim 1, wherein the IgG1 heavy chain constant region contains three amino acid substitutions, wherein the substitutions are L234A, L235E and G237A (EU numbering), and wherein the C-terminal residue in the IgG1 heavy chain constant region is absent. 9. The antibody according to claim 2, wherein the IgG1 heavy chain constant region contains three amino acids - 50 046142 new replacements, where the replacements are L234A, L235E and G237A (EU numbering). 10. The antibody of claim 2, wherein the IgG1 heavy chain constant region contains three amino acid substitutions, wherein the substitutions are L234A, L235E and G237A (EU numbering), and wherein the C-terminal residue in the IgG1 heavy chain constant region is present. 11. The antibody of claim 2, wherein the IgG1 heavy chain constant region contains three amino acid substitutions, wherein the substitutions are L234A, L235E and G237A (EU numbering), and wherein the C-terminal residue in the IgG1 heavy chain constant region is absent. 12. An isolated antibody that specifically binds to TREM-1 containing CDR1, CDR2, CDR3 heavy chain; CDR1, CDR2, CDR3 light chain; and an IgG1 heavy chain constant region, in which the heavy chain CDR1, CDR2 and CDR3 comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively, in which CDR1, CDR2 and the light chain CDR3 comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively, and in which the IgG1 heavy chain constant region contains three or more amino acid substitutions including L234A , L235E and G237A (EU numbering), and in which the C-terminal residue in the constant region of the IgG1 heavy chain is absent. 13. The antibody of claim 12, comprising a heavy chain variable region and a light chain variable region, wherein VH comprises SEQ ID NO: 14 and VL comprises VL SEQ ID NO: 15. 14. The antibody of claim 13, comprising a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NO: 85 and wherein the light chain contains SEQ ID NO: 54. 15. An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NO: 85 and wherein the light chain contains SEQ ID NO: 54. 16. A heavy chain for producing an antibody that specifically binds to TREM-1, comprising SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 85. 17. The heavy chain according to claim 16, comprising SEQ ID NO: 50. 18. The heavy chain of claim 16, comprising SEQ ID NO: 85. 19. An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NO: 50 and wherein the light chain contains SEQ ID NO: 54. 20. An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NO: 51 and wherein the light chain contains SEQ ID NO: 54. 21. An isolated antibody that specifically binds to TREM-1, comprising a heavy chain and a light chain, wherein the heavy chain contains SEQ ID NO: 52 and wherein the light chain contains SEQ ID NO: 54. 22. A bispecific molecule comprising an antibody according to any one of claims 1-15 or 19-21 linked to a molecule having a second binding specificity. 23. Nucleic acid encoding an antibody according to any one of claims 1-15 or claims 19-21. 24. An expression vector containing the nucleic acid according to claim 23. 25. Cell - antibody producer according to any of claims 1-15 or claims 19-21, containing the vector according to claim 24. 26. An immunoconjugate containing an antibody according to any one of claims 1-15 or claims 19-21, linked to a therapeutic agent. 27. A composition for the treatment of an inflammatory disease or an autoimmune disease, containing an antibody according to any one of claims 1-15 or claims 19-21, a bispecific molecule according to claim 22, a nucleic acid according to claim 23, a vector according to claim 24 or an immunoconjugate according to clause 26 and carrier. 28. A kit for the treatment of an inflammatory disease or an autoimmune disease, containing an antibody according to any one of claims 1-15 or 19-21, a bispecific molecule according to claim 22, a nucleic acid according to claim 23, a vector according to claim 24, or an immunoconjugate according to claim 28. 26 and instructions for use. 29. A method of inhibiting TREM-1 activity in an individual who needs it, comprising administering to the individual an antibody according to any one of claims 1-15 or claims 19-21, a bispecific molecule according to claim 22, a nucleic acid according to claim 23, a vector according to claim 24 or an immunoconjugate according to claim 26. 30. A method of treating an inflammatory disease or an autoimmune disease in an individual in need thereof, comprising administering to the individual an antibody according to any one of claims 1-15 or claims 19-21, a bispecific molecule according to claim 22, a nucleic acid according to claim 23, the vector according to claim 24 or the immunoconjugate according to claim 25. 31. The method of claim 30, 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, -