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

Description

Link to the list of sequences presented in electronic form on the EFS-WEB website

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

Prerequisites for creating the invention

TREM-1 is an activating receptor that is 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 once bound, 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. In addition, TREM-1 signaling synergistically interacts with numerous Toll-like receptors (TLRs), further enhancing proinflammatory signals. In turn, this causes up-regulation of TREM-1 expression, leading to a vicious cycle that increases inflammation (see Bouchon et al., J. Immunol., 164, 2000, pp. 4991-4995). Increasing evidence suggests that TLRs contribute to the onset and progression of chronic inflammatory diseases such as RA and IBD.

Humanized MAbs to TREM-1, which inhibit the function of TREM-1 in both humans and cynomolgus monkeys, are described in various documents (see WO 2013/120553 A1 and WO 2016/009086 A1). However, these antibodies either have a viscosity profile that hinders the manufacturing process or lead to 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 without causing the problems associated with previously developed anti-TREM-1 antibodies.

Summary of the invention

The present invention provides isolated antibodies, such as monoclonal antibodies, primarily human (eg, monoclonal) antibodies, that specifically bind to trigger receptor expressed on myeloid cells-1 (TREM-1) and have 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 contains one or more amino acid substitutions relative to the wild IgG1 heavy chain constant region type (SEQ ID NO: 9). In some embodiments, the antibody cross-competes with mAb 0318 for binding to TREM-1, blocking it, and contains a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, wherein the heavy chain constant region IgG1 contains 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 containing one or more amino acid residues selected from the group consisting of D38 , V39, K40, C41, D42, Y43, T44, L45, E46, K47, F48, A49, S50, S51, Q52, K53, A54, W55, Q56, Y90, H91, D92, H93, G94, L95 and L96 SEQ ID NO: 1. In some embodiments, the antibody specifically binds to the 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 heavy chain constant region of an IgG1 antibody as provided herein contains 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 IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M (EU numbering). In some embodiments, the IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S (EU numbering). In some embodiments, the IgG1 heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S (EU numbering) .

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In some embodiments, an antibody provided herein comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein 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 provided herein comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein 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 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 provided herein comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein 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 contains RASNLES (SEQ ID NO: 28) or RASNLES (SEQ ID NO: 28) except for one or two substitutions. In some embodiments, the light chain CDR3 contains 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 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%, is 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 In embodiments, the VL antibody 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 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: 15. In some embodiments, inventions VH and VL contain 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 Carrying out the invention, the light chain contains SEQ ID NO: 54.

The present invention provides 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, wherein CDR1, CDR2 and CDR3 of the heavy chain contain TYAMH (SEQ ID NO: 24).

RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) _and DMGIRRQFAY (SEQ ID NO: 26), respectively, where the light chain CDR1, CDR2 and CDR3 contain 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 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 containing

CDR1, CDR2, CDR3 heavy chain;

CDR1, CDR2, CDR3 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 CDR1, CDR2, and CDR3 light chain contain 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 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 heavy chain;

CDR1, CDR2, CDR3 light chain; and an IgG1 heavy chain constant region, wherein CDR1, CDR2 and CDR3 of the heavy chain contain TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) with DMGIRRQFAY (SEQ ID NO: 26) _{corresponding to} ^v and and respectively, where CDR1 , CDR2 and CDR3 of the light chain contain 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 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 containing

CDR1, CDR2, CDR3 heavy chain;

CDR1, CDR2, CDR3 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 CDR1, CDR2 and CDR3 light chain 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 one or more amino acid substitutions selected from the group that consists 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 provided herein has lower binding affinity for FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), FcyRIIIB (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 binding affinity for FcyRI ( CD64), lower 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-fold, at least nine-fold, or at least 10-fold 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, presented in SEQ ID NO: 54.

In some embodiments, an antibody provided herein is less immunogenic than 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 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 expression of a pro-inflammatory cytokine in immature dendritic cells (iDCs) when the cells are incubated in the presence of the antibody and in the absence of a stimulant, compared to an antibody containing a heavy chain that consists of the amino acid sequence set forth in SEQ ID NO: 76 , and a light chain which consists of the amino acid sequence shown in SEQ ID NO: 54.

In some embodiments, an antibody provided herein blocks the production of a pro-inflammatory cytokine in a cell when the cells are activated in the presence of both the antibody and the stimulant. In some embodiments, the stimulator is a ligand for TREM-1. In some embodiments, the proinflammatory cytokine is selected from the group consisting of IL-6, TNF-α, IL-8, ΙΕ-1β, IL-12, chitinase-3-like protein 1 (CHI3L1), and combinations thereof.

In some embodiments, an antibody of the present invention binds to human FcRn, cynomolgus monkey FcRn, and/or murine FcRn in a pH-dependent manner. In some embodiments, an antibody provided herein has greater thermal stability compared to a reference 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 , presented in SEQ ID NO: 54, according to

- 3 046142 measurements using a capillary differential scanning calorimeter (CAP-DSC). In some embodiments, when heated to 77°C, about 10 to 20%, about 20 to 30% (eg, 24%), or about 30 to 40% of the antibodies are reversible. In some embodiments, the antibody has a higher melting point ( _Tm ) 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 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 antibody has a viscosity of less than 10 cP (eg, 9 cP) at a concentration of 130 mg/ml.

In some embodiments, antibody binding to human TREM-1 has a KD value of less than 4 nM (eg, 3.4 nM) as measured by the Biacore method. In some embodiments, antibody binding to cynomolgus TREM-1 has a KD value of less than 1 nM (eg, 0.91 nM) as measured by the Biacore method.

In some embodiments, the antibody is monomeric as determined by gel filtration-high performance liquid chromatography (GP-HPLC) analysis. In some embodiments, the antibody has minimal risk of fragmentation as determined by two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC/MS) analysis or liquid chromatography-tandem mass spectrometry (LC/MS) analysis of intact protein mass. In some embodiments, the antibody has an isoelectric point of 8 to 9 (eg, 8.75).

In some embodiments, the antibody is stable in a composition containing histidine, sucrose, arginine and NaCl. In some embodiments, the antibody is stable for at least 2 months in a composition containing 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.

Also provided herein are bispecific molecules comprising an anti-TREM-1 antibody as provided herein, which is linked to a molecule having a second binding specificity.

Provided herein are nucleic acids encoding the antibody described herein, vectors containing the nucleic acids, and cells transformed with the vectors.

Provided herein are immunoconjugates containing the anti-TREM-1 antibodies described herein that are linked to an agent.

Provided herein are compositions containing anti-TREM-1 antibodies or antigen-binding fragments thereof, bispecific molecules or immunoconjugates as provided herein, and a carrier. Also provided herein are kits containing anti-TREM-1 antibodies or antigen-binding fragments thereof, bispecific molecules or immunoconjugates described herein, and instructions for their use.

Provided herein is a method of 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 defined herein.

Provided herein is a method of treating an inflammatory disease or an autoimmune disease in an individual in need thereof, comprising administering an anti-TREM-1 antibody, bispecific molecule, nucleic acid, vector, cell or immunoconjugate as defined 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, Sjögren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, other autoimmune diseases, which are the result

- 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.

In fig. 1A and 1B provide data demonstrating that all variants of mAb 0318 bind to human and cynomolgus monkey TREM-1 with an affinity similar to that of the parent mAb 0318 antibody (IgG4). In fig. 1A shows the binding affinity of the 318-IgG1.3f variant to human TREM-1 (top row) and cynomolgus monkeys (bottom row). In fig. Figure 1B shows the binding affinity of several different mAb 0318 variants to human TREM-1. The various variants presented include (I) 318-IgG1.1f, (II) 318-IgG1.3f, (III) 318-IgG4-Aba, (IV) 318-IgG1-Aba, and (V) 318-IgG1.1f (mutants with M replaced by Q). In fig. For comparison purposes, Figure 1B presents data on the binding affinity of the antibody mAb 0318 (IgG4 variants A and B).

In fig. 2A and 2B show internalization of mAb 0318-IgG1.3f after binding to TREM-1 expressed on CD14 ⁺ monocytes at various time points after binding to TREM-1. In fig. 2A shows the results of TREM-1 receptor expression analysis at 0, 6 and 24 hours after binding. In fig. 2B - results of analysis of TREM-1 receptor expression 0, 4 and 20 hours after binding. In fig. Figure 2B also shows data obtained using the TREM26 antibody, which does not compete with variants of the 0318 antibody for binding to TREM-1. The TREM26 antibody was used to assess the fate of the TREM-1 receptor after internalization (eg, whether it is degraded or returned back to the surface).

In fig. 3 - Demonstration that mAb 0318 variants do not have an agonistic effect on TREM-1 signaling as determined by the BWZ/hTREM-1 reporter cell assay in which the reporter cell line was incubated with (both) or without CHO-CD32 (BWZ 36 only) . Antibody variants presented 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). Antibody 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.

In fig. Figure 4 presents data on the effectiveness of various variants of mAb 0318 in blocking TREM-1-mediated production of proinflammatory cytokines in various human cells. The mAb 0318 antibody variants shown in FIG. 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 (namely, most red blood cells were removed per protocol RBC precipitation using dextran according to the procedure described in the examples). To produce proinflammatory cytokines, cells were stimulated with either plate-immobilized PGRP1 and soluble peptidoglycan lacking TLR2 activity (PGRP+PGN-Ecndss) or phorbol-12-myristate-13acetate (PMA)-stimulated neutrophils expressing PGRP1 (PMA-Stim Neutrophil Endogenous PGRP (endogenous PGRP of PMA-stimulated neutrophils)). The antibody variants presented 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 specific proinflammatory cytokine was not determined.

In fig. 5A and 5B show the effectiveness 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. In fig. 5A shows inhibition data obtained using a whole blood pharmacodynamic assay according to the method described in the Examples. In fig. 5B shows inhibition data obtained using an intracellular cytokine assay in whole blood according to the method described in the Examples.

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

In fig. 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 sweep for each point at constant temperature. The solid line represents Cree

- 5 046142 vyu obtained by nonlinear approximation of data using the best fit method. The dotted line indicates the maximum permissible viscosity level from an efficiency point of view.

In fig. 8 provides 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 (snail lymph hemocyanin) were used as positive controls. Avastin was used as a negative control.

In fig. 9A and 9B provide data demonstrating that all variants of mAb 0318 have the ability to bind to FcRn (human (black), cynomolgus monkey (white) and mouse (gray)) in a pH-dependent manner. In fig. 9A shows FcRn binding data as % Rmax (maximum ability to bind to FcRn). In fig. Figure 9B presents data on binding to FcRn in the form of sensorgrams. Presented variants of antibody 0318 include (I) IgG1-Aba, (II) IgG4-Aba, (III) IgG1.1f and (IV) IgG1.3f. For comparison purposes, data for mAb 0318 (IgG4) is also presented.

In fig. 10A and 10B provide data demonstrating that all variants of mAb 0318 have a reduced ability to bind to one or more human FcyRs (namely CD64, variant CD32a-H131, variant CD32a-R131, CD32b, variant CD16a-V158 and variant CD16B-NA2 ). In fig. 10A shows binding affinity data as %Rmax (maximum ability to bind FcyR). In fig. 10B shows sensorgrams. Presented variants of antibody 0318 include (I) IgG1-Aba, (II) IgG4-Aba, (III) IgG1.1f and (IV) IgG1.3f. For comparison purposes, data are presented for mAb 0318 (IgG4). Antibody lF4-hIgG1, which is known to bind to several FcyRs, was used as a control.

In fig. 11A-11I provide data demonstrating that the 0318 antibody variants alone (i.e., in the absence of a stimulus) are not agonistic, as demonstrated by measurements of the release of various proinflammatory cytokines (eg, IL-6, TNF-α, and IL-6). 12) when cultivated with immature dendritic cells. In fig. 11A, 11B and 11B show the amounts of IL-6 produced by immature dendritic cells isolated from donors D179, D276 and D341, respectively. In fig. 11D, 11E, and 11E show the amounts of TNF-α produced by immature dendritic cells isolated from donors D179, D276, and D341, respectively. In fig. 11G, 11H and 11I show the amounts of IL-12 produced by immature dendritic cells isolated from donors D179, D276 and D341, respectively. Presented antibody variants 0318 include (I) IgG1.1f, (II) IgG1.3f, and (III) IgG1-Aba. The doses (μg/ml) of antibodies are indicated in parentheses. Immature dendritic cells were cultured with anti-TREM-1 antibody variants in the presence and absence of CHO cells expressing membrane CD32a (to stimulate Fc cross-linking). PGRP+PGN, CD40L trimer (Trimer), and Pfizer CD40 agonist antibody (CP870) were used as positive controls. An isotype antibody (CHIL-6) was used as a negative control. The dotted line indicates the lower limit of detection of the assay.

In fig. 12 - pharmacokinetic characteristics of the mAb 0318-IgG1.3f variant in the body of the cynomolgus monkey after subcutaneous administration of a single dose. Each animal was administered 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 kg (n=3) (3) or (IV) 10 mg/kg (n=4) (4). Data are presented as mean ± standard deviation.

In fig. 13 shows a target-mediated drug distribution (TMDD) diagram used to describe pharmacokinetics (PK), pharmacodynamics (PD), and receptor occupancy (RO) data in cynomolgus monkeys.

In fig. 14A and 14B show the total TREM-1 receptor density on monocytes (FIG. 14A) and granulocytes (FIG. 14B) following administration (subcutaneous (sc) or intravenous (iv)) of a single dose of mAb 0318-IgG1.3f cynomolgus variant to monkeys. TREM-1 receptor density is presented as a percentage of TREM-1 receptor density before antibody administration. Each of the animals 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) (D) Data are presented as mean ± standard deviation.

In fig. 15A and 15B show TREM-1 receptor occupancy on monocytes (FIG. 15A) and granulocytes (FIG. 15B) following administration of a single dose of the mAb 0318-IgG1.3f variant to cynomolgus monkeys. Receptor occupancy data are presented as a percentage of the total number of TREM-1 receptors expressed on cells. Each animal was administered one of the following doses: (a) 0.1 mg/kg (sc) (n=4) (A), (b) 0.5 mg/kg (n=4) (sc) (B), (c) 2 mg/kg (n=3) (sc) (C), (d) 2 mg/kg (n=3) (iv) (D) or (e) 10 mg/kg (n=4) (sc) (D). Data are presented as mean ± standard deviation. The dotted line corresponds to 85% receptor occupancy.

In fig. 16A and 16B show experimental (open circles) and model-predicted (solid line) estimates of PK (upper left panel), PD (upper right panel), RO (lower left panel), and total surface expression of the TREM- receptor. 1 (lower right panel) following single dose administration of the mAb 0318-IgG1.3f variant to cynomolgus monkeys using a 2-compartment PK model with TMDD for theoretical evaluation.

- 6 046142 in the central compartment. In fig. 16A shows data obtained when each monkey was administered a single dose of antibody subcutaneously at 0.1 mg/kg. In fig. 16B presents data obtained from subcutaneous injection of a single dose of antibody of 10 mg/kg into animals.

Detailed Description of the Invention

To facilitate understanding of the present description, definitions of certain concepts are first given. Additional definitions are provided in the detailed description text.

It should be understood that the use of an element in the singular implies its use in the plural; for example, the term nucleotide sequence is understood to mean 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.

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

Thus, it should be borne in mind that the concept and/or 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). Likewise, it should be kept in mind that the concept and/or used in an expression such as A, B and/or C includes the following variants: A, B and C; A, B or C; A or B; A or B; B or C; A and B; A and B; B and C; A (individually); B (individually) and C (individually).

It should be understood that when the term comprising is used with respect to any aspects herein, it also includes similar aspects otherwise described by the terms consisting of and/or substantially consisting of.

Unless otherwise indicated, all technical and scientific terms used in this description have the meaning known to one of ordinary skill in the field to which the present invention relates. 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 the Oxford Dictionary Of Biochemistry And Molecular Biology, updated edition, Oxford University Press, 2000, are widely known dictionaries in which many of the terms used herein can be found by one skilled in the art.

Units, prefixes and symbols are used in a form consistent with the International System of Units (SI). Numeric ranges include numbers that define the boundaries of the range. Unless otherwise noted, nucleotide sequences are written from left to right in a 5'^3' orientation. Amino acid sequences are written from left to right in an amino-terminal to carboxy-terminal orientation. The headings given in this description do not limit the scope of the various objects of the invention, which is determined by the entire application as a whole. Accordingly, the concepts defined immediately below are more fully defined by the application as a whole.

The term approximately, as used herein, means approximately, roughly, about, or in the area thereof. When the concept is approximately used in relation to a numerical range, it modifies the specified range by expanding the boundaries of the above and below specified numerical values. In general, the concept can roughly modify a numerical value to increase or decrease the specified value by a difference of, for example, 10%, up or down (making it higher or lower).

The term trigger receptor expressed on myeloid cells 1 (also known as TREM1, TREM-1 and CD354) refers to a receptor that is expressed on monocytes, macrophages and neutrophils. The main ligand for TREM-1 is peptidoglycan recognition protein 1 (PGLYRP1), which belongs to the peptidoglycan (PGN)-binding proteins (PGRP) family. Once activated, TREM-1 associates with the ITAM-containing signaling adapter protein, DAP12. Downstream signaling may involve activation of the transcription factor NFAT, causing upregulation of proinflammatory cytokine production. The term TREM-1 includes any variants or isoforms of TREM-1 that are naturally expressed by cells. Therefore, in some embodiments, antibodies provided herein may cross-react with TREM-1 from non-human species (eg, TREM-1 from cynomolgus monkeys).

Three isoforms of TREM-1 have been identified. Isoform 1 (registration number NP_061113.1; SEQ ID NO: 1) consists of 234 amino acids and is a canonical sequence. Isoform 2 (registration number NP_001229518.1; SEQ ID NO: 2) consists of 225 amino acids and differs from the canonical sequence by amino acid residues 201-234. The amino acid residues encode part of the transmembrane domain and the cytoplasmic domain. Isoform 3 (registration 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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Below are the amino acid sequences of the three known isoforms of human TREM-1.

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

KAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVE DSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKA LCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKS LVFSVLFAVTLRSFVP (signal sequence is underlined);

(b) Human TREM-1 isoform 2 (accession number NP_001229518.1; SEQ ID NO: 2; encoded by the nucleotide sequence having 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 accession number NM_001242590; SEQ ID NO: 6):

MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGOTLDVKCDYTLEKFASSO

KAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVE

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

The TREM-1 protein of 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 is underlined).

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

Antibodies to TREM-1 provided herein block TREM-1 signaling through one or a combination of several different mechanisms, blocking TREM-1 directly or indirectly. In one embodiment, the antibodies prevent the natural ligand for TREM-1, peptidoglycan recognition protein 1 (PGLYRP1), from forming a functional complex with TREM-1. In another embodiment, 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 caused by anti-TREM-1 antibodies having the ability to bind to the portion of TREM-1 that would otherwise be at the interface in the TREM-1 dimer, thereby preventing the association of individual TREM-1 molecules with each other. In other embodiments, the reduction or prevention of TREM-1 dimerization or multimerization is caused by anti-TREM-1 antibodies that interfere with the interaction of TREM-1 with its ligand.

In some embodiments, antibodies to TREM-1 can block PGLYRP1-induced activation of TREM-1. PGLYRP1, a highly conserved 196 amino acid protein that consists of a signal peptide and a peptidoglycan binding domain, is expressed on neutrophils and released upon their activation. Below is the amino acid sequence of PGLYRP1 (registration number NP_005082.1; SEQ ID NO: 8):

- 8 046142

MSRRSMLLAWALPSLLRLGAAQETEDPACCSPIVPRNEWKALASECAOHLSLPL RYVVVSHTAGSSCNTPASCQQQARNVQHYHMKTLGWCDVGYNFLIGEDGLVY EGRGWNFTGAHSGHLWNPMSIGISFMGNYMDRVPTPQAIRAAQGLLACGVAQ GALRSNYVLKGHRDVQRTLSPGNQLYHLIQNWPHYRSP sequence is underlined).

Thus, in some embodiments, the anti-TREM-1 antibodies provided herein down-regulate or block the release of pro-inflammatory cytokines from myeloid cells (eg, dendritic cells and monocytes). In some embodiments, anti-TREM-1 antibodies block the release of TNF-α, MIP-1 beta, MCP-1, IL-1 beta, GM-CSF, IL-6, and/or IL-8 from macrophages, neutrophils, synovial tissue cells and/or reporter cells, as demonstrated herein.

Although the controlled release of proinflammatory cytokines in response to foreign antigens can be beneficial (eg, induce an effective adaptive immune response), excessive release of proinflammatory cytokines can have severe consequences. For example, one common and toxic complication that has been observed with the in vivo administration of certain antibodies against immune receptors on the surface of immune cells (e.g., anti-human CD3 antibodies such as OKT3) is cytokine release syndrome (CRS), which is associated with excessive release of various cytokines (eg, TNF-alpha, IFN-gamma and IL-2) into the bloodstream. CRS can result from simultaneous binding of antibodies to their cognate antigen (eg, CD3 on T cells) (via the antibody variable region) and Fc receptors (eg, FcyR) and/or complement receptors (via the antibody constant region) on supporting cells (for example, on antigen presenting cells). This interaction results in the activation of cells (eg, T cells and/or support cells) and the release of various cytokines, which cause 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 provided herein, in addition to blocking PGLYRP1-induced production of pro-inflammatory cytokines, result in a reduction in or elimination of the occurrence of cytokine release syndrome when administered to an individual in need thereof. In some embodiments, antibodies to TREM-1 do not induce expression of pro-inflammatory cytokines by cells (eg, dendritic cells) when the cells are incubated in the presence of the antibody alone, compared to an antibody containing 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, anti-TREM-1 antibodies have a reduced ability to bind to one or more FcyRs, thereby reducing 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, TREM-1 includes any naturally occurring form of TREM-1, which may be derived 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 TREM-1 (such as a human, chimpanzee, cynomolgus monkey, or rhesus monkey); 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, TREM-1 has the sequence SEQ ID NO: 1 (human TREM-1, isoform 1). TREM-1 may be a mature form of TREM-1, such as TREM-1 protein, which has been post-translationally processed in the appropriate cell. Such a mature TREM-1 protein may, for example, be glycosylated. TREM-1 may be the full-length TREM-1 protein

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

In some embodiments, the anti-TREM-1 antibodies provided in the infusion

- 9 046142 in the general description, are recombinantly expressed in prokaryotic or eukaryotic cells. In some embodiments, the prokaryotic cell is E.coH. In specific embodiments, the eukaryotic cell is a yeast, insect, or mammalian cell, such as a cell derived from an organism that is a primate (such as a human, chimpanzee, cynomolgus monkey, or rhesus monkey); 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. Antibodies to TREM-1 presented herein can also be produced using other methods known to one skilled in the art, such as phage display or yeast display. Once produced, 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 WO 2013/120553.

The term antibody as used herein refers to a protein derived from a germline immunoglobulin sequence that has the ability to specifically bind to an antigen (TREM-1) or a portion thereof. The concept 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 part thereof may bind only to that antigen or part thereof, or it may bind to a limited number of homologous antigens or parts thereof. Full-length antibodies typically contain at least four polypeptide chains: two heavy (H) chains and two light (L) chains, which are linked together by disulfide bonds.

One subclass of immunoglobulins of pharmaceutical interest is the IgG family. In humans, the IgG class can be divided into 4 subclasses: IgG1, IgG2, IgG3 and IgG4 based on the sequences of their heavy chain constant regions. Light chains can be divided 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 of which is attached to the heavy chain by a disulfide bond. The heavy chain may contain a heavy chain variable region (VH) and up to three heavy chain constant regions (CH): CH1, CH2 and CH3. The light chain may comprise 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), between which are more conserved regions called framework regions (FRs). The VH and VL regions typically consist of three CDRs and four FRs, which are arranged in the amino-terminal to carboxy-terminal direction 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 binding of the immunoglobulin to host tissues or factors, including (but not limited to) various cells of the immune system (effector cells) , Fc receptors and the first component (C1q) of the classical complement system. The antibodies of the present invention may be in isolated form. The term isolated antibody refers to an antibody that is separated and/or isolated 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. Certain antigen-binding antibody fragments may be considered within the context of the present invention if it is demonstrated that the full-length antibody fragments can perform the antigen-binding function of the antibody.

The term antigen-binding antibody fragment refers to one or more antibody fragment(s) that retain the ability to specifically bind an antigen, such as TREM-1, as provided 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 the antibody), single-chain Fv- (scFv; see, for example, Bird et al., Science, 242, 1988, pp. 42S-426; Huston et al., PNAS, 85, 1988, pp. 5879-5883), dsFv-, Fd - (usually VH- and CH1-domains) and dAb- (usually VH-domain) fragments; VH, VL, VhH and V-NAR domains; monovalent molecules containing one VH and one VL chain; miniantibodies, diabody, triabody, tetrabody and kappa chain antibodies (see, for example, I11 et al., Protein Eng., 10, 1997, pp. 949-957); camel IgG; IgNAR; and one or more isolated CDRs or functional paratope, wherein the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked to each other to form a functional antibody fragment. Various types of antibody fragments are described or reviewed, for example, in Holliger and Hudson, Nat. Biotechnol., 2S, 2005, p. 1126-1136; in international patent application publication WO 2005/040219 and in US patent publications 2005/0238646 and 2002/0161201. These antibody fragments can be prepared 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 region and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, then the constant region is also derived from human germline immunoglobulin sequences. Antibodies to TREM-1 provided herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced through random or site-directed mutagenesis in vitro or through somatic mutation in vivo). However, as used herein, the term human antibody does not include antibodies in which CDR sequences derived from the germ line of other mammalian species, such as mice, are grafted into human framework sequences. The terms human antibodies and fully human antibodies are used interchangeably.

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 an immunoglobulin of a non-human species. Thus, a humanized antibody is a human immunoglobulin (recipient antibody) in which at least residues from the hypervariable region of the recipient antibody are replaced by residues from the hypervariable region of an antibody of a non-human species (donor antibody), such as mice, rats, rabbits or non-human primates, which have the required specificity, affinity, sequence composition and functionality. In some cases, human immunoglobulin FR residues are replaced with corresponding non-human residues. An example of such a modification is the introduction of one or more so-called reverse mutations, which are usually amino acid residues originating from the donor antibody. Humanization of an antibody can be accomplished using recombination techniques known to one skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. S. Lo). Suitable human recipient framework regions for both light and heavy chain variable domains can be identified, for example, based on sequence homology or structural homology. Alternatively, fixed recipient scaffolds can be used, for example based on structural knowledge, biophysical and biochemical properties. The recipient framework regions may be derived from the germ line or from the mature antibody sequence. CDR regions from a donor antibody can be transferred by CDR transplantation. The CDR-grafted humanized antibody can be further optimized, for example, with respect to affinity, functionality and biophysical properties by identifying critical positions in the framework region at which reintroduction (back mutation) of an amino acid residue from the donor antibody has a beneficial effect on the properties of the humanized antibody. . In addition to the use of donor antibody-derived back mutations, a humanized antibody can be generated by the introduction of germline residues into CDRs or framework regions, elimination of immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

In addition, 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 characteristics of the antibody. In general, a humanized antibody will contain at least one, usually 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 are residues from a human immunoglobulin sequence. The humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically human immunoglobulin. The term humanized antibody derivative refers to any modified form of a humanized antibody, such as a conjugate containing an 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 by recombination techniques, such as (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transchomosomal with human genes. immunoglobulin or derived from hybridoma;

(b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectome;

(c) antibodies isolated from a recombinant combinatorial library of human antibodies; and (d) antibodies produced, expressed, created or isolated by any other method that involves splicing 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 include subsequent transformations and mutations occurring, for example, during antibody maturation. As is known in the art (see, for example, Lonberg, Nature Biotech., 23(9), 2005, pp. 1117-1125), variable regions contain an antigen binding domain encoded by various genes, which is converted to produce an antibody specific for foreign antigen. In addition to transformation, the variable region can be further modified by several single amino acid substitutions (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody for a foreign antigen. The constant region can be changed to further respond to the antigen (ie, by isotype switching). Thus, nucleic acid molecules that encode immunoglobulin light chain and heavy chain polypeptides that have been converted 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. .identical at least 80%).

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, for example, an antibody in which the variable regions are derived from a murine antibody and the constant regions are derived from a human antibody.

In one embodiment, the anti-TREM-1 antibodies described herein are in the form of 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 the naturally occurring IgG antibody of the same subclass.

For example, the anti-TREM-1 IgG1 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 similarly arranged disulfide bridges as in naturally occurring antibody in the form of IgG1 (unless the antibody has been mutated to modify disulfide bridges).

As used herein, the term isotype refers to a class of antibodies (eg, an antibody in the form of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE) that is encoded by heavy chain constant region genes.

The term allotype refers to naturally occurring variants in a particular isotype group, where the variants differ by several amino acids (see, for example, Jefferis et al., mAbs 1, 2009, p. 1). Antibodies to TREM-1 presented herein can be of any allotype. In some embodiments, anti-TREM-1 antibodies have an IgG1.3f' allotype that contains 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 (eg SEQ ID NO: 9). In other embodiments, anti-TREM-1 antibodies have an IgG1.1f allotype that contains one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S (EU numbering), compared to isotype Wild-type IgG1 (eg, SEQ ID NO: 9). In specific embodiments, anti-TREM-1 antibodies have an IgG1-Aba allotype that contains one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S (EU numbering), compared to the wild IgG1 isotype type (eg SEQ ID NO: 9). In other embodiments, anti-TREM-1 antibodies have an IgG4-Aba allotype that contains the CH1 domain of the wild-type IgG4 isotype (eg, SEQ ID NO: 10) and the CH2 and CH3 domains of IgG1. In some embodiments, an IgG4-Aba allotype antibody contains one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S (EU numbering) , compared to the wild type IgG1 isotype (eg, SEQ ID NO: 9).

The terms antigen-recognizing antibody and antigen-specific antibody are used interchangeably herein with the term antibody that specifically binds an antigen.

As used herein, the term isolated antibody refers to an antibody that is separated and/or isolated from/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 was obtained.

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 resulting therefrom. 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 down-regulation.

- 12 046142 properties (for example, B-cell receptor; BCR). Such effector functions typically require association of an Fc region with a binding domain (eg, an antibody variable domain). In one embodiment, the anti-TREM-1 antibodies provided herein contain Fc regions that do not bind one or more FcyRs and therefore do not have effector function (ie, are not effectors).

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

The term Fc region (a region that is a crystallizable fragment) or Fc domain or Fc refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulin to host tissues or factors, including binding to Fc receptors found on various cells of the immune system (for example, on effector cells) or with the first component (C1q) of the classical complement system. Thus, the Fc region contains the antibody constant region excluding the first domain of the immunoglobulin constant region (eg, CH1 or CL).

In IgG, the Fc region contains the immunoglobulin domains CH2 and CH3 and the hinge between the CH1 and CH2 domains. Although the definition of the boundaries of the immunoglobulin heavy chain Fc region may vary, for purposes of this disclosure, the human IgG heavy chain Fc region is defined as the segment extending from amino acid residue D221 in the case of IgG1, V222 in the case of IgG2, L221 in the case of IgG3, and P224 in the case of IgG4. , to the carboxy end 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 on the C-terminal side of the CH2 domain in the Fc region, i.e. it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is missing), or 445 (if the C-terminal glycine and lysine residues are missing) of IgG. As used herein, an Fc region may be a native sequence Fc, including any allotype variants, or a variant Fc (eg, a non-naturally occurring Fc). The term Fc may also refer to the specified region, considered individually, or in the context of an Fc polypeptide-containing protein, such as an Fc region-containing binding protein, also referred to as an Fc fusion protein (eg, an antibody or immunoadhesive agent).

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

having the native sequence of a human IgG2 Fc region;

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

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

An Fc region sequence variant or non-naturally occurring Fc contains a modification, typically serving 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 lack thereof. In some embodiments, the anti-TREM-1 antibodies provided herein may be modified chemically (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, the anti-TREM-1 antibody is an IgG1 isotype antibody and carries a modified Fc domain containing one or more and possibly all of the following mutations that result in reduced affinity for certain Fc receptors (L234A, L235E and G237A ) and to reduced C1q-mediated complement fixation (A330S and P331S) respectively

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

The terms hinge, hinge domain, hinge region, and hinge region of an antibody refer to the heavy chain constant region domain 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., 161 , 1998, p. 4083). The hinge allows for varying degrees of flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide binding of the constant regions of the two heavy chains. As used herein, the hinge begins at Glu216 and ends at Gly237 in all IgG isotypes (Roux et al., J. Immunol., 161, 1998, p. 4083). The wild-type IgG1, IgG2, IgG3 and IgG4 hinge sequences are known in the art (see, for example, international patent application publication WO 2017/087678). In one embodiment, the CH1 hinge region of anti-TREM-1 antibodies is modified to change, for example, increase or decrease, the number of cysteine residues in the hinge region. This approach is described in more detail, for example, in US No. 5677425.

The constant region can be modified to stabilize the antibody, for example, to reduce the risk of the divalent antibody separating into two monovalent VH-VL fragments. For example, in the IgG4 constant region, residue S228 (residues numbered according to the EU index) can be mutated to a proline (P) residue to stabilize the formation of a disulfide bridge at the heavy chain hinge (see, e.g., 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 regions of an antibody in which amino acid residues involved in binding to antigen are located. The hypervariability 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 Cabot 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 light chain variable domain and 31 -35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) of the heavy chain variable 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, CDRs can be defined as regions containing residues from the hypervariable loop (residues 26-33 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53- 55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk, J. Mol. Biol., 196, 1987, pp. 901-917).As a rule, the numbering of amino acid residues in this region is carried out with using the method described in Kabat et al., supra. Expressions such as Cabot position, Cabot residue, and Cabot residue, as used herein, refer to the specified numbering system for heavy chain variable domains and light chain variable domains. When using the system Cabot numbering, the actual linear amino acid sequence of the peptide may contain fewer amino acids or additional amino acids, corresponding to a truncation or insertion in the framework region (FR) or CDR of the variable domain. For example, the heavy chain variable domain may include amino acid insertions (residues 52a, 52b and 52c according to Cabot) after residue 52 of CDR H2 and residues inserted by insertion (for example, residues 82a, 82b and 82c, etc. according to Cabot) after residue 82 of the heavy chain FR. Cabot residue numbers can be determined for a subject antibody by comparative analysis of regions of homology between the antibody sequence and a standard Cabot numbered sequence.

The term epitope or antigenic determinant refers to a site on an antigen (eg, TREM-1) to which an immunoglobulin or antibody specifically binds, as determined, for example, by a specific method designed to identify it. Epitopes can be formed by both continuously located amino acids (usually in the case of a linear epitope) and non-continuously located amino acids that are brought closer to each other as a result of tertiary folding of the protein (usually in the case of a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when exposed to denaturing solvents. An epitope typically comprises 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 bind to the antibody of interest (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays that test for overlapping or continuous peptides (e.g., from TREM-1) to assess reactivity against the antibody in question (eg, anti-TREM-1 antibody). Methods for determining the spatial conformation of epitopes include methods known in the art and methods presented herein, for example, X-ray crystallography, antigen mutational 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 provided herein include, for example, epitope mapping techniques such as X-ray crystal analysis of antigen:antibody complexes, which provides epitope-level resolution atoms, and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of an antibody to antigen fragments or mutant variants of the antigen, where loss of binding ability due to modification of an amino acid residue in the antigen sequence is often taken as an indication of the presence of an epitope component. In addition, computational combinatorial methods can also be used for epitope mapping. These methods rely on the ability of the antibody of interest to isolate specific short peptides based on affinity from combinatorial phage display peptide libraries. Antibodies having the same VH and VL sequences or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

The term antibodies that compete with another antibody for binding to a target refers to antibodies that inhibit (partially or completely) the binding of another antibody to a target. The question is whether two antibodies compete with each other for binding to the target, i.e. Whether and to what extent one antibody inhibits the binding of another antibody to its target can be determined using known competition experiments, such as the BIACORE® surface plasmon resonance (SPR) assay. In some embodiments, the 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 (ie, the cold antibody that is first incubated with the target). Competition assays can be performed according to methods described, for example, in Cold Spring Harb Protoc, ed. Harlow and David Lane, 2006, DOI: 10.1101/pdb.prot4277; or in Chapter 11 Using Antibodies, ed. Harlow and David Lane, 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 is first brought into contact with the antigen in a competition assay.

As used herein, the terms specific binding, selective binding, selectively binding, and specifically binding refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, antibody binding is characterized by (I) a dissociation reaction equilibrium constant (K _D ) of less than about 10 ^-7 M, for example, less than about 10 ^-8 , 10 ^-9 or 10 ^-10 M or even less, at its determination, for example, using surface plasmon resonance (SPR) technology using a BIACORE® 2000 device using a predefined antigen, for example, recombinant human TREM-1, as an analyte and an antibody as a ligand, or an antibody binding assay with positive antigen by cells using the Scatchard method; and (II) a binding affinity for a predetermined antigen that is at least twice its binding affinity for a nonspecific antigen (eg, 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 value 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 TREM-1 refers to an antibody that binds to cynomolgus TREM-1 with a KD value 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 have substantially undetectable levels of binding to the 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 fragment thereof, with a single antigen or with a limited number of highly homologous antigens (or epitopes). Conversely, antibodies that have the ability to specifically bind to TREM-1 do not have the ability to bind to molecules dissimilar to it. Antibodies of the invention may not have the ability to bind to Nkp44, a natural killer cell p44-related protein.

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

- 15046142 The abilities of ligands (such as antibodies) to bind to their targets are known in the art and include, for example, ELISA, Western blotting, RIA and flow cytometry analysis. The binding kinetics and binding affinity of an antibody can also be assessed using standard assays known in the art, such as SPR.

Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include a competition binding assay for myeloid cells expressing TREM-1, for example, using a flow cytometry technique such as that described in the Examples section. Other methods include SPR (eg, BIACORE®), direct or indirect radioimmunoassay (RIA), 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 assay using a labeled compound, solid-phase direct sandwich assay using a labeled compound (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988); solid phase direct RIA using a labeled compound 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 RIA using a labeled compound (Moldenhauer et al., Scand. J. Immunol., 32, 1990, p. 77).

In the context of the present description, the concept group is defined in relation to the reference entity. If the second antibody cannot bind to an antigen at the same time as the reference antibody, then the second antibody is considered to be in the same group as the reference antibody. In this case, the reference antibody and the second antibody bind to the same part of the antigen and are designated as competing antibodies. If the second antibody is able to bind to the antigen at the same time as the reference antibody, then the second antibody is considered to be in a different group. In this case, the reference antibody and the second antibody do not compete to bind to the same part of the antigen and are referred to as non-competing antibodies.

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

As used herein, the term binding affinity refers to the measurement of the strength of the non-covalent interaction between two molecules, for example, an antibody or fragment thereof and an antigen. The concept of binding affinity is used to describe monovalent interactions (activities inherent in molecules).

The binding affinity of two molecules, for example, an antibody or fragment thereof and an antigen, due to a monovalent interaction, can be quantified 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 formation and dissociation of the complex, for example, using the SPR method. The rate constants corresponding to the association and dissociation of a monovalent complex are denoted as the association reaction rate constant k _a (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 KD values for individual antibody/antigen complexes.

As used herein, the term high affinity for a target antigen for an IgG antibody refers to an antibody having a K _D value of 10 ^-8 M or less, 10 ^-9 M or less, or 10 ^-10 M or less. However, the high binding affinity may differ from that reported for other antibody isotypes. For example, high binding affinity for an IgM isotype refers to an antibody having a K _D value of 10 ^-10 M or less or 10 ^-8 M or less.

The concept of 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 which induces a response of 50% of the maximal response, i.e. is midway between the maximum response and the baseline level.

As used herein, the term naturally occurring when applied to an object reflects the fact that the object may be present in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified by humans in the laboratory is a naturally occurring sequence.

- 16 046142

The term polypeptide refers to a chain containing at least two sequentially linked amino acid residues, and there is no upper limit on the length of the chain. One or more amino acid residues in a protein may contain a modification such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A protein may contain one or more polypeptides.

As used herein, the term nucleic acid molecule includes DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded and may be a cDNA.

Conservative amino acid substitutions refer to replacements of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. 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, for example, 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).

In the context of nucleic acids, the term substantial homology means that two nucleic acids or sequences representing them, when optimally aligned and compared, taking into account corresponding nucleotide insertions or deletions, have at least about 80% of the nucleotides, at least about 90 to 95%, or about 98 to 99.5% of nucleotides are identical. Alternatively, significant homology occurs when the segments hybridize under selective hybridization conditions with the complement of the chain.

In the context of polypeptides, substantial homology means that two polypeptides or the sequences designating them, when optimally aligned and compared, taking into account corresponding amino acid insertions or deletions, have at least about 80% of the nucleotides, at least about 90 to 95%, or about 98 to 99.5% of 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 . Comparison of sequences 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 percentage identity of two nucleotide sequences can be determined using the GAP program included in the GCG software package (available on the Internet site web.gcg.com), using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and weights of length 1, 2, 3, 4, 5 or 6. The percentage 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 into the ALIGN program (version 2.0) using a table of residue weights, a gap length penalty of 12 and a gap penalty of 4. Additionally, 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 included in the GCG software package (available on the Internet site web.gcg.com), using either a Blossum 62 matrix or a RAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6.

The nucleotide and protein sequences presented herein can also be used as query sequences to search public databases, for example, to identify related sequences. Such searches can be performed using the programs NBLAST and XBLAST (version 2.0) described in Altschul et al., J. Mol. Biol., 215, 1990, p. 403-410. Nucleotide sequence searches using the BLAST algorithm can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules presented herein. BLAST searches for protein sequences can be performed using the XBLAST program, score=50, word length=3, yielding amino acid sequences homologous to the protein molecules presented herein. To perform alignments using gaps for comparison purposes, you can use

- 17 046142 use the Gapped BLAST program according to the procedure described in Altschul et al., Nucleic Acids Res., 25(17), 1997, p. 3389-3402. When using the BLAST and Gapped BLAST programs, you can use the default settings of the corresponding programs (for example, XBLAST and NBLAST) (see worldwideweb.ncbi.nlm.nih.gov).

The nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially purified form. A nucleic acid is isolated or considered to be substantially pure if it is purified from other cellular components or other impurities, such as other nucleic acids present in the cell (for example, from other parts of a chromosome) or proteins, by standard methods, including alkali treatment. SDS, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art (see Current Protocols in Molecular Biology, edited by F. Ausubel et al., Greene Publishing and Wiley Interscience, New York, 1987).

Nucleic acids, such as cDNA, can be mutated according to standard methods to obtain gene sequences. In the case of coding sequences, these mutations may affect the amino acid sequence, if desired. In particular, DNA sequences may be considered that are substantially homologous to or derived from native segments of the V, D, J genes, constant sequences, switches, and other similar sequences presented herein (where derived means that the sequence is identical to or derived from another sequence from it by modification).

As used herein, the term vector refers to a nucleic acid molecule that is 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 in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal vectors for mammalian cells). Other vectors (eg, non-episomal vectors for mammalian cells) can be integrated into the host cell genome by introduction into the host cell, where they replicate along with the host genome. In addition, 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. In the present description, the terms plasmid and vector can be used interchangeably, since plasmid is the most widely used form of vector. However, the concept also includes other forms of expression vectors, such as viral vectors (eg, retroviruses, adenoviruses and replication-defective adeno-associated viruses) that perform equivalent functions.

As used herein, the term recombinant host cell (or simply host cell) is intended to mean 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 concepts refer not only to the particular cell in question, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations, either due to mutations or due to environmental influences, such progeny may not actually be identical to the parent cell, but it still falls within the scope of the concept of host cell as used herein.

As used herein, the term linked refers to the association of two or more molecules. The bond may be covalent or non-covalent. The link may also be due to genetic reasons (ie, obtained by recombinant fusion). Such bonds can be created using a wide variety of techniques accepted in the art, 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 the body of an individual using any of the various methods and delivery systems known to those skilled in the art. Various routes of administration of the anti-TREM-1 antibody provided herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes, such as 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 lar, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as electroporation in vivo. Alternatively, the antibody provided herein can be administered by a non-parenteral route, such as topical, epidermal, or mucosal administration, such as intranasally, orally, vaginally, rectally, sublingually, or topically. The 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 inhibit or block (eg, in relation to inhibiting/blocking TREM-1 ligand binding to TREM-1 on cells) are used interchangeably and include both partial and complete inhibition/blocking. In some embodiments, the anti-TREM-1 antibody inhibits the binding of a ligand for TREM-1 to TREM-1 by at least about 50%, such as about 60, 70, 80, 90, 95, 99, or 100% as assessed according to the methods presented later in this description. In some embodiments, the anti-TREM-1 antibody inhibits the binding of the ligand for TREM-1 to TREM-1 by no more than 50%, such as about 40, 30, 20, 10, 5, or 1%, as assessed in the following. this description of the methods.

The terms treat, treatment and treatment as used herein refer to any type of intervention or process carried out on an individual, or the administration of an active substance to an individual, for the purpose of reversing, alleviating, reducing, inhibiting or slowing down, or preventing progression, development, severity or relapse a symptom, complication, condition or biochemical parameter associated with a disease, or an increase in overall survival. Treatment may be given to an individual having a disease or to an individual who does not have a disease (eg, for prophylaxis).

The term effective dose or effective dosage means 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 drug that, when administered alone or in combination with another therapeutic agent, enhances disease regression as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or preventing 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 a drug that, when administered alone or in combination with another therapeutic agent to an individual at risk of developing a disease or suffering from a relapse of a disease, inhibits development or relapse of the disease. The ability of a therapeutic agent to enhance disease regression or inhibit disease progression or recurrence can be assessed by various methods known to the practitioner, for example, through human clinical trials, animal model systems to predict efficacy in humans, or by analysis of agent activity in in vitro assays.

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

As used herein, the term individual includes any human or non-human animal. For example, the methods and compositions presented herein can be used to treat an individual having 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.

As used herein, 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 the present description are described in more detail.

I. Antibodies to TREM-1.

Provided herein are antibodies, for example fully human antibodies, that have specific functional characteristics or properties. For example, the antibodies provided herein specifically bind to human TREM-1 and more specifically to a specific domain (eg, 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 (eg, PGLYRP1) binds. In some embodiments, the antibodies are antagonistic antibodies, i.e. they inhibit or suppress TREM-1 activity (ie, do not have agonistic activity upon binding) on cells such as monocytes, macrophages and neutrophils. In some embodiments, anti-TREM-1 antibodies cross-react with TREM-1 from one

- 19 046142 or several non-human primates, such as TREM-1 cynomolgus monkeys. In some embodiments, anti-TREM-1 antibodies block the production of proinflammatory 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 contain 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 (eg, dendritic cells) and thereby reduce or prevent the onset of a cytokine storm following administration of the anti-TREM-1 antibody to an individual in need thereof.

In some embodiments, the specific anti-TREM-1 antibodies provided 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, 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 provided herein are, in certain embodiments, included in the same group as mAb 0318.

Antibody mAb 0318 has a heavy chain variable region (VH) containing SEQ ID NO: 14 and a light chain variable region (VL) containing SEQ ID NO: 15 (see International Patent Application Publication 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. CDR1, CDR2 and CDR3 of the light chain of mAb 0318 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 VH and VL having SEQ ID NO: 14 and SEQ ID NO: 15, respectively. In another embodiment, the VH of an anti-TREM-1 antibody comprises a CDR1 sequence consisting of amino acids 31-35 (TYAMH) SEQ ID NO: 14, in which one of the amino acids may be replaced by another amino acid. In certain embodiments, the VH of an anti-TREM-1 antibody comprises a CDR2 sequence consisting of amino acids 50-68 (RIRTKSSNYATYYAASVKG) SEQ ID NO: 14, in which one, two or three amino acids may be replaced by 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) SEQ ID NO: 14, in which one, two or three amino acids may be replaced by another amino acid.

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

Methionine residues in antibody CDRs can become oxidized, leading to possible chemical degradation and subsequent reduction in antibody effectiveness. Therefore, in the anti-TREM-1 antibodies provided herein, one or more methionine residues in the heavy and/or light chain CDRs may be replaced by amino acid residues that are not subject to oxidative degradation. In some embodiments, the methionine residues in the heavy chain CDR1 and CDR3 are replaced with amino acid residues that are not subject to oxidative degradation (eg, 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) SEQ ID NO: 81 or amino acids 101-110 (DLGTRRQFAY) SEQ ID NO: 82

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

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 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 presented in WO 2017/152102, a CDR2 sequence selected from the group consisting of SEQ ID NO : 28-40 presented in 2017/152102, and/or a CDR3 sequence selected from the group consisting of SEQ ID NO: 41-119 presented in WO 2017/152102. In one embodiment of the invention, VH antibodies to TREM

- 20 046142 contains a CDR1 sequence selected from the group that consists of SEQ ID NO: 120-143 presented in WO 2017/152102, a CDR2 sequence selected from the group that consists 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 contain 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%, or at least 99%) of 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 contains 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 presented in WO 2017/152102, and/or a light chain variable region (VL) selected from the group consisting of SEQ ID NO: 316-395 presented in WO 2017/152102.

In some embodiments, the anti-TREM-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain and light chain comprise the amino acid sequences set forth in Table 1. 7. In some embodiments, the 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 contains 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 contains the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments In carrying out the invention, 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, the antibody TREM-1 comprises a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence set forth in SEQ ID NO: 53 and the light chain contains the amino acid sequence set forth in SEQ ID NO: 54.

Heavy and light chains containing 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 presented herein, for example, in SEQ ID NO: 50-54 can be used to create antibodies to TREM-1 having the desired characteristics, for example, those presented below in the present description.

In some embodiments, an anti-TREM-1 antibody provided herein comprises a heavy chain and a light chain, wherein the heavy chain contains an amino acid sequence that is at least 80% identical, at least 85% identical, or at least 90% identical. %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100% of the amino acid sequence shown in SEQ ID NO: 50, 51 , 52 or 53, and where the light chain contains an amino acid sequence that is at least 80, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100% of the amino acid sequence set forth in SEQ ID NO: 54.

In some embodiments, an anti-TREM1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region contains 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, the anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region contains one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, L358M and any combination thereof (EU numbering). In some embodiments, an anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region contains 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, the anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region contains 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, the 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 by e.g. by surface plasmon resonance method. In another embodiment, the 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., as characterized by a KD value as assessed by BIACORE™ (e.g., according to the method described in section Examples) 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. In some embodiments, anti-TREM-1 antibodies represented by as used herein, are associated with TREM-1 of cynomolgus monkeys (supo), for example, with a KD value that is assessed by BIACORE™ (for example, according to the method described in the Examples section) of 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, from 10 ^-10 to 10 ^-7 M or from 10 ^-9 to 10 ^-7 M.

In one embodiment, the antibodies provided herein bind to the same one or the same multiple epitopes on TREM-1 as mAb 0318. In some embodiments, the anti-TREM-1 antibody has the ability to specifically bind with (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, 1115, Y116, Q117, P118, P119, and any combination thereof, 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 to F48 of SEQ ID NO: 1 (human TREM-1) as determined by analysis, for example, by NX-MS or X-ray diffraction. In some embodiments, the epitope for an anti-TREM-1 antibody comprises one, two, three, four, five, six, seven, or all of 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 e.g. , NX-MS or X-ray diffraction. In specific embodiments, the epitope for an anti-TREM-1 antibody comprises one, two, three, or all of amino acid residues selected from the group consisting of D42, E46, D92, H93 of SEQ ID NO: 1 (human TREM-1), according to analysis using variants of TREM-1 and the surface plasmon resonance method.

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

In one embodiment, an anti-TREM-1 antibody has the ability to specifically bind to human TREM-1, wherein the epitope for the antibody contains one, two, three, four, five, six, seven, eight, nine, or all of the amino acid residues selected from the group which consists of V39, K40, C41, D42, Y43, L45, E46, K47, F48 and A49 from SEQ ID NO: 1.

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

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V39, C41, D42, Y43, L45 of SEQ ID NO: 1. In the following embodiments, the epitope for the antibody may comprise E46, K47, and A49 of SEQ ID NO: 1. In a specific embodiment, the epitope for the antibody to TREM-1 may further comprise F48 from 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 (eg, 9 cP) at a concentration of 130 mg/mL.

In some embodiments, the anti-TREM-1 antibody provided herein contains mutations that replace one or more negatively charged residues in the CDR1 and CDR3 regions of the antibody's light chain with uncharged residues. In some embodiments, the anti-TREM-1 antibody comprises replacing one or more of 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 provided herein contains mutations in the Fab-Fab interaction region having SEQ ID NO: 14 to reduce Fab-Fab dimerization. Previously, for the mAb 0318 antibody, it was found that since the antibodies contain two Fabs, multimerization may affect viscosity. Such mutations are referred to as Fab-Fab interaction mutations. In specific embodiments, the anti-TREM-1 antibody contains a mutation that replaces 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 per 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 provided herein contains a mutation at position 32 of SEQ ID NO: 15 whereby phenylalanine is replaced by 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 the 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 to enhance the Fab-TREM-1 interaction is referred to as a Fab-TREM-1 interaction mutation. Provided herein are anti-TREM-1 antibodies whose variable regions are linked (e.g., covalently linked or fused) to an Fc, e.g., 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, for example, 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 a non- or substantially non-effector-functioning Fc, such as IgG1. In some embodiments, the variable regions of anti-TREM-1 antibodies are linked to an Fc that has reduced or no binding ability to one or more FcyRs.

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

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ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP , AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP 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 provided herein may comprise the amino acid sequence of any VH domain provided herein fused to a non-effector constant region, such as the following amino acid sequences: effector function of the human IgG1 constant domain:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

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

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

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

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

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

- 24 046142 as used herein, contains the amino acid sequence of any VH domain described herein fused to an IgG1 constant domain containing the following amino acid sequences:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTS

PPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: AND;

IgG1-Aba containing 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 substitutions S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S (EU numbering, which are underlined).

The VL domain provided herein may be fused to a human kappa or lambda light chain constant domain. For example, the VL domain of an anti-TREM-1 antibody may comprise the amino acid sequence of any VL domain provided herein fused to the following human IgG1 kappa light chain amino acid sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13)

In certain embodiments of the invention, the heavy chain constant region contains a lysine or other amino acid at the C-terminus, for example, it contains the following amino acids: LSPGK (SEQ ID NO: 48) in the heavy chain. In certain 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, the variable region of the anti-TREM-1 antibody is linked to a non- or substantially non-effector-functioning Fc. In specific embodiments of the invention, the variable region of the anti-TREM-1 antibody is linked to an Fc selected from the group consisting of IgG1.1f, IgG1.3f, IgG1-Aba and IgG4-Aba presented herein.

In general, the variable regions provided herein may be linked to an Fc containing one or more modifications typically made to alter one or more functional properties of the antibody, such as Fc receptor binding, proinflammatory cytokine release, half-life in serum, complement fixation and/or antibody-dependent cellular cytotoxicity. In addition, the antibody provided herein may be chemically modified (eg, 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 such embodiments of the invention is described in more detail below. The numbering of residues in the Fc region corresponds to the Cabot EU index.

The Fc region includes domains derived from the immunoglobulin constant region (eg, 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. An immunoglobulin constant region is a naturally occurring or synthetically produced polypeptide homologous to the C-terminal region of an immunoglobulin, and may include a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a 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 FcyRI, FcyRII and FcyRIII. Data have been published that sequences that are important for the binding of IgG to FcYR receptors are localized in the CH2 and CH3 domains. The ability of the antibody to bind to the Fc receptor (FcR) affects the half-life of the antibody in serum.

In one embodiment, the Fc region of anti-TREM-1 antibodies is a variant Fc region, e.g., has an Fc sequence that is modified (e.g., by amino acid substitution, deletion, and/or insertion) from the parent Fc sequence (e.g. , an unmodified Fc polypeptide that is then modified to create a variant) to provide the 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 will generally contain at least one amino acid modification in the Fc region. It is believed that a combination of amino acid modifications is particularly preferred. For example, a variant Fc region may include two, three, four, five, etc. substitutions, for example, at specific positions in the Fc region specified herein.

The Fc region variant may also have a sequence change whereby the amino acids involved in the formation of the disulfide bond 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 provided herein. Even when cysteine residues are removed, single-chain Fc domains can still form a dimeric Fc domain in which they are present together through non-covalent interactions. In other embodiments, the Fc region can be modified to be more compatible with the host cell of choice. For example, a PA sequence present near the N-terminus of a typical native Fc region that can be recognized by a digestive enzyme in E. coH, such as proline-specific immunopeptidase, can be removed. In other embodiments, one or more glycosylation sites in the Fc domain can be removed. Residues that are typically glycosylated (eg, asparagine) may mediate a cytolytic response. Such residues can be removed or replaced with non-glycosylated residues (eg 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 EKK sequence can be removed by deletion or replaced. In particular embodiments of the invention, sites that affect Fc receptor binding, preferably sites other than salvage receptor binding sites, can be removed. In other embodiments, the Fc region can be modified to remove the 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, the Fc hinge region is modified to change, for example increase or decrease, the number of cysteine residues in the hinge region. This approach is described in detail in US No. 5,677,425 to Bodmer et al. The number of cysteine residues in the Fc hinge region is varied, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 interface of the Fc hinge domain such that the antibody has a reduced ability to bind Staphylococcyl protein A (SpA) compared to the SpA binding ability of an antibody having a native hinge fragment. Fc. This approach is described in more detail in US No. 6165745 to Ward et al.

In further embodiments, the Fc region is modified by replacing at least one amino acid residue with another amino acid residue to change 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 another amino acid residue such that the antibody has an altered affinity for the effector ligand , but retained the antigen-binding ability of the parent antibody. The effector ligand for which the affinity is changed may be, for example, an Fc receptor or a C1 component of the complement system. This approach is described in more detail in US patents No. 5624821 and US No. 5648260,

- 26 046142 both addressed to Winter and others.

In another example, one or more of the amino acid residues at positions 329, 331, and 322 may be replaced with another amino acid residue so as to alter the ability of the antibody to bind C1q and/or reduce or eliminate complement-dependent cytotoxicity (CDC). This approach is described in more detail in US No. 6194551 to Idusogie et al.

In another example, one or more amino acid residues at amino acid positions 231 and 239 may be replaced to thereby alter the complement fixation ability of the antibody. 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 reduce antibody-dependent cell-mediated cytotoxicity (ADCC) and/or to reduce 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. Example 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 FcyR 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 presented in Strohl, Current Opinion in Biotechnology 20, 2009, p. 685-691.

Other Fc modifications that can be made to the Fc are modifications designed to reduce or eliminate the ability to bind to FcyR and/or complement proteins, thereby reducing or eliminating 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 EU- index). Examples of replacements include, but are not limited to, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S and 331S (e.g. 330S and 331S) (numbered according to EU index). The Fc variant may contain 236R/328R. Other modifications designed to reduce interactions with FcyR and complement include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S, 229S, 238S, 233P and 234V, and also eliminating glycosylation at position 297 by mutation or enzymatically, or by production in organisms such as bacteria in which protein glycosylation does not occur. A review of these and other modifications is presented in Strohl, Current Opinion in Biotechnology, 20, 2009, p. 685-691.

Optionally, the Fc region may contain a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, for example, US No. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; International Application Publications No. patent 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 05/070 963 ; WO 05/040217, WO 05/092925 and WO 06/0201 14).

The affinities and binding abilities of the Fc region to its ligand can be determined using various in vitro assays (biochemical and immunoassays) known in the art, including but not limited to equilibrium methods (eg, enzyme-linked immunosorbent assay (ELISA) ) or radioimmunoassay (RIA)), or kinetic (eg, BIACORE assay) and other methods such as indirect binding assay, competitive inhibition assay, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (eg, gel filtration). These and other methods may employ a label on one or more of the components to be examined and/or employ a variety of detection techniques 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., ed. Paul W.E., Lippincott-Raven, Philadelphia, 1999, which focuses on antibody-immunogen interactions.

In certain embodiments, the anti-TREM-1 antibodies provided herein contain an Fc that has reduced or no ability to bind to FcyR. In some embodiments, an anti-TREM-1 antibody has reduced binding affinity to FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), FcyRIIIB (CD 6b), or any combination thereof, compared to an antibody containing 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 anti-TREM-1 antibody has binding affinity

- 27 046142 tion 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 by 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 set forth in SEQ ID NO: 76 and a light chain consisting from the amino acid sequence shown in SEQ ID NO: 54.

In some embodiments, anti-TREM-1 antibodies comprise an IgG1 Fc variant containing (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, the anti-TREM-1 antibody provided herein is of (a) an IgG1 isotype and contains 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 or Cabot numbering, or has an amino acid deletion in the Fc region at a position corresponding to glycine 236;

(b) an IgG2 isotype and contains 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, where the numbering of the residues corresponds to the EU numbering or Cabot numbering; or (c) an IgG4 isotype and contains 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 numbering of the residues corresponds to the EU numbering or Cabot numbering.

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

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

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

In particular embodiments, an Fc is selected that has substantially no effector function, i.e. characterized by reduced ability to bind to FcyR and reduced ability to fix complement. An example of an Fc, such as an IgG1 Fc, that does not have effector function is an Fc containing the following five mutations: L234A, L235E, G237A, A330S, and P331S.

II. Physical properties of antibodies.

Antibodies to TREM-1, such as those presented herein, have some or

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

Glycosylation sites, primarily present in variable regions, can cause increased immunogenicity of an antibody or a change in the pK value of the antibody due to altered binding to antigen (Marshall et al., Annu. Rev. Biochem., 41, 1972, pp. 673-702; Gala et al. 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 in motifs containing the sequence N-X-S/T. In some embodiments, anti-TREM-1 antibodies provided herein have no or reduced levels of variable region glycosylation. 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 set forth in SEQ ID NO: 76 and a light chain that consists of the amino acid sequence shown in SEQ ID NO: 54.

In some embodiments, anti-TREM-1 antibodies do not contain asparagine isomerization sites. Deamination of asparagine can occur on the N-G or D-G sequences and results in the formation of an isoaspartic acid residue, which causes the polypeptide chain to bend and reduce its stability (isoaspartic acid effect).

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

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

In some embodiments, anti-TREM-1 antibodies provided herein are not rapidly degraded. 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, anti-TREM-1 antibodies provided herein have minimal aggregation properties that may result in the initiation of an undesirable immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable at an aggregation level 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 gel filtration columns (SEC), high performance liquid chromatography (HPLC), and dynamic light scattering (DLS). In some embodiments, the anti-TREM-1 antibodies provided herein are monomers as determined by gel filtration-high performance liquid chromatography (GP-HPLC) analysis. In some embodiments, anti-TREM-1 antibodies have a minimal risk of fragmentation as assessed by two-dimensional liquid 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 is nucleic acid molecules encoding antibodies to TREM-1, presented in the present description. Nucleic acids may be present in whole cells, in cell lysates, 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 impurities, such as other nucleic acids present in the cell (for example, other chromosomal DNA, such as chromosomal DNA naturally linked to isolated DNA) or proteins , using standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, restriction enzyme digestion, agarose gel electrophoresis, and other methods well known in the art (see Current Protocols in Molecular Biology, ed. F. Ausubel et al., Greene Publishing and Wiley Interscience, New York, 1987). The nucleic acid referred to herein may be, for example, DNA or RNA, and may or may not contain intronic sequences. In some embodiments, the nucleic acid is a cDNA molecule.

Nucleic acids presented herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (for example, hybridomas produced 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 methods cDNA cloning. For antibodies obtained from an immunoglobulin gene library (eg, 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. Examples of 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 of producing the anti-TREM-1 antibody provided herein may involve expressing the heavy chains and light chains in a cell line containing nucleotide sequences encoding the heavy and light chains with a signal peptide, for example SEQ ID NO: 58-61 and SEQ ID NO: 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, the DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody genes, Fab fragment genes, or scFv genes. In these manipulations, a 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, operably linked means that two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in reading 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 with another DNA molecule encoding the heavy chain constant regions (hinge, CH1, CH2 and/or CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., E. A. Kabat 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 using standard PCR amplification. The heavy chain constant region may 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 a Fab fragment heavy chain gene, the VH encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL encoding DNA with another DNA molecule encoding the light chain constant region, CL. Human light chain constant region gene sequences 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 using standard PCR amplification. The light chain constant region may be a kappa or lambda chain constant region.

Another object of the present invention are cells (eg, host cells) expressing (eg, recombinantly) antibodies to TREM-1 presented in the present description, and accordingly

- 30 046142 corresponding polynucleotides and expression vectors. Also provided herein are vectors containing polynucleotides that contain nucleotide sequences encoding antibodies to TREM-1 or a fragment thereof. In some embodiments, vectors can be used to recombinantly express TREM-1 antibodies provided 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 transcription and translation of the inserted coding sequence, or in the case of a viral RNA vector, elements necessary for replication and translation after introduction into the appropriate host cell. Expression vectors may include plasmids, phagemids, viruses and derivatives thereof.

Expression vectors provided herein may include polynucleotides encoding an antibody or antigen binding fragment thereof as provided herein. In some embodiments, the coding sequences of an antibody or an antigen-binding fragment thereof are operably linked to an expression control sequence. As used herein, 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 retains its functionality. A coding sequence and a gene expression control sequence are said to be operably linked if they are covalently linked such 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 considered to be functionally linked if induction of a promoter in the 5' region of the expressed gene sequence 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 such that the resulting transcript is 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; viruses like SV40; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; cowpox virus; poliovirus; and an RNA virus such as a retrovirus. It is acceptable to use other vectors well known in the art. Specific viral vectors are based on noncytopathic eukaryotic viruses in which non-critical genes are replaced by a gene of interest. Noncytopathic viruses include retroviruses whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses are allowed for experiments in therapy using human genes. The most suitable are retroviruses that are replication deficient (i.e., they are able to control the synthesis of the required proteins, but are not able to produce an infectious particle). Such genetically modified retroviral expression vectors are widely used to carry out highly efficient gene transduction in vivo. Standard protocols for the production of replication-deficient retroviruses (including the steps of insertion of exogenous genetic material into a plasmid, transfection of a packaging cell line with the plasmid, production of recombinant retroviruses by a packaging cell line, collection of viral particles from tissue culture medium, and infection of 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 double-stranded DNA virus. An adeno-associated virus can be engineered to be a replication-deficient virus and be able to infect a wide range of cell types and species. It has additional advantages such as thermal stability and stability in lipid solvent; high frequencies of transduction in cells of various lineages, including hematopoietic cells; and lack of inhibition of superinfection, which

- 31 046142 allows for several series of transduction. Data have been published that an adeno-associated virus can integrate into the DNA of a human cell in a site-specific manner, which minimizes the possibility of insertional mutagenesis and variability in the expression characteristics of the integrated gene during infection with a retrovirus. In addition, wild-type adeno-associated virus infections in tissue culture were followed over more than 100 subcultures in the absence of selective pressure, suggesting that adeno-associated virus integration into the genome is a relatively stable event. The adeno-associated virus can also function outside the chromosome.

IV. Immunoconjugates.

Also provided herein are immunoconjugates containing any of the anti-TREM-1 antibodies described herein. In some embodiments, the immunoconjugate comprises an antibody or an antigen-binding fragment thereof coupled to an agent. In some embodiments, the immunoconjugate comprises a bispecific molecule as provided herein coupled to an agent (eg, such as a therapeutic agent or 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-specific labels used for sample analysis. Detectable labels that can be associated with any anti-TREM-1 antibody provided herein can be any of the various types currently used in the in vitro diagnostic field, 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 N ₄ , chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., and enzymes tags that convert the substrate in question into a detectable marker, and polynucleotide tags that are detected after amplification, for example, by polymerase chain reaction. Suitable enzyme tags include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label may be the enzyme alkaline phosphatase, which is detected by assessing the presence or generation of chemiluminescence following the conversion of 1,2-dioxetane substrates such as adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), disodium-3-(4-(methoxyspiro{1,2-dioxetane- 3,2'-(5'chloro)tricyclo{3.3.1.1 3,7}decane}-4-yl)phenylphosphate) (CSPD), as well as CDP and CDP-STAR® or other luminescent substrates well known in the art , for example, 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 with the naked eye, in the case where the label is particulate and accumulates in appropriate quantities, or using instruments such as a spectrophotometer, luminometer, fluorimeter, etc., in all cases in accordance with standard practice .

In some embodiments, conjugation techniques produce bonds that are substantially (or nearly) non-immunogenic, such as peptide (ie, amide), sulfide, (sterically hindered), disulfide, hydrazone, and ether bonds. These bonds are essentially non-immunogenic and are characterized by acceptable stability in serum (see, for example, 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 antibody, different conjugation strategies can be used. If the fragment is a naturally occurring or recombinant fragment consisting of 50-500 amino acids, textbooks on the chemistry of protein conjugate synthesis describe standard procedures that can be easily carried out 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). Some embodiments of the invention employ the interaction of a maleimide moiety with a cysteine residue present in the antibody or fragment thereof. This interaction is the most suitable chemical coupling reaction when using, for example, a Fab or Fab' fragment of an antibody. Alternatively, in some embodiments, coupling is performed 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, p. 3361-3371).

In general, site-specific interaction and covalent coupling are 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 particular 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 using the specific enzymatic reactivity of certain enzymes towards a naturally occurring amino acid in the context of the sequence in question (see, for example, Taki M. et al., Prot. Eng. Des. Sel., 17, 2004, pp. 119-126; Gautier A. et al., Chern.

- 32 046142

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

The reactivity of the N-terminal cysteine toward benznitriles can be used to effect site-specific covalent coupling (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 No. 6,437,095 B1 describes a conjugation method based on the rapid interaction of cysteine present in the segment of negatively charged amino acids with cysteine located in the segment of positively charged amino acids.

The fragment may also be a synthetic peptide or a peptide mimetic. In the case where the polypeptide is synthesized chemically, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, for example, de Graaf A.J. et al., Bioconjug. Chem., 20, 2009, 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 tag, the conjugate with a 1:1 stoichiometry can be separated from other conjugation byproducts using chromatography. This procedure can be facilitated by the use of a dye-labeled mating partner and a charge-carrying linker. By using this kind of labeled and highly negatively charged mating partner, monoconjugated polypeptides can be easily separated from unlabeled polypeptides and polypeptides that carry more than one linker, since 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, the moiety attached to the anti-TREM-1 antibody is selected from the group consisting of a binding moiety, a tagging moiety, and a biologically active moiety.

The anti-TREM-1 antibodies provided herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove 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. ADC can be obtained according to the method described in US No. 7087600; 6989452 and 7129261; PCT publications WO 02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO 08/103693; US patent publications No. 20060024317; 20060004081; and 20060247295.

Antibodies to TREM-1, such as those presented herein, can also be used to detect TREM-1, such as human TREM-1, such as human TREM-1, in tissues or tissue samples. Antibodies can be used, for example, in an ELISA assay or a flow cytometry assay. In some embodiments, the anti-TREM-1 antibody is contacted with cells, eg, cells in a tissue, for a period of time sufficient to allow specific binding to occur, and then a reagent, eg, an antibody that detects the anti-TREM-1 antibody, is added. Examples of analyzes are presented in the Examples section. The anti-TREM-1 antibody may be a fully human antibody, or it may 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, e.g., human TREM-1, in a sample (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. 1 with TREM-1 in the sample, and (II) contacting the sample with a detection reagent, e.g., an antibody that specifically binds to an anti-TREM-1 antibody, e.g., the Fc region of an anti-TREM-1 antibody, for detection TREM-1 associated with anti-TREM-1 antibody. After incubation with the antibody and/or detection reagent, washing steps can be applied. The anti-TREM-1 antibodies used in these methods do not require conjugation to a tag or detection agents because the detection agent can be used alone.

- 33 046142

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

V. Bispecific molecules.

Antibodies to TREM-1 presented herein can be used to create bispecific molecules. An anti-TREM-1 antibody or antigen-binding fragments thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (eg, another antibody or ligand for a receptor), 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 coupled 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 provided herein (e.g., anti-IP-10 antibodies or TNF-α). In fact, an antibody provided 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; it is intended that such multispecific molecules also fall within the term bispecific molecule as used herein. To create a bispecific molecule as described herein, an antibody provided 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, fragment antibodies, peptide or binding mimetic to produce a bispecific molecule.

Thus, provided herein are 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 disclosed herein, in which the bispecific molecule is multispecific, the molecule may additionally have a third binding specificity.

In some embodiments, the bispecific molecules provided herein contain as binding specificity at least one antibody or antibody fragment, 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 an Fv or single chain construct as described in US 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 provided herein.

The bispecific molecules provided herein can be prepared by conjugating its 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 each other. When the binding specificities are proteins or peptides, a variety of binding or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2- pyridylthio)propionate (SPDP) and sulfosuccinimidyl-4-(K-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., J. Exp. Med., 160, 1984, p. 1686; Liu M.A. et al., Proc. Nail. Acad. Sci., USA, 82, 1985, p. 8648). Other methods include those described in Paulus, Behring Ins. Mitt., No. 78, 1985, p. 118-132; Brennan et al., Science, 229, 1985, p. 81-83; and Glennie et al., J. Immunol., 139, 1987, p. 2367-2375). Examples of conjugating agents are SATA and sulfo-SMCC, both commercially available from Pierce Chemical Co. (Rockford, Illinois).

When the binding specificities are antibodies, they can be conjugated through sulfhydryl binding of 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 sulphihydryl 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 MAtxMAt, MAtxFab, MAtx(scFv) ₂ , FabxF(ab') ₂ or ligandxFab. The bispecific antibody may comprise an antibody containing 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 contain at least two single-chain molecules. Methods for preparing bispecific molecules are described, e.g.

- 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 techniques known in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (eg, 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 (eg, antibody) specific for the complex of interest.

VI. Sets.

Provided herein are kits containing one or more of the anti-TREM-1 antibodies described herein, or antigen-binding fragments thereof, bispecific molecules, or immunoconjugates thereof. In some embodiments of the invention, a pharmaceutical package or kit is provided comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions provided herein, such as one or more antibodies provided herein or antigen binding agents thereof. fragments, not necessarily instructions for use. In some embodiments, the kits contain a pharmaceutical composition as provided herein and any prophylactic or therapeutic agent, such as those provided herein.

VII. Compositions and preparative forms.

Also provided herein are compositions (e.g., pharmaceutical compositions) and formulations containing one or more anti-TREM-1 antibodies (including polynucleotides, vectors, and cells that encode and/or express anti-TREM-1 antibodies) described herein. . For example, in one embodiment, the invention provides a pharmaceutical composition comprising one or more anti-TREM-1 antibodies provided 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, isotonicizing and absorption retarding agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the route of administration to the active compound, i.e. The antibody, immunoconjugate, or bispecific molecule may be coated with a material that protects the compound from 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 improves the stability of the anti-TREM-1 antibody and thereby ensures its long-term storage. In some embodiments, the pharmaceutical formulation provided herein comprises (a) an anti-TREM-1 antibody;

(b) a buffering agent;

(c) stabilizer;

(d) salt;

(e) filler; and/or (f) a surfactant. In some embodiments, the pharmaceutical formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 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.

Buffer agents that can be used in accordance with the present invention may be a weak acid or base used to maintain the 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 a controlled pH of the formulation. Suitable buffering agents also make it possible to ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties may 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 contains a histidine (eg, L-histidine) with isotonicity agents and can adjust the pH value with an acid or

- 35 046142 base known/known in the art. In specific embodiments of the invention, the buffering agent is L-histidine. In specific embodiments of the invention, 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 stabilize proteins in many different ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine or threonine, carbohydrates such as glucose, sucrose, trehalose, raffinose or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrins or dextrans of any type and having any molecular weight, or PEG. In one aspect of the invention, a stabilizer is selected to maximize the stability of the FIX polypeptide in lyophilized preparations. In specific embodiments of the invention, the stabilizer is sucrose and/or arginine.

Filler

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

Surface-active substance.

Surfactants are amphipathic substances having lyophilic and lyophobic groups. The surfactant may 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 dodecyldimethylamine 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 (eg, 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 further contain one or more buffering systems, a preservative, a tonicity control 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 one 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 means a formulation containing at least 50% by weight water. Likewise, the term aqueous solution refers to a solution containing at least 50% by weight water, and the term aqueous suspension refers to a suspension containing at least 50% by weight water.

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

The pharmaceutical compositions presented herein can be administered as part of combination therapy, i.e. in combination with other agents. For example, combination therapy may 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 may include other compounds, drugs and/or agents used to treat a disease or disorder (eg, an inflammatory disorder). Such compounds, drugs and/or agents may include, for example, anti-inflammatory drugs or antibodies that block or reduce the production of pro-inflammatory cytokines. In some embodiments, the 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 (eg, AVONEX®, REBIF®), interferon beta-W (eg, BETASERON®, EXTAVIA®), gati

- 36 046142 ramer acetate (eg COPAXONE®, GLATOPA®), mitoxantrone (eg NOVANTRONE®), non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids and combinations thereof.

The pharmaceutical compounds provided 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 produce any undesirable toxicological effects (see, for example, 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 those formed from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, 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, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The desired 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 achieved by sterilization procedures (see above) and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, etc. It may be advantageous to include isotonicity agents such as sugars, sodium chloride and the like in the compositions. In addition, it is possible to ensure prolonged absorption of the injectable pharmaceutical form 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 sterile injectable solutions or dispersions for immediate administration. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in pharmaceutical compositions is envisaged except in cases where any/any medium or agent is incompatible with the active substance. The pharmaceutical composition may or may not contain a preservative. Additional active ingredients may be included in the composition.

Therapeutic compositions generally must be sterile and stable under conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for incorporating the drug at high concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. The desired fluidity can be maintained, for example, by the use of materials suitable for coating, 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 isotonicity agents such as 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 retards absorption, for example, monostearates and gelatin.

Sterile injection solutions can be prepared by incorporating the active substance in the required quantity into an appropriate diluent, if necessary, together with one ingredient or a combination of ingredients listed above, and subsequent sterilization according to

- 37 046142 microfiltration agent. Typically, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains the basic dispersion medium and the necessary other ingredients listed herein. In the case of sterile powders intended for the preparation of sterile solutions for injection, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active substance plus any additional required ingredient from a previously sterilized solution containing them by filtration.

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 particular route of administration. The amount of active ingredient that can be combined with the carrier material to form a single dose dosage form will generally be the amount of the composition that produces a therapeutic effect. Typically, this amount, unless it is 100%, may vary 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 substance in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus dose may be administered, multiple divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased as required by the therapeutic situation. It is most preferable to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, the term unit dosage form refers to physically discrete units suitable as unitary dosages for individuals to be treated; wherein each unit contains the active substance in a predetermined amount, which is calculated to achieve the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification of the unit dosage forms presented herein is dictated by and is directly related to (a) the unique characteristics of the active substance and the specific therapeutic effect to be achieved; and (b) limitations existing in the field of formulations using the active substance for the treatment of susceptible individuals.

To administer an anti-TREM-1 antibody, such as that provided herein, dosages ranging from about 0.0001 to 100 mg/kg or more, typically 0.01 to 5 or 10 mg/kg of host body weight, are used. . For example, doses may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, or 10 mg/kg body weight, or may be 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 a dose of 1 mg/kg body weight every three weeks.

In some embodiments, the anti-TREM-1 antibody is administered at a constant dose (continuous 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 based on body weight.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within specified ranges. The antibody is usually administered several times. The intervals between administrations of individual doses may be, for example, one week, one month, three months or one year. The intervals may also be non-uniform and may be determined depending on the measured levels of antibody to the target antigen in the patient's blood. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μg/ml, and in some methods about 25-300 μg/ml.

The antibody can be administered in a sustained release formulation, in which case less frequent administration is required. The dose and frequency of administration vary depending on the half-life of the antibody in the patient. 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 case of prophylactic use

- 38 046142 they are administered at a relatively low dose at relatively infrequent intervals over a long period of time. Some patients continue to undergo treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer a relatively high dose at relatively short intervals until the progression of the disease is reduced or stopped and until the patient experiences partial or complete improvement in the symptoms of the disease. After this, the patient can be transferred to a prophylactic regimen.

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

The composition presented herein can be administered through one or more routes of administration using one or more of a variety of methods known in the art. As will be apparent to one skilled in the art, the route and/or form of administration will vary depending on the results desired. Routes of administration of the anti-TREM-1 antibodies provided herein may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. The term parenteral administration as used herein refers to routes of administration other than enteral and topical 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, an antibody provided herein can be administered by a non-parenteral route, such as the topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The 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, edited by 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 of the invention, the therapeutic composition presented herein can be administered using a needleless hypodermal injection device, such as the devices described in US No. 5,399,163; 5383851; 5312335; 5064413; 4941880; 4790824; or 4,596,556. Examples of well-known implants and modules for use using anti-TREM-1 antibodies disclosed herein include those set forth in US Pat. No. 4,487,603, which describes an implantable microinfusion pump for controlled rate release of drug; US No. 4486194, which describes a therapeutic device for administering drugs through the skin; US No. 4447233, which describes a drug infusion pump for delivering a drug by infusion at a precise rate; US No. 4,447,224, which describes an implantable variable flow infusion device for continuous drug delivery; US No. 4439196, which describes an osmotic drug delivery system having multi-chamber compartments; and US No. 4475196, 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 of the invention, antibodies to TREM-1 presented in the present description can be produced in a form that provides the desired distribution in vivo. For example, the blood-brain barrier (BBB) prevents the passage of many highly hydrophilic compounds. In order to ensure passage of the therapeutic compounds presented herein across the BBB (if necessary, for example in the case of brain cancers), they can be included, for example, in liposomes. Methods for preparing liposomes are described, for example, in US No. 4522811;

- 39 046142

5374548; 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, for example, V.V. Ranade, J. Clin. Pharmacol., 29, 1989, p. 685 ). Examples of targeting moieties include folate or biotin (see, for example, US 5416016 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 receptor A (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 anti-TREM-1 antibodies provided herein and formulations containing such antibodies (eg, pharmaceutical composition, formulations, polynucleotides, vectors and cells) can be used to treat an inflammatory disease (eg, by inhibiting TREM-1 activity).

Thus, one aspect 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 anti-TREM-1 antibody. Examples of inflammatory diseases that can be treated with anti-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, Sjögren'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, antibodies to TREM-1 can be used to treat individuals with inflammatory bowel disease. Inflammatory bowel disease (IBD) is a disease that can affect any part of the gastrointestinal tract from the mouth to the anus, 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: patients with ulcerative colitis (UC) and patients with Crohn's disease (CD). CD typically affects the ileum and colon; it can affect any area of the intestine, but the involvement is often focal (the affected areas spread in patches throughout the intestine). UC always affects the rectum (ie, refers to the large intestine) and the involvement 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 controlling symptoms, maintaining remission, and preventing relapse. The effectiveness of treatment of inflammatory bowel disease in a clinical setting can be assessed by the reduction in 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, but also by a disease activity index (DAI) calculated from a combination of stool consistency, body weight, and the presence of blood in the stool.

In one embodiment, the anti-TREM-1 antibodies provided 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 inflammation of the joint, causing pain, stiffness, heat, 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, said inflammation can lead to, among other physiological effects, severe damage to bone and cartilage and joint destruction and severe pain. The affected joint may lose its shape and 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 reminiscent of human rheumatoid arthritis

- 40 046142 Rt. Since CIA is characterized by immunological and pathological features similar to RA, this makes it a suitable model for screening potential anti-inflammatory compounds in humans. The effectiveness of this model is assessed by the reduction in joint swelling.

Effectiveness in RA in the 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 provided 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 treatment agents, more than one million people worldwide require ultraviolet light treatment or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks associated with ultraviolet radiation and the toxicity inherent in many therapies limit their long-term use. In addition, patients typically experience a recurrence of psoriasis and, in some cases, it occurs soon after stopping immunosuppressive therapy. A model of psoriasis 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 useful for the treatment of psoriasis (Davenport et al., Internal Immunopharmacol., 2, 2002, c 653-672). The effectiveness of this model is assessed using a scoring system for the reduction of skin pathology. Similarly, efficacy for patients is assessed by the reduction of skin pathology.

In one embodiment, antibodies to TREM-1 can be used to treat individuals with psoriatic arthritis. Psoriatic arthritis (RA) is a type of inflammatory arthritis that occurs in a subpopulation of patients with psoriasis. In these patients, the skin pathology/symptoms are accompanied by joint swelling, similar to the swelling seen in rheumatoid arthritis. It is characterized by the presence of patchy, raised, reddened areas of skin inflammation characterized by peeling. Psoriasis often affects the creases 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.

From the point of view of the present description, prophylactic, palliative, symptomatic and/or radical treatment can be considered as separate objects of the invention. The antibody of the invention can be administered parenterally, for example intravenously, for example intramuscularly, for example subcutaneously. Alternatively, the antibody of the invention may be administered by a non-parenteral route, for example, orally or topically. The antibody of the invention can be administered for prophylactic purposes. The antibody of the invention can be administered for therapeutic purposes (on demand).

The following examples are given to illustrate the invention and not 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 variants 318 with human TREM-1 and cynomolgus monkeys using the surface plasmon resonance method.

The binding kinetics of mAb 0318 variants to human TREM-1-Fc (hTREM-1) and cynomolgus monkey TREM-1-Fc (cTREM-1) were determined. The binding study was carried out on a ProleOn analyzer (BioRad), which measured molecular interactions in real time based on 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 in direct correlation with the mass on the surfaces of individual sensor chips in six parallel flow cells. Anti-human Fc monoclonal antibody or anti-mouse Fc polyclonal antibody from Biacore human or mouse Fc capture kits were immobilized horizontally onto the flow cells of the GLM sensor chip according to the manufacturer's instructions. The final level of capture antibody immobilization corresponded to approximately 2600-6000 RU in each experiment. Capture of purified mouse monoclonal or recombinantly expressed anti-hTREM-1 antibodies was accomplished 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), after which they were injected in the vertical direction at a rate of 30 μl/min for 60 s, as a result of which intermediate reference spots were created adjacent to all flow cells in which only the anti-Fc antibody was immobilized. This typically resulted in final capture levels of test antibodies corresponding to approximately

- 41 046142

100-300 RU and Rmax values of the analyte 30-90 RU. Binding of hTREM-1 or cTREM-1 proteins was accomplished by injecting the analyte (antigen) into all flow cells in a horizontal direction 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, covering 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 with two injections over 18 s of 10 mM glycine, pH 1.7, and 50 mM NaOH at a rate of 100 μL/min. This regeneration step removed the TREM-1 antibody and any bound TREM-1 protein from the capture antibody-immobilized surface and allowed subsequent binding of the next pair of interaction assay samples. The regeneration procedure did not directly remove the immobilized Fc capture antibody from the chip surface.

The binding affinities of antibodies to antigen were quantified by determining the equilibrium constant of the dissociation reaction (KD), which was determined by measuring the kinetic characteristics of complex formation and dissociation. Rate constants corresponding to 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 for estimation. 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 double controls (subtracting signals from the reference surface as well as levels obtained by injecting blank buffer onto captured TREM-1 antibodies). This made it possible to correct for instrumental noise, shift and drift of the array during sample injection.

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

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

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

Next, the TREM26 antibody (catalog number 314902, Biolegend; see US No. 20150274825, paragraph [0005]) was used for comparison to determine the fate of the TREM-1 receptor after its internalization. Antibody TREM26 does not compete with antibody variants 0318 presented herein. As shown in FIG. 2B, at time point 20 hours there was a significant decrease in TREM26+ expression (~51%) compared to time point 0 hours. There was also a significant decrease (4-fold) in the level of MFI (mean fluorescence index) of TREM26+ at time point 20 hours (after treatment with mAb 0318-IgG1.3f), suggesting that TREM-1 was cleaved after internalization. This decrease in TREM-1 receptor expression after exposure to the antibody was reversible, however, this occurred after removal of the antibody (data not shown).

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

The ability of anti-TREM-1 mAb 0318 variants to inhibit human TREM-1 signaling was determined by assay using the BWZ.36/hTREM-1DAP12:NFATLacZ cell line (also referred to herein as the BWZ/hTREM-1 reporter cell) described for example, in US No. 9550830 B2 and the publication of international patent application WO 2016/009086 A1. The overall method was as follows: approximately 40,000 hTREM-1/BWZ.36 cells/well were seeded onto 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 (cat. no. tlrl-kipgn, Invivogen, San Diego, CA, USA) to provide a positive signal at half maximal intensity, or alternatively in the presence of a submaximal level (1 µg/ml) monoclonal antibody to TREM-1 (catalog number MAB1278, R&D Systems, Minneapolis, MN, USA) adsorbed onto the plastic to provide a positive signal.

During the assay, mAb 0318 variants (i.e., 0318-IgG1.3f; 0318-IgG1.1f; 0318-IgG1-Aba; and 0318-IgG4-Aba) were titrated starting at a concentration of 10 μg/ml using 5 serial 2 - multiples of divorce

- 42 046142 ni. The test sample was incubated overnight at 37°C, then developed using Beta Glo (catalog number E4740, Promega, Madison, WI, USA) according to the Beta Glo protocol and luminescence was recorded. Based on the results, a graph of the dependence of Beta Glo luminescence intensity (in relative units) on the antibody concentration was plotted. Each assay plate was tested with non-neutralizing mIgG1 as a negative control (cat. no. MAB002, R&D Systems, Minneapolis, MN, USA) and as a positive control with neutralizing goat polyclonal anti-hPGLYRP1 antibody (cat. no. AF2590, R&D Systems, Minneapolis, MN, USA). Antibody MAB1278 (catalog number MAB1278, R&D Systems; see US No. 20150274825, paragraph [0005]), a known agonist of TREM-1 signaling, was also used as a positive control (see rectangular inset).

As shown in FIG. 3, all variants of mAb 0318 were potent inhibitors of human TREM-1 signaling, as previously described for mAb 0318 IgG4 antibody in international patent application publication WO 2016/009086 A1.

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

To further evaluate the antagonistic properties of TREM-1 mAb 0318 variants, their effectiveness in blocking the release of various proinflammatory cytokines (eg, TNF-α, IL-6, or IL-8) by activated human primary cells was analyzed. Primary monocytes, neutrophils, and peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood and stimulated with plate-immobilized PGRP1 and soluble peptidoglycan (PGN-ECndss; a form of peptidoglycan without TLR2 activity).

As shown in FIG. 4, all variants of mAb 0318 (i.e. IgG1.3f, IgG1.1f, IgG1-Aba and IgG4-Aba) were effective in inhibiting (IC ₅₀ values in the range of ~10-20 pM) TREM-1-mediated release TNF-α from PBMCs and monocytes. The efficacy of these mAb 0318 variants was similar to that of mAb 0318-IgG4. Antibody mAb 0318-IgG1.3f was also effective in inhibiting IL-6 production (IC ₅₀ value ~32 pM). In the case of neutrophils, mAb 0318-IgG1.3f was superior to mAb 0318-IgG4 in blocking TREM-1-mediated IL-8 production by neutrophils (see FIG. 4).

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

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

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

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

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

To further demonstrate the antagonistic 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, the method consisted of the following: human whole blood collected in tubes with EDTA from three healthy volunteers (donors No. 126, 290 and 322) was stimulated with pre-complexed hPGRP1 (50 μg/ml) and PGN-ECndss (10 μg/ml) ( Invivogen, catalog number tlrl-ksspgn) overnight in the presence of mAb 0318-IgG1.3f in various

- 43 046142 concentrations (0-1nM). After stimulation, plasma was removed and frozen for measurement of cytokine levels. mRNA was isolated from 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 Superscript VILO Master Mix (Thermo Fisher, 11755250). qPCR was then carried out using the following probes: HPRT1 (Hs99999909_ml) (Thermo Fisher, 4351370), CHI3L1 (Hs01072228_ml) (Thermo Fisher, 4331182), IL1p (Hs00174097_ml) (Thermo Fisher, 4331182) and IL6 (H s00985639_ml) ( Thermo Fisher, 4331182) and universal master mix TaqMan Fast (2x) (Thermo Fisher, 4366072). Gene expression levels were standardized against the level of the HPRT1 probe and AAST values were obtained. The results were then plotted and IC ₅₀ values were determined.

As shown in FIG. 6A-6B, and consistent with the results presented in the examples above, mAb 0318-IgG1.3f had the ability to inhibit TREM-1-mediated expression of various inflammatory mediators in human whole blood. The inhibition was found to be dose dependent. The IC50 values are given in the table below. 1.

Taken together, the results presented above demonstrate that the mAb 0318 antibody variants provided herein are antagonistic and can effectively block TREM-1-mediated production of proinflammatory cytokines by various human cells.

Table 1

Cell type Pro-inflammatory mediator Stimulus mAb 0318-IgG1.3f 1C ₅₀ (pM) whole blood CHI3L1 RNA Soluble PGRP + Soluble PGN-Ecndss 4.58±5.60 (N=3) RNA ILlb 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 using the selected composition (20 mM histidine, 150 mM sucrose, 25 mM arginine, 50 mM sodium chloride, pH 6.0), and then concentrated using Amicon Ultra molecular weight cutoff centrifugal filters. The aggregation state of the samples was assessed using gel filtration to monitor possible changes in monomerity, if any, after concentration. The dependence of viscosity on concentration was determined using a RheoSense m-VROC solution viscometer, measuring increasing shear forces at 3 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 a similar viscosity profile. At a concentration of approximately 130 mg/ml, variant 0318-IgG1.3f had a viscosity value of approximately 9 cP. This viscosity profile is close to the profile previously determined for mAb 0318-IgG4 (see international patent application publication WO 2016/009086).

Example 8. Immunogenic potential of mAb 0318 variants.

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

table 2

Analysis Cohort Option0318 END (mg/ml) Host Cells 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 by surface plasmon resonance (SPR).

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

- 44 046142

FcRn, FcRn receptors (mice, humans and cynomolgus monkeys) were immobilized on the CM5 BIAcore biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) through amine coupling until a level corresponding to 400 response units (RU) was achieved. The assay was performed at room temperature using PBS, 0.05% Tween-20™, pH 6.0 (GE Healthcare Bioscience) as running and dilution buffers. Various 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, the dissociation phase (also at pH 6.0) took 360 s. Regeneration of the chip surface until it returned to the original level was carried out by a short-term injection of 50 mM Tris, pH 8.0 and 150 mM NaCl. Analysis of SPR data was performed by comparing the height of the biological response signal 180 s after injection and 300 s after injection. The relevant parameters were the maximum level in RU (RU max) (180 s after injection) and late stability level (300 s after completion of injection).

As shown in FIG. 9A and 9B, all variants of mAb 0318 (IgG1-Aba mod, IgG4-Aba mod, IgG1.1f and IgG1.3f) had the ability to bind to human, mouse and cynomolgus monkey FcRn 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 stated above, in vivo administration of antibodies to certain receptors on the surface of immune cells can induce the release of cytokines, 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 could result in possible TREM-1 agonist activity, mAb 0318-IgG4 was converted to one of the format options provided herein (i.e., IgG1.1f, IgG1. 3f, IgG1-Aba or IgG4-Aba). The ability of the 0318 antibody variants to bind to various FcyRs was then assessed.

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

Example 11. Analysis of the induction of pro-inflammatory cytokines by mAb 0318 variants.

To further determine whether in vivo treatment with mAb 0318 variants leads to a 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 plating monocytes (4x106 cells/well) and culturing them in differentiation medium containing IL-4 and GM-CSF (100 ng/ml). Approximately 2-3 days 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 cells using a flow cytometer. Immature dendritic cells were then seeded onto a flat bottom plate (0.8 x 105 cells/well) and mAb 0318 variants (containing or not containing 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 next day, supernatants from the wells were collected and the amount of TNF-α, IL-6 and IL-12 produced was assessed by ELISA.

Results obtained for representative donors are presented in FIG. 11A-11I. Addition of various variants of mAb 0318 (0318-IgG1.1f, 0318-IgG1.3f and 0318-IgG1.1 Aba) resulted in minimal production of IL-6 (Fig. 11A, 11B and 11B), TNF-α (Fig. 11D, 11D and 11E) and IL-12 (Figs. 11G, 113 and 11K) 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 given in table below. 3.

- 45 046142

Table 3

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

Property Method Result Identity lx-ms Intact mass confirmed Peptide mapping by LC-MS/MS Sequence confirmed by peptide map using Lys-C (95.4% yield; degree of glycosylation 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 main bands -50 kDa and 25 kDa Under non-reducing conditions: the main band is -150 kDa with minor fragments -98 kDa, 75 kDa, 50 kDa, 25 kDa (artifacts due to 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) Thermal stability 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) Demonstrated reduced binding ability to all FcyRs Expected pH-dependent FcRn binding is comparable to wild-type IgGl MAb binding

For clinical development, the biophysical characteristics of the mAb 0318-IgG1.3f variant are preferred. The identity of the antibody was confirmed by mass spectrometric analysis (intact mass analysis and peptide mapping). According to gel filtration analysis, >96% of the antibody was in monomer form. The presence of one N-glycosylation site on N301 of the heavy chain was confirmed, with a glycan profile consistent with that of CHO-expressed monoclonal antibodies (G0F, G1F and G2F). Thermal stability (T _w 1=66.2°C; T _w 2=78.4°C; T _w W=83.2°C) and reversibility when heated (24% at 77°C) mAb 0318-IgG1.3f were in the range characteristic of a typical human monoclonal antibody IgG1.3.

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

- 46 046142

Table 4

Stability of 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 identified Profile solubility/concentration UV, SEC >150 mg/ml in formulation buffer (20mM histidine, 150mM sucrose, 50mM sodium chloride, 25mM arginine, pH 6.0) Selecting a pH value that ensures conformational and colloidal stability Optim2 (melting temperatures and onset of aggregation Tsh and Tagg) Optimal conformational stability at pH 6-8 Optimal colloidal stability at pH 5-6 Selection of Buffer and Excipients Optim2 (melting temperatures and onset of aggregation Tsh and Tagg) Stabilizers: sucrose, sorbitol, arginine (high concentration), glycerin Accelerated stability test 150 mg/ml 12 weeks at 4°C, 25°C and 40°C in formulation buffer (20mM histidine, 150mM sucrose, 50mM sodium chloride, 25mM arginine, pH 6.0) SEC, cIEF, HIC, LC-MS/MS (peptide mapping), LC/MS (intact mass), 2DLC/MS (fragments), Biacore, bioanalysis, UVVis, DLS 12 weeks at 40°C: increase in HMW content by 1.2%/month increase in LMW content by 2.4%/month 12 weeks at 40°C: increase in Rh and Pd 12 weeks at 40°C: increase in acidic variants by 11%/month 12 weeks at 40°C: VSNK deamination by 3%/month Viscosity rating Determination of the concentration-viscosity profile The solution viscosity reaches 9 cP at -130 mg/ml, the predicted viscosity is 12 cP at 150 mg/ml

There were no problems associated with physical stability when conducting a freeze-thaw stress test (3 cycles) at a concentration of 150 mg/ml in a buffer of the described composition (20 mM histidine, 150 mM sucrose, 50 mM sodium chloride, 25 mM arginine, pH 6.0). Tests for accelerated degradation of the composition in question at a concentration of 150 mg/ml were carried out at 4, 25 and 40°C (over a period of time up to 3 months). Chemical modifications of the CDR remained at low levels at all temperature conditions and did not affect activity as determined by SPR analysis. VSNK deamination was lower than expected compared to other IgG1.3f scaffold monoclonal antibodies (3%/month increase when stored at 40°C), and changes were time and temperature dependent. All other chemical modifications (oxidation, deamination, isomerization) also remained at low levels when observed during stability tests. 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 observed changes were dependent on time and temperature, such that the amount of HMW increased by 0.25%/month, and the amount of LMW remained unchanged under storage conditions at 4°C during the study period. The low production of LMW under storage conditions at 40°C was classified based on 2O-LC/\1(' measurements and accurate high-resolution mass measurements as a result of the accumulation of species with the loss of 1 Fab, as well as the Fab arm, presumably in a conserved sequence in upper hinge area.

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) characteristics of the antibody to TREM-1 0318-IgG1.3f were studied when administered in a single dose to cynomolgus monkeys. Some animals (n=3) were administered intravenously the 0318-IgG1.3f antibody at a dose of 2 mg/kg. Other animals were injected subcutaneously with the 0318-IgG1.3f antibody at one of the following doses: (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), TREM-1 receptor occupancy (RO) and ex vivo pharmacodynamic responses were analyzed at predefined time points.

Pharmacokinetic characteristics (PK).

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

- 47 046142 in animal serum using a ligand binding assay using biotinylated recombinant TREM-1 protein as the capture reagent and a commercial polyclonal goat anti-TREM-1 antibody as the detection reagent.

As shown in FIG. 12, it was found that the pharmacokinetic characteristics of the antibody 0318-IgG1.3f were non-linear in the concentration range from 0.1 to 10 mg/kg, mainly due to target-mediated clearance (i.e. internalization and degradation of the antibody after binding to the TREM-receptor 1; see example 2).

The PK parameters obtained using non-compartmental analysis are presented in Table. 5 (intravenous 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 plasma volume, indicating limited extravascular distribution. The half-life of the 0318-IgG1.3f antibody increased from 2 days with a dose of 0.1 mg/kg to 10 days with doses of 2 and 10 mg/kg (single dose subcutaneous administration) (see Table 6). The bioavailability of antibody 0318-IgG1.3f after subcutaneous administration (2 mg/kg) was high and amounted to 84%.

Table 5

PK parameters of mAb 0318-IgG1.3f after IV administration of a single dose 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 Т1/2 (days) 10 ± 2 AUCiast (µMhh) 116 ± 20 ADA-positive/all analyzed 3/3 (1/3)*

* Number of monkeys with persistent ADA among all analyzed.

Table 6

PK parameters of mAb 0318-IgG1.3f after SC administration of a single dose 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 Stach (nM) 13 ± 4 50 ± 3 189 ± 22 1419 ± 93 Ttah (h) 24 48 168 ± 96 84 ± 24 AUCiast (µMhh) 1±0.2 15 ± 1 94 ± 13 649 ± 64 Т1/2 (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 with persistent ADA among all analyzed.

Anti-drug antibodies (ADA).

While ADAs were detected in the serum of most monkeys (16 of 18) following a single dose (regardless of route of administration), most ADA-positive animals showed no negative effect on 0318-IgG1.3f antibody exposure and receptor occupancy TREM-1 (RO) (see Tables 4 and 5). An accelerated decline in the final exposure of 0318-IgG1.3f antibody, which was accompanied by the formation of ADA, was detected in only two monkeys (one in the 0.1 mg/kg group on day 7 and one in the 0.0 mg/kg group). 5 mg/kg, on day 21). Due to the fact that ADAs affected exposure in these two monkeys at the end phase, the corresponding data were excluded from the PK analysis.

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

The occupancy of TREM-1 receptors expressed on peripheral blood monocytes and granulocytes was then assessed. As shown in FIG. 15A and 15B, for all doses tested, the percentages of TREM-1 receptors occupied (ie, associated with anti-TREM-1 antibody) on monocytes and granulocytes were similar. In addition, it turned out that the duration of receptor occupancy depended on the dose of antibody 0318-IgG1.3f administered to the animals. At the 0.5 mg/kg dose, RO levels of >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 of >85% were maintained for at least 1 month after administration.

Following dosing of 0318-IgG1.3f antibody, total TREM-1 receptor levels decreased as

- 48 046142 noocytes and granulocytes (see Fig. 14A and 14B). As described previously, this decrease is primarily due to increased receptor turnover following antibody binding. The reduction in TREM-1 receptor surface expression was reversible and the duration of surface receptor 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 single-dose administration of mAb variant 0318 mAb-IgG1.3f in all dose groups tested (data not shown). It remains unclear whether the increase in sTREM-1 levels is due to the administration of anti-TREM-1 antibody or to the general conditions of the animals during the study. In any case, since drug concentrations were much higher (>1000-fold) than sTREM-1 levels, it cannot be assumed that the soluble target influenced the binding of the mAb 0318 mAb-IgG1.3f variant to TREM-1 at the cell surface.

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

Data on PK, RO, total receptor and PD levels in monkeys were analyzed using a 2-compartment PK model with saturable target-mediated drug distribution (TMDD) in the central compartment to accommodate them to the experimentally observed nonlinear PKs (see Example 12 and Fig. 13). The direct action inhibition model was used to describe the PD response. This model was able to effectively describe the time dependence of FC/RO/PD observed in monkeys. Study observed (open circles) and model-predicted endpoints (solid line) are presented in Fig. 16A (0.1 mg/kg, subcutaneous dosing) and 16B (10 mg/kg, subcutaneous dosing). In table Figure 7 shows the established PK/PD parameters. When serum exposure data for the mAb 0318-IgG1.3f variant and TREM-1 RO were combined 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 in vivo EC ₅₀ RO value was found to be 1.6 ± 0.2 nM.

Table 7

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

PK parameters Description Cynomodel-based estimation (CV%) Predicted parameter estimates for humans 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) Rate constant for elimination 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 after SC administration 80 (5) 80 Kdeg(h-1) Circulation rate constant 0.09 (20) 0.09 Kint (h-1) Rate constant for internalization of the target-MAb complex 0.39 (18) 0.39 R0 (nmol/kg) Baseline target level in the central compartment 0.024 (22) 0.024 KD(hM) Affinity of MAb to TREM-1 0.64 (12) 0.24 Koff (h-1) MAb dissociation rate from TREM-1 0.37 (7) 0.30 IC50(hM) Concentration required to inhibit ex-vivo PGN+PGRP-mediated TREM-1 stimulation by 50% 1.9 (92) 0.1 E0 (%) Baseline 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, -