BR112020017605B1 - HEAVY CHAIN, ANTI-TREM-1 ANTIBODIES, BIESPECIFIC MOLECULE, IMMUNOCONJUGATE, COMPOSITION AND KIT THEREOF - Google Patents

Abstract

The present invention relates to antibodies, or antigen-binding portions thereof, that specifically bind to and inhibit TREM-1 signaling, wherein the antibodies do not bind to one or more Fc^Rs and do not induce cell myeloids to produce inflammatory cytokines. Uses of such antibodies, or antigen-binding portions thereof, in therapeutic applications, such as treatment of autoimmune diseases, are also provided.

Description

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY THROUGH EFS-WEB

[001] The content of the sequence listing electronically submitted in ASCII text file (Name: 3338_092PC01_SeqListing.txt; Size: 106,162 bytes; and Creation Date: March 27, 2019) deposited with the application is incorporated into this document by reference in its entirety.

BACKGROUND OF THE INVENTION

[002] TREM-1 is an activation receptor expressed on monocytes, macrophages and neutrophils. These cells play a central role in chronic inflammatory diseases by releasing cytokines and other mediators that drive inflammation. TREM-1 mRNA and protein expression are upregulated in patients with rheumatic arthritis (RA) and inflammatory bowel disease (IBD), and TREM-1 positive cells accumulate at sites of inflammation, correlating with severity of the disease. See Bouchonet al., Nature 410:1103-1107 (2001); Schenk et al., Clin Invest 117:3097-3106 (2007); and Kuaiet at al., Rheumatology 48:1352-1358 (2009). Peptidoglycan recognition protein 1 (PGLYRP1) expressed mainly by activated neutrophils is a ligand for TREM-1 and mediates TREM-1 signaling upon binding.

[003] In vitro, TREM-1 engagement triggers secretion of inflammatory pro-cytokines including TNF, IL-8, and monocyte chemoattractant protein 1. Furthermore, TREM-1 signaling synergizes with multiple Toll-like receptors (TLRs) to also foster pro-inflammatory signals. In turn, this down-regulates TREM-1 expression, leading to a vicious cycle of increasing inflammation. See Bouchonet al., J. Immunol 164:4991-4995 (2000). Increasing evidence indicates that TLRs contribute to the development and progression of chronic inflammatory diseases such as RA and IBD.

[004] Humanized anti-TREM-1 mAbs that inhibit both human and cynomolgus TREM-1 function have been described elsewhere. See WO 2013/120553 A1 and WO 2016/009086 A1. However, such antibodies either have a viscosity profile that can complicate the manufacturing process or have other problems that can limit their therapeutic potential (e.g., cytokine storm and ADCC). See Shire et al., J. Pharm. Sci. 93:1390-1402 (2004); and Warnckeet al., J Immunol. 188:4405-11 (2012). Consequently, there is a need for an anti-TREM-1 antibody that can specifically bind to and inhibit the function of TREM-1 but without the issues of previous anti-TREM-1 antibodies.

SUMMARY OF THE INVENTION

[005] Isolated antibodies are provided herein, such as monoclonal antibodies, in particular, human antibodies (e.g., monoclonal), which specifically bind triggering receptor expressed on myeloid cells 1 (TREM-1) and have desirable functional properties. In some embodiments, the antibody comprises a heavy chain variable region (VH), a light chain variable region (VL), and an IgG1 heavy chain constant region, wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions compared to a wild-type IgG1 heavy chain constant region (SEQ ID NO: 9). In some embodiments, the antibody cross-competes with mAb 0318 for binding to blocking TREM-1 and comprises a heavy chain variable region (VH), a light chain variable region (VL), and a heavy chain constant region. IgG1, wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions compared to a wild-type IgG1 heavy chain constant region (SEQ ID NO: 9).

[006] In some embodiments, the antibody specifically binds to the same TREM-1 epitope as mAb 0318. In some embodiments, the antibody specifically binds to an epitope of TREM-1 comprising one or more amino acid residues selected from the group consisting on D38, V39, K40, C41, D42, Y43, T44, L45, E46, K47, F48, A49, S50, S51, Q52, K53, A54, W55, Q56, Y90, H91, D92, H93, G94, L95 , and L96 of SEQ ID NO: 1. In some embodiments, the antibody specifically binds to an epitope of TREM-1 comprising amino acids D38 to L45, E46 to Q56, and/or Y90 to L96 of SEQ ID NO: 1.

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

[008] In some embodiments, the antibody described herein comprises a CDR1, CDR2, and CDR3 heavy chain and a CDR1, CDR2, and CDR3 light chain, wherein the CDR3 heavy chain comprises DMGIRRQFAY (SEQ ID NO: 26) or DMGIRRQFAY (SEQ ID NO: 26) except one or more substitutions. In some embodiments, the CDR3 heavy chain comprises DQGIRRQFAY (SEQ ID NO: 72).

[009] In some embodiments, the antibody described herein comprises a CDR1, CDR2, and CDR3 heavy chain and a CDR1, CDR2, and CDR3 light chain, wherein the CDR2 heavy chain comprises RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) or RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) except one or more substitutions.

[0010] In some embodiments, the antibody described herein comprises a CDR1, CDR2, and CDR3 heavy chain and a CDR1, CDR2, and CDR3 light chain, wherein the CDR1 heavy chain comprises TYAMH (SEQ ID NO: 24) or TYAMH (SEQ ID NO: 24) except one or more substitutions.

[0011] In some embodiments, the antibody described herein comprises a CDR1, CDR2, and CDR3 heavy chain and a CDR1, CDR2, and CDR3 light chain, wherein the CDR1 light chain comprises RASQSVDTFDYSFLH (SEQ ID NO: 27) or RASQSVDTFDYSFLH (SEQ ID NO: 27) except one or more substitutions. In some embodiments, the CDR2 light chain comprises RASNLES (SEQ ID NO: 28) or RASNLES (SEQ ID NO: 28) except one or more substitutions. In some embodiments, the CDR3 light chain comprises QQSNQDPYT (SEQ ID NO: 29) or QQSNQDPYT (SEQ ID NO: 29) except one or more substitutions.

[0012] In some embodiments, the VH of the antibody described herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence referred to as SEQ ID NO: 14. In somoe embodiments , the VL of the antibody described herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96 %, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence referred to as SEQ ID NO: 15. In some embodiments, the VH and VL comprise SEQ ID NOs: 14 and 15, respectively.

[0013] In some embodiments, the antibody of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. In some embodiments, the light chain comprises SEQ ID NO: 54.

[0014] Provided herein is an isolated antibody that specifically binds to TREM-1, comprising a CDR1, CDR2, CDR3 heavy chain; a CDR1, CDR2, CDR3 light chain; and an IgG1 heavy chain constant region, wherein the CDR1, CDR2, and CDR3 heavy chain comprises TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively ; wherein the CDR1, CDR2, and CDR3 light chain comprises RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; and wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, and G237A, by EU numbering.

[0015] Provided herein is an isolated antibody that specifically binds to TREM-1, comprising a CDR1, CDR2, CDR3 heavy chain; a CDR1, CDR2, CDR3 light chain; and an IgG1 heavy chain constant region, wherein the CDR1, CDR2, and CDR3 heavy chain comprises TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively ; wherein the CDR1, CDR2, and CDR3 light chain comprises RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; and wherein the IgG1 heavy chain constant region comprises amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S, by EU numbering.

[0016] Provided herein is an isolated antibody that specifically binds to TREM-1, comprising a CDR1, CDR2, CDR3 heavy chain; a CDR1, CDR2, CDR3 light chain; and an IgG1 heavy chain constant region, wherein the CDR1, CDR2, and CDR3 heavy chain comprises TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively ; wherein the CDR1, CDR2, and CDR3 light chain comprises RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; and wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S, by EU numbering.

[0017] Provided herein is an isolated antibody that specifically binds to TREM-1, comprising a CDR1, CDR2, CDR3 heavy chain; a CDR1, CDR2, CDR3 light chain; and an IgG1 heavy chain constant region, wherein the CDR1, CDR2, and CDR3 heavy chain comprises TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25), and DMGIRRQFAY (SEQ ID NO: 26), respectively ; wherein the CDR1, CDR2, and CDR3 light chain comprises RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28), and QQSNQDPYT (SEQ ID NO: 29), respectively; and wherein the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S, by EU numbering .

[0018] In some embodiments, TREM-1 comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

[0019] In some embodiments, the antibody of the present invention has a reduced binding affinity to FCYRI (CD64), FcYRIIA (CD32), FCYRIIB (CD32), FCYRIIIA (CD16a), FCYRIIIB (CD16b), or any combination thereof in comparison with an antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 54. In some embodiments, the antibody has an affinity reduced binding to FCYRI (CD64) by at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, or at least 10 times compared to an antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 54.

[0020] In some embodiments, the antibody described herein is less immunogenic compared to an antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned mentioned in SEQ ID NO: 54. In some embodiments, the antibody does not agonize 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 an inflammatory cytokine in immature dendritic cells (iDCs) when the cells are incubated in the presence of the antibody and in the absence of a stimulator compared to an antibody comprising a heavy chain consisting of the sequence of amino acid as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 54.

[0021] In some embodiments, the antibody described herein blocks the production of an inflammatory cytokine in a cell when the cells are activated in the presence of both the antibody and the stimulator. In some embodiments, the stimulator is a TREM-1 ligand. In some embodiments, the inflammatory cytokine is selected from the group consisting of IL-6, TNF-α, IL-8, IL-1β, IL-12, chitinase-3-like protein 1 (CHI3L1), and combinations thereof.

[0022] In some embodiments, the antibody of the present invention binds to human FcRn, cynomolgus FcRn, and/or mouse FcRn in a pH-dependent manner. In some embodiments, the antibody described herein is more thermally stable compared to a reference antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned in mentioned in SEQ ID NO: 54, when measured by a Capillary Differential Scanning Calorimeter (CAP-DSC). In some embodiments, about 10% to 20%, about 20% to 30% (e.g., 24%), or about 30% to 40% of the antibody is reversible when it is heated to 77°C. In some embodiments, the antibody has a higher melting temperature (Tm) compared to an antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 54.

[0023] In some embodiments, the antibody described 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 less than 10 cP (e.g., 9 cP) at a concentration of 130 mg/mL.

[0024] In some embodiments, the antibody binds to human TREM-1 with a KD of less than 4 nM (e.g., 3.4 nM) when measured by Biacore. In some embodiments, the antibody binds to cynomolgus TREM-1 with a KD of less than 1 nM (e.g., 0.91 nM) when measured by Biacore.

[0025] In some embodiments, the antibody is monomeric when observed by size exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, the antibody exhibits minimal risk of fragmentation when observed by two-dimensional liquid chromatography tandem mass spectrometry (2D-LC/MS) or intact mass analysis using liquid chromatography tandem mass spectrometry (LC/MS). In some embodiments, the antibody has an isoelectric point of 8 to 9 (e.g., 8.75).

[0026] In some embodiments, the antibody is stable in a formulation comprising histidine, sucrose, arginine, and NaCl. In some embodiments, the antibody is stable for at least 2 months in a formulation comprising 20 mM histidine, 150 mM sucrose, 25 mM arginine, and 50 mM NaCl. In some embodiments, the formulation is at a pH of 6.0 and/or the formulation is stored at 4°C, 25°C, or 40°C.

[0027] Bispecific aquimolecules comprising an anti-TREM-1 antibody of the present invention, linked to a molecule having a second binding specificity, are also provided.

[0028] Nucleic acids encoding the antibody described herein, vectors comprising the nucleic acids, and cells transformed with the vectors are provided herein.

[0029] Provided herein are immunoconjugates comprising the anti-TREM-1 antibodies described herein, linked to an agent.

[0030] Compositions comprising anti-TREM-1 antibodies, or antigen-binding portions thereof, bispecific molecules, or immunoconjugates described herein, and a carrier are provided herein. Also provided herein are kits comprising the anti-TREM-1 antibodies, or antigen-binding portions thereof, bispecific molecules, or immunoconjugates described herein, and instructions for use.

[0031] Provided herein is a method of inhibiting TREM-1 activity in an individual in need thereof, comprising administering the anti-TREM-1 antibody, the specific molecule, the nucleic acid, the vector, the cell, or the immunoconjugate of the present invention.

[0032] Provided herein is a method of treating an inflammatory disease or an autoimmune disease in an individual in need thereof, comprising administering the anti-TREM-1 antibody, the specific molecule, the nucleic acid, the vector, the cell, or the immunoconjugate of the present invention. In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of an inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA) , psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), nephritis lupus nephritis, vasculitis, sepsis, systemic inflammatory response syndrome (SIRS), type I diabetes, Grave's disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft versus host disease, Sjogrens syndrome, autoimmune nephritis, Goodpasture syndrome, inflammatory demyelinating polyneuropathy chronic, allergy, asthma, other autoimmune diseases that are a result of acute or chronic inflammation, and any combinations thereof. In some embodiments, the method also comprises administering one or more additional therapeutic products. In some embodiments, additional therapeutics are an anti-IP-10 antibody or an anti-TNF-α antibody.

BRIEF DESCRIPTION OF DRAWINGS/FIGURES

[0033] FIGS. 1A and 1B show that all mAb 0318 variants bind to human TREM-1 and cynomolgus TREM-1 like the original mAb 0318 antibody (IgG4). FIG. 1A shows binding adinity data for the 318-IgG1.3f variant for both human (top row) and cynomolgus (bottom row) TREM-1. FIG. 1B shows binding affinity data for several different varieties of human TREM-1 mAb 0318 variants. The different variants shown include: (i) 318-IgG1.1f, (ii) 318-IgG1.3f, (iii) 318-IgG4-Aba, (iv) 318-IgG1-Aba, and (v) 318-IgG1. 1f (M to Q mutant). Binding affinity data for the mAb 0318 antibody (IgG4 A and B) has been provided for comparison purposes in FIG. 1B.

[0034] FIGS. 2A and 2B show the internalization of mAb 0318-IgG1.3f after binding to TREM-1 expressed on CD14+ monocytes at various times after binding to TREM-1. In FIG. 2A, TREM-1 receptor expression 0, 6, and 24 hours after binding. In FIG. 2B, TREM-1 receptor expression was analyzed at 0, 4, and 20 hours after ligation. FIG. 2B also provides data using the TREM26 antibody, which does not compete with the 0318 antibody variants for binding to TREM-1. The TREM26 antibody was used to assess whether the TREM-1 receptor was internalized (e.g., whether it was degraded or recycled back to the surface).

[0035] FIG. 3 shows that mAb 0318 variants do not agonize TREM-1 signaling when measured by the BWZ/hTREM-1 Reporter Cell Assay when a reporter cell line is incubated with ("Both") or without CHO-CD32 ("Both") or without CHO-CD32 ("Both") or without CHO-CD32 ("Both") or without CHO-CD32 ("Both") BWZ 36 only"). Variant antibodies shown include: (i) 318-IgG1.1f ("IgG1.1f"), (ii) 318-IgG1.3f ("IgG1.3f"), (iii) 318-IgG4-Aba ("FcAba- 4"), (iv) 318-IgG1- Aba ("FcAba-1"). The antibody MAB1278, a known agonist of TREM-1 signaling, was used as a positive control antibody (see inset box). antibody 5C8 (isotype control) was used as a negative control.

[0036] FIG. 4 shows the potency of different mAb 0318 variants in blocking TREM-1-mediated production of inflammatory cytokines by different human cells. The mAb 0318 antibody variants shown in FIG. 4 inhibit TREM-1-mediated release of inflammatory cytokines (e.g., TNF-α, IL-6, or IL-8) from different human cell types: PBMC, monocytes, neutrophils, and RBC pelleted whole blood (i.e. , majority of red blood cells removed using the dextran-based RBC sedimentation protocol as described in the Examples). To produce inflammatory cytokines, cells were stimulated with plaque-bound PGRP1 and soluble peptidoglycan that lacks TLR2 activity ("PGRP + PGN-Ecndss") or phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils expressing PGRP1 (" PMA-Stim Neutrophil Endogenous PGRP"). Variant antibodies shown include: (i) 0318-IgG4, (ii) 0318-IgG1.3f, (iii) 0318-IgG1.1f, (iv) 0318-IgG1-Aba, and (v) 318-IgG4-Aba. "N/A" indicates that the expression level of the particular inflammatory cytokine has not been determined.

[0037] FIGS. 5A and 5B show the potency of mAb 0318-IgG1.3f to block whole blood IL-8 production following stimulation with PGN with or without PGRP1 in the presence of NOD2 inhibitor. FIG. 5A provides inhibition data generated using the whole blood pharmacodynamics assay as described in the Examples. The IG. 5B provides inhibition data generated using the whole blood intracellular cytokine assay as described in the Examples.

[0038] FIGS. 6A to 6C show the RNA expression levels of chitinase-3-like protein 1 ("CHI3L1") (FIG. 6A), IL-1β (FIG. 6B), and IL-6 (FIG. 6C) from stimulated whole blood in the presence of varying concentrations of mAb 0318-IgG1.3f (0-1 nM). Whole blood was collected from three different donors (#126, #290, and #322) and stimulated with soluble PGRP1 and soluble peptidoglycan that does not have TLR2 activity ("Soluble PGRP + Soluble PGN-Ecndss"). In each of FIGs. 6A to 6C, RNA expression levels (y-axis) are shown as both % inhibition (right column) and ΔΔCt (difference between the value of a reference gene and the test sample value) (left column). The different concentrations of the 0318-IgG1.3f antibody are shown on the x-axis.

[0039] FIG. 7 shows the viscosity concentration profile of both the 318-IgG1.1f (square) and the 318-IgG1.3f (circle) variant antibodies. The viscosity profile was generated from the dilution scheme using the Rheosense m-VROC dynamic viscometer using a 3-point shear sweep for each point at constant temperature. The solid line provides the best nonlinear curve fit to the data shown. The dotted line shows the maximum viscosity level allowed for effectiveness.

[0040] FIG. 8 shows that both variant antibodies 318-IgG1.1f and 318-IgG1.3f have a low to medium risk of immunogenicity. VL6 (immunogenic IL-21RmAb) and KLH (keyhole keyhole limpet hemocyanin) were used as positive controls. Avastin was used as a negative control.

[0041] FIGS. 9A and 9B show that all mAb 0318 variants are capable of binding to FcRn (human (black), cino (white), and black (gray)) in a pH-dependent manner. FIG. 9A provides FcRn binding % of Rmax (maximum FcRn binding capacity). FIG. 9B provides FcRn binding as sensorgrams. Antibody 0318 variants shown include: (i) IgG1-Aba, (ii) IgG4-Aba, (iii) IgG1.1f, and (iv) IgG1.3f. The mAb 0318 (IgG4) antibody is also shown for comparison purposes.

[0042] FIGS. 10A and 10B shows that all mAb 0318 antibody variants demonstrate decreased binding to one or more of the human FCYRS (i.e., CD64, CD32a-H131 variant, CD32a-R131 variant, CD32b, CD16a-V158 variant, and CD16B-NA2 variant ). FIG. 10A shows the binding affinity as % of Rmax (maximum FCYR capacity). FIG. 10B shows the sensorgrams. Antibody 0318 variants shown include: (i) IgG1-Aba, (ii) IgG4-Aba, (iii) IgG1.1f, and (iv) IgG1.3f. The mAb 0318 (IgG4) antibody is shown for comparison purposes. Antibody 1F4-hIgG1f, which is known to bind to multiple FCYRS, was used as a control.

[0043] FIGS. 11A to 11I show that the 0318 variant antibodies are non-agonistic in themselves (i.e., in the absence of a stimulus), as measured by the release of different inflammatory cytokines (e.g., IL-6, TNF-α, and IL-12 ) when cultured with immature dendritic cells. FIGS. 11A, 11B, and 11C give the amount of IL-6 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. FIGS. 11D, 11E, and 11F give the amount of TNF-α produced by immature dendritic cells isolated from donors D179, D276, and D341, respectively. FIGS. 11G, 11H, and 11I give the amount of IL-12 produced for immature dendritic cells isolated from donors D179, D276, and D341, respectively. The 0318 antibody variants shown include: (i) IgG1.1f, (ii) IgG1.3f, and (iii) IgG1-Aba. The dosages (μg/mL) of the antibodies used are shown in parentheses. Immature dendritic cells were cultured with anti-TREM-1 antibody variants with and without membrane CD32a expressing CHO cells (to stimulate Fc cross-linking). PGRP+PGN trimer, CD40L ("Trimer"), and Pfizer anti-CD40 agonist antibody ("CP870") were used as positive controls. Ispotype antibody ("CHIL-6") was used as a negative control. The dotted line shows the lower limit of detection for the assay.

[0044] FIG. 12 shows the pharmacokinetics of a single dose of mAb 0318-IgG1.3f variant in a cynomolgus monkey following subcutaneous administration. Each of the animals received one of the following doses: (i) 0.1 mg/kg (n=4) ("1"), (ii) 0.5 mg/kg (n=4) ("2"), (iii) 2 mg/kg (n=3) ("3"), or (iv) 10 mg/kg (n=4) ("4"). Data are shown as the mean ± standard deviation.

[0045] FIG. 13 shows the target-mediated drug disposition scheme used to describe pharmacokinetic (PK), pharmacodynamic (PD), and receptor occupancy (RO) data in cynomolgus monkeys.

[0046] FIGS. 14A and 14B show the density of total TREM-1 receptor on monocytes (FIG. 14A) and granulocytes (FIG. 14B) after a single dose (subcutaneous (sc) or intravenous (iv) administration) of variant mAb 0318-IgG1.3f in cynomolgus macaque. TREM-1 receptor density is shown as a percentage of the TREM-1 receptor density before antibody administration. Each of the animals received one of the following doses: (a) 0.1 mg/kg (sc) (n=4) ("A"), (b) 0.5 mg/kg (n=4) (sc) ("B"), (c) 2 mg/kg (n=3) (sc) ("C"), (d) 2 mg/kg (n=3) (iv) ("D"), or ( e) 10 mg/kg (n=4) (sc) ("E"). Data are shown as the mean ± standard deviation.

[0047] FIGS. 15A and 15B show the occupancy of the TREM-1 receptor on monocytes (FIG. 15A) and granulocytes (FIG. 15B) after a single dose of mAb 0318-IgG1.3f variant in cynomolgus monkey. Receptor occupancy data is shown as a percentage of total TREM-1 receptor expressed in cells. Each of the animals received one of the following: (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 (and ) 10 mg/kg (n=4) (sc) ("E"). Data are shown as the mean ± standard deviation. The dotted line shows 85% receiver occupancy.

[0048] FIGS. 16A and 16B show the observed (open loop) and model predicted (solid line) PK (top left panel), PD (top right panel), RO (base left panel), and total surface TREM-1 receptor expression (bottom right panel) after a single dose of variant mAb 0318-IgG1.3f in cynomolgus monkey described by a 2-compartment PK model with TMDD in central compartment. In FIG. 16A, each of the monkeys received a single dose of 0.1 mg/kg of the antibody subcutaneously. In FIG. 16B, animals received a single dose of 10 mg/kg of the antibody subcutaneously.

DETAILED DESCRIPTION OF THE INVENTION

[0049] So that the present description can be more easily understood, certain terms are first defined. Additional definitions are mentioned throughout the detailed description.

[0050] It should be noted that the term "a" or "a" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. Therefore, the terms "one" (or "one"), "one or more," and "at least one" may be used interchangeably herein.

[0051] Furthermore, "and/or" where used herein should be taken as a specific description of each of the specific features or components with or without the other. Accordingly, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and " B" (alone). Likewise, the term "and/or" when used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0052] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art, to which this description relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of experience with a general dictionary of many terms used in this description.

[0054] Units, prefixes, and symbols are denoted in their accepted International System of Units (SI) form. Numerical variations are inclusive of the numbers defining the variation. Unless otherwise noted, nucleotide sequences are written left to right in a 5' to 3' orientation. Amino acid sequences are written left to right in an amino to carboxy orientation. The titles provided herein are not limitations on the various aspects of the description, which may be taken by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined with reference to the specification in its entirety.

[0055] The term "about" is used herein to mean approximately, around, around, or in the regions of. When the term "about" is used in conjunction with a numerical variation, it modifies that range extending the limits above and below the numerical values presented. In general, the term "about" can modify a numerical value above and below the value established by a variance of, for example, 10 percent, above or below (greater or smaller).

[0056] The term "activating receptor expressed on myeloid cells" (also known as TREM1, TREM-1, and CD354) refers to a receptor that is expressed on monocytes, macrophages, and neutrophils. Primary ligand for TREM-1 includes peptidoglycan recognition protein 1 (PGLYRP1), which belongs to a family of peptidoglycan (PGN) binding proteins (PGRPs). When activated, TREM-1 associates with the ITAM-containing signaling adapter protein, DAP12. Downstream signaling may include activation of the transcription factor NFAT, causing an upregulation of pro-inflammatory cytokine production. The term "TREM-1" includes any variants or isoforms of TREM-1 that are naturally expressed by cells. Accordingly, in some embodiments, the antibodies described herein may cross-react with TREM-1 from non-human species (e.g., TREM-1 from cynomolgus).

[0057] Three isoforms of human TREM-1 have been identified. Isoform 1 (Accession Number NP_061113.1; SEQ ID NO: 1) consists of 234 amino acids and represents the canonical sequence. Isoform 2 (Accession Number NP_001229518.1; SEQ ID NO: 2) consists of 225 amino acids and differs from the canonical sequence at amino acid residues 201-234. The amino acid residues encode part of the transmembrane domain and the cytoplasmic domain. Isoform 3 (Accession No. 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.

[0058] Below are the amino acid sequences of the three known isoforms of human TREM-1. (A) Human TREM-1 isoform 1 (Accession No. NP_061113.1; SEQ ID NO: 1; encoded by the nucleotide sequence having Accession No. NM_018643; SEQ ID NO: 4): MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEK FASSQKAWQIIRDGEMPKTLACTERPSKNSHPV QVGRIILEDYHDHG LLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDIRLVVTKGFSGTP GSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSE INLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP (the signal sequence is underlined); (B) Human TREM-1 isoform 2 (Accession No. NP_001229518.1; SEQ ID NO: 2; encoded by the nucleotide sequence having Accession No. NM_001242589; SEQ ID NO: 5): MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEK FASSQKAWQIIRDGEMPKTLAC TERPSKNSHPVQVGRIILEDYHDHG LLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDIRLVVTKGFSGTP GSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSE INLTNVTDIIRYSFQVPGPLVWTLSPLFPSLCAERM (the signal sequence is underlined); (C) Human TREM-1 isoform 3 (Accession No. NP_001229519; SEQ ID NO: 3; encoded by the nucleotide sequence having Accession No. NM_001242590; SEQ ID NO: 6): MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEK FASSQKAWQIIRDGEMPKTLACTERPS KNSHPVQVGRIILEDYHDHG LLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFRCST LSFSWLVDS (a signal sequence is underlined).

[0059] The cynomolgus TREM-1 protein (Accession No. XP_001082517; SEQ ID NO: 7) is predicted to have the following amino acid sequence: MRKTRLWGLLWMLFVSELRATTELTEEKYEYKEGQTLEVKCDYALEK YANSRKAWQKMEGKMPKILAKTERPSENSHPVQVGRITLEDYPDHG LLQVQMTNLQV EDSGLYQCVIYQHPKESHVLFNPICLVVTKGSSGTP GSSENSTQNVYRTPSTTAKALGPRYTSPRTVTQAPPESTVVVSTPGS EINLTNVTDIIRVPVFNIVIIVAGGFLSKSLVFSVLFAVTLRSFGP (the signal sequence is underlined).

[0060] The present description refers to antibodies that specifically bind and block the function of TREM-1. The antibodies block the function of TREM-1 by reducing/blocking the activation of TREM-1 and downstream signaling.

[0061] The anti-TREM-1 antibodies of the present description 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 of TREM-1, peptidoglycan recognition protein 1 (PGLYRP1), from creating 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, dimerization or multimerization of TREM-1 is reduced or prevented by anti-TREM-1 antibodies that are capable of binding to a portion of TREM-1 that would otherwise be present at the interface of a TREM dimer. -1, thereby preventing individual TREM-1 molecules from associating with each other. In other embodiments, the dimerization or multimerization of TREM-1 is reduced or prevented by anti-TREM-1 antibodies that interfere with the interaction of TREM-1 with its ligand.

[0062] In some embodiments, anti-TREM-1 antibodies can block PGLYRP1-induced activation of TREM-1. PGLYRP1, a highly conserved 196 amino acid long protein consisting of a signal peptide and a peptidoglycan binding domain, is expressed in neutrophils and released upon their activation. The amino acid sequence of PGLYRP1 (Accession No. NP_005082.1; SEQ ID NO: 8) is provided below: VPTPQAIRAAQGLLACGVAQGALRSNYVLKGHRDVQRTLSPGNQL YHLIQNWPHYRSP (the signal sequence is underlined).

[0063] Consequently, in some embodiments, the anti-TREM-1 antibodies of the present description down-regulate or block the release of proinflammatory cytokines from myeloid cells (e.g., dendritic cells and monocytes). In some embodiments, anti-TREM-1 antibodies block the release of TNF-α, MIP-1beta, MCP-1, IL-1beta, GM-CSF, IL-6 and/or IL-8 from macrophages, neutrophils, cells of synovial tissue/or a reporter cell, as described herein.

[0064] While the controlled release of inflammatory cytokines in response to foreign antigens can be beneficial (e.g., mounting an effective adaptive immune response), the release of excess inflammatory cytokines can have dire consequences. For example, a common toxic clinical complication that has been observed with in vivo administration of certain antibodies against cell surface immune receptors (e.g., anti-human CD3 antibodies such as OKT3) is cytokine release syndrome (CRS). , which is associated with the excessive release of several cytokines (e.g., TNF-alpha, IFN-gamma, and IL-2) into the circulation. CRS can occur as a result of simultaneous binding of antibodies to their cognate antigen (e.g., CD3 on T cells) (via the variable region of the antibody) and the Fc receptors (e.g., FCYRS) and/or complement receptors. (via the constant region of the antibody) on accessory cells (e.g., antigen-presenting cells). The interaction results in the activation of cells (e.g., T cells and/or accessory cells) and the release of various cytokines that produce a response systemic inflammatory disease characterized by hypotension, pyrexia and rigors. Other symptoms of CRS include fever, chills, nausea, vomiting, and dyspnea.

[0065] In addition to blocking PGLYRP1-induced production of inflammatory cytokines, in one embodiment, the anti-TREM-1 antibodies of the present disclosure reduce the incidence of or result in no incidence of cytokine release syndrome when administered to an individual in need for these. In some embodiments, anti-TREM-1 antibodies do not induce expression of inflammatory cytokines by cells (e.g., dendritic cells) when the cells are incubated in the presence of the antibody alone, compared to an antibody comprising a heavy chain consisting of the amino acid sequence as set forth in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as set forth in SEQ ID NO: 54. In some embodiments, anti-TREM-1 antibodies have decreased binding to one or more FcyRs, which may help to reduce the onset of CSR.

[0066] In some embodiments, the anti-TREM-1 antibodies of the present description equally bind to human TREM-1 and TREM-1 from other species. The term "TREM-1", as used herein, thus encompasses any naturally occurring form of TREM-1, which may be derived from any suitable organism. For example, TREM-1 for use as described herein may be vertebrate TREM-1, such as mammalian TREM-1, such as TREM-1 from a primate (such as a human, a chimpanzee, a cynomolgus monkey, or a 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 certain embodiments, TREM-1 is SEQ ID NO: 1 (human TREM-1, isoform 1). TREM-1 may be a mature form of TREM-1, such as a TREM-1 protein that has undergone post-translational processing within a suitable cell. Such as a mature TREM-1 protein can, for example, be glycosylated. TREM-1 may be a full-sized TREM-1 protein.

[0067] In some embodiments, the anti-TREM-1 antibodies of the present disclosure are monoclonal antibodies, in the sense that they are directly or indirectly derived from a single clone of a B lymphocyte. 1 are produced, analyzed and purified using, for example, the methods described in Examples of International Publication No. WO 2013/120553. In summary, a suitable mouse such as a TREM-1 or TREM-1/TREM-3 knockout (KO) mouse is immunized with TREM-1, a TREM-1 expressing cell, or a combination of both. In another embodiment, the anti-TREM-1 antibodies are polyclonal antibodies, in the sense that they are mixtures of monoclonal antibodies as described herein.

[0068] In some embodiments, the anti-TREM-1 antibodies of the current description are recombinantly expressed in prokaryotic or eukaryotic cells. In some embodiments, the prokaryotic cell is E. coli. In certain embodiments, the eukaryotic is a yeast, insect, or mammalian cell, such as a cell derived from an organism that is a primate (such as a human, a chimpanzee, a cynomolgus monkey, or a 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. Anti-TREM-1 antibodies, as described herein, can also be produced by other methods known to the person skilled in the art, such as phage display or yeast display. Once produced, antibodies can be evaluated by binding to, for example, full-size TREM-1 or mutants thereof in the Examples of International Publication No. WO 2013/120553.

[0069] The term "antibody" as used herein refers to a protein, derived from a germline immunoglobulin sequence, that is capable of specifically binding to an antigen (TREM-1) or a portion thereof. The term includes full-sized antibodies of any class or isotype (i.e., IgA, IgE, IgG, IgM and/or IgY) and any single chain or fragment thereof. An antibody that specifically binds to an antigen, or portion thereof, may bind exclusively to that antigen, or portion thereof, or it may bind to a limited number of homologous antigens, or portions thereof. Full-size antibodies typically comprise at least four polypeptide chains: two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. One immunoglobulin subclass of particular pharmaceutical interest is the IgG family. In humans, the IgG class can be subdivided into 4 subclasses: IgG1, IgG2, IgG3, and IgG4, based on the sequence of their heavy chain constant regions. Light chains can be divided into two types, kappa and lambda, based on differences in their sequence composition. IgG molecules are composed of two heavy chains, linked by two or more disulfide bonds, and two light chains, each linked to a heavy chain by a disulfide bond. A heavy chain may comprise a heavy chain variable region (VH) and up to three heavy chain constant regions (CH): CH1, CH2 and CH3. A light chain can comprise a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can also be subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called structure regions (FR). The VH and VL regions are typically composed of three CDRs and four FRs, organized from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy and light chains form a binding domain that is capable of interacting with an antigen, while the constant region of an antibody can mediate binding of the immunoglobulin to host factors or tissues, including, but not limited to, several immune system cells (effector cells), Fc receptors and the first component (C1q) of the classical complement system. The antibodies of the current invention can be isolated. The term "isolated antibody" refers to an antibody that has been separated and/or recovered from other component(s) in the environment in which it was produced and/or that has been purified from a mixture of components present in the environment in which it was produced. Certain antigen-binding fragments of antibodies may be suitable in the context of the current invention, as it has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-size antibody.

[0070] The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen, such as TREM-1, as described herein. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)S, Fv (typically the VL and VH domains of a single branch of an antibody), Fv single-chain (scFv; see, e.g., Bird et al., Science 242:42S-426 (1988); Huston et al., PNAS 85:58795883 (1988)), dsFv, Fd (typically the VH and CH1 domains ), and dAb fragments (typically a VH domain); VH, VL, VhH, and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 10:949-57 (1997)); camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, wherein the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, for example, Holliger and Hudson, Nat Biotechnol 2S:1126-1136 (2005); International Publication No. WO 2005/040219, and United States Publication Nos. 2005/0238646 and 2002/0161201. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be evaluated for utility in the same manner as intact antibodies.

[0071] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequence. The anti-TREM-1 antibodies described herein may include amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by site-specific or random mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibodies and "fully human" antibodies are used synonymously.

[0072] A "humanized" antibody refers to a human/non-human chimeric antibody that contains one or more sequences (CDR regions or parts thereof) that are derived from a non-human immunoglobulin. human immunoglobulin (recipient antibody) wherein at least the residues of a hypervariable region of the recipient are replaced by residues of a hypervariable region of an antibody from a non-human species (donor antibody) such as from a mouse, rat, rabbit or non-human primate human, which possesses the desired specificity, affinity, sequence composition and functionality. In some cases, human immunoglobulin FR residues are replaced by corresponding non-human residues. An example of such a modification is the introduction of one or more so-called reverse mutations, which are typically amino acid residues derived from the donor antibody. Humanization of an antibody can be accomplished using recombinant techniques known to the person skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. C. Lo). A suitable human container structure for both light and heavy chain variable domains can be identified by, for example, structural or sequence homology. Alternatively, fixed container structures can be used, for example, based on knowledge of the structure, biophysical and biochemical properties. The recipient structures may be germline derived from a mature antibody sequence. The CDR regions of the donor antibody can be transferred by CDR grafting. The CDR-grafted humanized antibody can also be optimized for, for example, affinity, functionality and physical properties by identifying critical framework positions where reintroduction (mutation reverse) of the amino acid residue of the donor antibody has a beneficial impact on the properties of the humanized antibody. In addition to the donor antibody derived from the reverse mutations, the humanized antibody can be designed by introducing germline residues into the framework or CDR regions, elimination of immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0073] Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications are made to also improve the performance of the antibody. In general, a humanized antibody will comprise at least one - typically two - variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and wherein all or substantially all of the FR residues are those of a sequence of human immunoglobulin. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The term "humanized antibody derivative" refers to any modified form of the humanized antibody, such as a conjugate of the antibody and another agent or antibody.

[0074] The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant methods, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for the human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectome, (c) antibodies isolated from a library of combinatorial, recombinant human antibody, and (d) antibodies prepared, expressed, created or isolated by any other methods that involve linking human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that utilize germline particular human immunoglobulin sequence are encoded by germline genes, but include subsequent recombinations and mutations that occur, for example, during antibody maturation. As known in the art (see, e.g., LonbergNature Biotech. 23(9): 1117-1125 (2005)), the variable region contains the antigen-binding domain, which is encoded by several genes that rearrange to form a antibody specific to a foreign antigen. In addition to reorganization, the variable region can also be modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region will change in response to an antigen (i.e., isotype change). Therefore, the somatically mutated and rearranged nucleic acid molecules encoding the light chain and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar (i.e., have at least 80% identity).

[0075] A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

[0076] In one embodiment, the anti-TREM-1 antibodies of the current description are IgG antibodies. An "IgG antibody", e.g., a human IgG1, as used herein, has, in certain embodiments, the structure of a naturally occurring IgG antibody, that is, it has the same number of heavy and light chains and linkages. disulfide as a naturally occurring IgG antibody of the same subclass. For example, the TREM-1 IgG1 antibody consists of two heavy chains (HCs) and two light chains (LCs), in which the two heavy chains and light chains are linked by the same number and location of disulfide bonds that occur in the antibody. Naturally occurring IgG1 (unless the antibody has been mutated to modify the disulfide bonds).

[0077] As used herein, "isotype" refers to the class of antibody (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that is encoded by the genes of the region heavy chain constant.

[0078] "Allotype" refers to naturally occurring variants within a specific isotype group, which variants differ in some amino acids (see, for example, Jefferis et al., mAbs 1:1 (2009)). anti-TREM-1 antibodies described herein may be of any allotype. In some embodiments, the anti-TREM-1 antibodies are of the "IgG1.3f" allotype, which comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, and G237A, by EU numbering, as compared to a wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In other embodiments, the anti-TREM-1 antibodies are of the "IgG1.1f" allotype, which comprises one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S, by EU numbering, when compared to a wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In certain embodiments, the anti-TREM-1 antibodies are of the "IgG1-Aba" allotype, which comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S, by EU numbering, when compared to a wild-type IgG1 isotype (e.g., SEQ ID NO: 9). In other embodiments, the anti-TREM-1 antibodies are of the "IgG4-Aba" allotype, which comprises the CH1 domain of a wild-type IgG4 isotype (e.g., example, SEQ ID NO: 10) and the CH2 and CH3 domains of IgG1. In some embodiments, the IgG4-Aba allotype antibody comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S, by EU numbering, when compared to a wild-type IgG1 isotype (e.g., SEQ ID NO: 9).

[0079] The phrases "an antibody recognizing an antigen" and "an antibody specific to an antigen" are used interchangeably herein with the term "an antibody that specifically binds an antigen."

[0080] An "isolated antibody," as used herein, is intended to refer to an antibody that has been separated and/or recovered from other component(s) in the environment in which it was produced and/or that it was purified from a mixture of components present in the environment in which it was produced.

[0081] An "effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom. Exemplary "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 the downregulation of a cell surface receptor (e.g., B-cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain). In one embodiment, the anti-TREM-1 antibodies of the current description comprise Fc regions that do not bind to one or more FCYRS and therefore lack effector function (i.e., no effector).

[0082] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody comprise receptors of the FCYR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activators (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and an inhibitory receptor (FcyRIIB). Several properties of human FcyRs are known in the art. Most innate effector cell types coexpress an activating FcyR and the inhibitory FcyRIIB, whereas natural killer (NK) cells selectively express an activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the types of activating Fc receptors it binds.

[0083] An "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g. effector cells) or to the first component (C1q) of the classical complement system. Thus, an Fc region comprises the constant region of an antibody excluding the first immunoglobulin domain of the constant region (e.g., CH1 or CL).

[0084] In IgG, the Fc region comprises CH2 and CH3 immunoglobulin domains and the hinge between the CH1 and CH2 domains. Although the definition of the boundaries of the Fc region of an immunoglobulin heavy chain must vary, as defined herein, the Fc region of human IgG heavy chain is defined to extend from an amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxy terminus of the heavy chain, wherein the numbering is according to the index I eat in Kabat. The CH2 domain of a human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is positioned to the C-terminal side of a CH2 domain in an Fc region, that is, it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of an IgG. As used herein, the Fc region may be a native sequence Fc, including any allotypic variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to this region in isolation or in the context of a protein polypeptide comprising Fc such as a "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesion).

[0085] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human lgG1 Fc region; human lgG2 Fc region of native sequence; human lgG3 Fc region of native sequence; and human IgG4 Fc region of native sequence as well as naturally occurring variants thereof. Native sequence Fc includes the various allotypes of Fcs (see, e.g., Jefferies and others), mAbs 1: 1 (2009)).

[0086] A "variant sequence Fc region" or "non-naturally occurring Fc" comprises a modification, typically to alter one or more of its functional properties, such as serum half-life, complement fixation, Fc receptor binding, stability protein and/or antigen-dependent cellular cytotoxicity, or lack thereof, among others. In some embodiments, the anti-TREM-1 antibodies of the present disclosure may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or be modified to alter their glycosylation, again to alter one or more functional properties of the antibody. . In one embodiment, the anti-TREM-1 antibody is an isotype of IgG1 and carries a modified Fc domain comprising one or more, and perhaps all of the following mutations that will result in decreased affinity for certain Fc receptors (L234A, L235E, and G237A) and in reduced C1q-mediated complement fixation (A330S and P331S), respectively (residue numbering according to the EU index).

[0087] The terms "hinge," "hinge domain," "hinge region," and "antibody hinge region" refer to the domain of a heavy chain constant region that connects the CH1 domain to the CH2 domain and includes the upper, middle and lower portions of the joint (Roux et al., J Immunol 161:4083 (1998)). The hinge provides varying levels of flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, a hinge starts at Glu216 and ends at Gly237 of all IgG isotypes (Roux et al., J Immunol 161:4083 (1998)). The joint sequence of IgG1, IgG2, IgG3, and wild-type IgG4 are known in the art (e.g., International PCT Publication No. WO 2017/087678). In one embodiment, the CH1 hinge region of anti-TREM-1 antibodies is modified such that the number of cysteine residues in the hinge region is changed, e.g., increased or decreased. This method is also described, for example, in United States Patent No. 5,677,425.

[0088] The constant region can be modified to stabilize the antibody, for example, to reduce the risk of a bivalent antibody separating into two monovalent VH-VL fragments. For example, in a constant region of IgG4, residue S228 (residue numbering according to the EU index) can be mutated to a proline residue (P) to stabilize inter-heavy chain disulfide bond formation at the joint ( see, for example, Angale et al., MolImmunol. 30: 105-8 (1995)). Antibodies or fragments thereof can also be defined in terms of their complementarity determining regions (CDRs). The term "complementarity determining region" or "hypervariable region", when used herein, refers to the regions of an antibody in which the amino acid residues involved in antigen binding are located. The hypervariability region or CDRs can be identified as the regions with the highest variability in the amino acid alignments of antibody variable domains. The database can be used for CDR identification such as the Kabat database, the CDRs, for example, being defined as comprising amino acid residues 24-34 (CDR1), 5059 (CDR2) and 89-97 ( CDR3) of the light chain variable domain, and 3135 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain; (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, CDRs can be defined as those residues of a "hypervariable cycle" (residues 26-33 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk, J. Mol. Biol 196: 901-917 (1987)). Typically, the numbering of the amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as "Kabat position", "Kabat residue", and "according to Kabat" in this document refer to this numbering system for the heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a decrease in, or insertion into, a structure ( FR) or CDR of the variable domain. For example, a heavy chain variable domain may include amino acid insertions (residues 52a, 52b, and 52c according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g., the residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of the residues can be determined by an antibody provided by aligning in regions of antibody sequence homology with a "standard" Kabat numbered sequence.

[0089] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., TREM-1) to which an immunoglobulin or antibody specifically binds, e.g., as defined by the specific method used to identify it. Epitopes can be formed equally from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by the tertiary duplication of a protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary duplication are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a single spatial conformation. Methods for determining which epitopes are bound by a provided antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoprecipitation and immunoblotting assays, in which the peptides contiguous or overlapping (e.g., of TREM-1) are tested by reactivity with a provided antibody (e.g., anti-TREM-1 antibody). Methods of determining special epitope conformation include techniques in the art and those described herein, e.g., TREM-1 crystallography. x-ray, antigen mutational analysis, two-dimensional nuclear magnetic resonance and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

[0090] The term "binds to the same epitope" with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a provided method. Techniques for determining whether antibodies bind to the "same epitope on TREM-1" as the antibodies described herein include, for example, epitope mapping methods, such as x-ray analysis of antigen complex crystals: antibody that provides atomic resolution of the epitope and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor antibody binding to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. Furthermore, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity-specific small peptides isolated from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

[0091] Antibodies that "compete with another antibody for binding to a target" refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. If two antibodies compete with each other for binding to a target, that is, whether and to what extent one antibody inhibits the binding of the other antibody to a target can be determined using known competitive experiments, for example, BIACORE® surface plasmon resonance (SPR) analysis. In certain embodiments, an antibody competes with, and inhibits the binding of, another antibody to a target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may be different depending of which the antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring HarbProtoc ; 2006; doi: 10.1101/pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross compete" if the antibodies block each other from same way by at least 50%, that is, regardless of whether one or the other antibody is first contacted with the antigen in the competition experiment.

[0092] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds," refer to antibody binding to an epitope on a predetermined antigen . Typically, antibody (i) binds with an equilibrium dissociation constant (KD) of approximately less than 10-7 M, such as approximately less than 10-8 M, 10-9 M or 10-10 M or even lower when determined by, for example, surface plasmon resonance (SPR) technology on a BIACORE® 2000 instrument using the predetermined antigen, e.g., recombinant human TREM-1, as the analyte and the antibody as the ligand, or Scatchard analysis of antibody binding to antigen-positive cells, and (ii) binds to the predetermined antigen with affinity that is at least twice as high as its affinity for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. Accordingly, an antibody that "specifically binds to human TREM-1" refers to an antibody that binds to cell-bound or soluble human TREM-1 with a KD of 10-7 M or less, such as approximately less than 10-8M, 10-9M or 10-10M or even lower. An antibody that "cross-reacts with cynomolgus TREM-1" refers to an antibody that binds to cynomolgus TREM-1 with a KD of 10-7 M or less, such as approximately less than 10-8 M, 10-9 M or 10-10 M or even smaller. In certain embodiments, such antibodies that do not cross-react with TREM-1 from a non-human species exhibit essentially undetectable binding against these proteins in standard binding assays.

[0093] The term "binding specificity" herein refers to the interaction of a molecule such as an antibody, or fragment thereof, with a unique unique antigen, or with a limited number of highly homologous antigens (or epitopes). Conversely, antibodies that are capable of specifically binding to TREM-1 are not capable of binding to dissimilar molecules. Antibodies according to the invention may not be capable of binding to Nkp44, the natural killer cell p44-related protein.

[0094] The specificity of an interaction and the value of an equilibrium binding constant can be determined directly by well-known methods. Standard assays for evaluating the ability of ligands (such as antibodies) to bind their targets are known in the art and include, for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. Antibody binding can also be assessed by standard assays known in the art, such as SPR.

[0095] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include: competition for binding to TREM-1 expressing myeloid cells, for example, by flow cytometry, as described in the Examples. Other methods include: SPR (e.g., BIACORE®), solid-phase indirect or direct radioimmunoassay (RIA), solid-phase indirect or direct enzyme immunoassay (EIA), sandwich competition assay (see, Stahlie et al, Methods in Enzymology 9:242 (1983)); Direct solid phase biotin-avidin EIA (see, Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); Solid phase direct label RIA using 1125 labels (see, Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0096] As used herein, the term "compartment" is defined using a reference antibody. If a second antibody is unable to bind to an antigen at the same time as the reference antibody, the second antibody is said to belong to the same "compartment" as the reference antibody. In this case, the reference and the second antibody competitively bind to the same part of an antigen and are called "competing antibodies". If a second antibody is able to bind to an antigen at the same time as the reference antibody, the second antibody is said to belong to a separate "compartment". In this case, the reference and the second antibody non-competitively bind to each other. same part of an antigen and are called "non-competing antibodies".

[0097] The "storage" antibody does not provide information about the epitope. Competition antibodies, that is, antibodies belonging to the same "compartment" may have identical epitopes, overlapping epitopes or even separate epitopes. The latter is the case in which the reference antibody bound to its epitope on the antigen takes up the space required for the second antibody to contact its epitope on the antigen ("steric block"). Non-competing antibodies generally have separate epitopes.

[0098] The term "binding affinity" in this document refers to an assessment of the strength of a non-covalent interaction between two molecules, for example, an antibody, or fragment thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).

[0099] The binding affinity between two molecules, for example, an antibody, or fragment thereof, and an antigen, through a monovalent interaction can be quantified by determining the equilibrium dissociation constant (KD). In turn, KD can be determined by measuring the kinetics of dissociation and complex formation, for example, by the SPR method. The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and dissociation rate constant kd (or k0ff), respectively. KD is related to ka and kd through the equation KD = kd / ka. Following the above definition, the binding affinities associated with different molecular interactions, such as comparing the binding affinity of different antibodies for a given antigen, can be compared by comparing the KD values for the individual antibody/antigen complexes.

[00100] As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a KD of 10-8 M or less, 10-9 M or less, or 10-10 M or less to a target antigen. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a KD of 10-10 M or less, or 10-8 M or less.

[00101] The term "EC50" in the context of an in vitro or in vivo assay using an antibody or antigen binding fragment thereof, refers to the concentration of an antibody or an antigen binding portion thereof that induces a response that be 50% of the maximum response, that is, halfway between the maximum response and the baseline.

[00102] The term "naturally occurring" as used herein, when applied to an object, refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by humans in the laboratory is naturally occurring.

[00103] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein may contain a modification such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation. A "protein" may comprise one or more polypeptides.

[00104] The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[00105] "Conservative amino acid substitutions" refer to substitutions 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 side chains aromatics (e.g. tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted non-essential amino acid residue in an anti-TREM-1 antibody is replaced with another amino acid residue from the same side chain family. Methods of identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[00106] For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or the designated sequences thereof, when ideally aligned and compared, are identical, with appropriate nucleotide deletions or insertions, by at least about 80% of the nucleotides, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments would hybridize under selective hybridization conditions, to the complement of the strand.

[00107] For polypeptides, the term "substantial homology" indicates that two polypeptides, or the designated sequences thereof, when optimally aligned and compared, are identical, with the appropriate amino acid deletions or insertions, in at least about 80% of the amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids.

[00108] The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of spaces , and the length of each space, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of the percentage of identity between the two sequences can be achieved using a mathematical algorithm, as described in the non-limiting examples below.

[00109] The percentage of identity between the two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using an NWSgapdna.CMP matrix and a space weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between the two nucleotide or amino acid sequences can also be determined using the algorithm by E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which was incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a space length penalty of 12 and a space penalty of 4. Additionally, the percent identity between the two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J. Mol. Biol. (48):444-453 (1970 )) which was incorporated into the GAP program in the GCG software package (available at worldwideweb.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a space weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[00110] The nucleic acid and protein sequences described herein can also be used as a "query sequence" to perform a search against the public database to, for example, identify related sequences. Such searches can be carried out using the NBLAST and XBLAST (version 2.0) programs by Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. Protein BLAST searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain nucleic acid sequences homologous to the protein molecules described herein. To obtain range alignments for comparison purposes, range BLAST can be used as described in Altschule et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Interval BLAST programs, the basic parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[00111] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., the other parts of the chromosome) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[00112] Nucleic acids, for example cDNA, can be mutated, according to standard techniques to provide gene sequences. For coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences substantially homologous to, or derived from, native V, D, J, constant, changes and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical to or modified from another sequence ).

[00113] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been 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 ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[00114] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular object cell, but to the progeny of such a cell. Because certain modifications may occur in succeeding generations, due to mutation or behavioral influences, such progeny may not, in fact, be identical to the cell of origin, but are still included within the scope of the term "host cell" as used in this document.

[00115] As used herein, the term "linked" refers to the association of two or more molecules. The bond can be covalent or non-covalent. The linkage may also be genetic (i.e., recombinantly fused). Such linkages can be obtained using a wide variety of recognized techniques, such as chemical conjugation and recombinant protein production.

[00116] As used herein, "administering" refers to the physical introduction of a composition comprising a therapeutic agent to an individual, using any of various delivery methods and systems known to those skilled in the art. Different administration routines for the anti-TREM-1 antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral administration routines, for example, by injection or infusion. The phrase "parenteral administration" as used herein means methods of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular infusion and injection. , intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal, as well as in vivo electroporation. Alternatively, an antibody described herein may be administered by a non-parenteral routine, such as a topical, epidermal or mucosal administration routine, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administration may also be carried out, for example, once, a plurality of times and/or for one or more extended periods.

[00117] As used herein, the terms "inhibits" or "blocks" (e.g., referring to the inhibition/blocking of TREM-1 ligand binding to TREM-1 in cells) are used interchangeably and equally encompass partial and complete inhibition/blocking. In some embodiments, the anti-TREM-1 antibody inhibits binding of the TREM-1 ligand to TREM-1 by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95 %, 99%, or 100%, determined, for example, as also described herein. In some embodiments, the anti-TREM-1 antibody inhibits binding of the TREM-1 ligand to TREM-1 by no more than 50%, e.g., by about 40%, 30%, 20%, 10%, 5% or 1%, determined, for example, as also described in this document.

[00118] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the individual with the aim of reversing, alleviate, ameliorate, inhibit or delay or prevent the progression, development, severity or recurrence of a symptom, complication, condition or biochemical clues associated with a disease or enhance general survival. Treatment may be for an individual having a disease or an individual who does not have a disease (e.g., for prophylaxis).

[00119] The term "effective dose" or "effective dosage" is defined as an amount sufficient to obtain, or at least partially obtain, a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of the disease as evidenced by a decrease in the severity of symptoms of the disease. disease, an increase in the frequency and duration of periods free from symptoms of the disease, or a prevention of impairment or disability due to the affliction of the disease. A therapeutically effective dosage or amount of a drug includes a "prophylactically effective amount" or a "prophylactically effective dosage", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to an individual at risk of developing a disease or from experiencing a recurrence of the disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit disease development or recurrence can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in predictive animal model systems of efficacy in humans, or testing the activity of the agent in in vitro assays.

[00120] The term "patient" includes humans and other mammalian individuals receiving prophylactic or therapeutic treatment.

[00121] As used herein, the term "individual" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat an individual having cancer. The term "non-human animal" includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[00122] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[00123] Various aspects described in this document are described in more detail in the following subsections.

I. Anti-TREM-1 Antibodies

[00124] Antibodies, for example, fully human antibodies, which are characterized by particular functional properties or characteristics, are described herein. For example, the antibodies of the present disclosure specifically bind to human TREM-1, and more specifically, a particular domain (e.g., a functional domain) within the extracellular domain of human TREM-1. In one embodiment, the antibodies specifically bind to the site on TREM-1 to which the TREM-1 ligand (e.g., PGLYRP1) binds. In certain embodiments, the antibodies are antagonistic antibodies, that is, they inhibit or suppress the activity of TREM-1 (that is, they do not agonize upon binding) on cells, for example, monocytes, macrophages and neutrophils. In some embodiments, anti-TREM-1 antibodies cross-react with TREM-1 from one or more non-human primates, such as cynomolgus TREM-1. In some embodiments, anti-TREM-1 antibodies block the production of inflammatory cytokines (e.g., IL-6, TNF-α, IL-8, IL-1β, IL-12, and combinations thereof) by cells (e.g. , macrophages, dendritic cells, neutrophils) in activation. In other embodiments, the anti-TREM-1 antibodies comprise Fc regions that do not bind to one or more FcYRs. In other embodiments, the anti-TREM-1 antibodies do not induce the release of proinflammatory cytokines by myeloid cells (e.g., dendritic cells) and thereby reduce or prevent the onset of the inflammatory cytokine storm following administration of anti-TREM-1 antibodies to an individual in need thereof.

[00125] In some embodiments, the particular anti-TREM-1 antibodies described herein are antibodies, e.g., monoclonal, recombinant and/or human antibodies, that cross-compete with mAb 0318 for binding to a human TREM-1. . In some embodiments, anti-TREM-1 antibodies also cross-compete with mAb 0318 for binding to cynomolgus TREM-1. In other words, the anti-TREM-1 antibodies of the present disclosure belong to the same "compartment" as mAb 0318 in certain embodiments.

[00126] The antibody mAb 0318 has a heavy chain variable region (VH) comprising SEQ ID NO: 14 and a light chain variable region (VL) comprising SEQ ID NO: 15. See International Publication No. 2016/009086. mAb 0318 also has a heavy chain CDR1, CDR2, and CDR3, which correspond to amino acids 31-35, 50-68, and 101-110 of SEQ ID NO: 14, respectively. The light chain CDR1, CDR2, and CDR3 of the mAb 0318 antibody correspond to amino acids 24-38, 54-60, and 93-101 of SEQ ID NO: 15.

[00127] Consequently, in some embodiments, the anti-TREM-1 antibody of the present description comprises VH and VL of SEQ ID NOs: 14 and 15, respectively. In another embodiment, the VH of the anti-TREM-1 antibody comprises a CDR1 sequence of amino acids 31-35 (TYAMH) of SEQ ID NO: 14, in which one of the amino acids can be replaced by a different amino acid. In certain embodiments, VH of the anti-TREM-1 antibody comprises a CDR2 sequence of amino acids 50-68 (RIRTKSSNYATYYAASVKG) of SEQ ID NO: 14, wherein one, two or three of the amino acids may be replaced by a different amino acid. In some embodiments, the VH of the anti-TREM-1 antibody comprises a CDR3 sequence of amino acids 101-110 (DMGIRRQFAY) of SEQ ID NO: 14, wherein one, two or three of the amino acids may be replaced by a different amino acid.

[00128] In some embodiments, the VL of the anti-TREM-1 antibody comprises a CDR1 sequence of amino acids 24-38 (QQSNQDPYT) of SEQ ID NO: 15, in which one, two or three of the amino acids may be replaced by a different amino acid. In other embodiments, the VL of the anti-TREM-1 antibody comprises a CDR2 sequence of amino acids 54-60 (RASNLES) of SEQ ID NO: 15, wherein one or two of the amino acids may be replaced with a different amino acid. In some embodiments, the VL of the anti-TREM-1 antibody comprises a CDR3 sequence of amino acids 93-101 (QQSNQDPYT) of SEQ ID NO: 15, wherein one or two of the amino acids may be replaced with a different amino acid.

[00129] Methionine residues in antibody CDRs can be oxidized, resulting in potential chemical degradation and consequent reduction in antibody potency. Accordingly, the anti-TREM-1 antibodies described herein may have one or more methionine residues in the heavy and/or light chain CDRs substituted with amino acid residues that do not undergo oxidative degradation. In some embodiments, methionine residues within heavy chain CDR1 and CDR3 are replaced with amino acid residues that do not undergo oxidative degradation (e.g., glutamine or leucine). Accordingly, in one embodiment, the VH of the anti-TREM-1 antibody comprises a CDR3 sequence of amino acids 101-110 (DQGIRRQFAY) of SEQ ID NO: 81 or amino acids 101-110 (DLGIRRQFAY) of SEQ ID NO: 82. In others In one embodiment, the VH of the anti-TREM-1 antibody comprises a CDR1 sequence of amino acids 31-35 (TYAQH) of SEQ ID NO: 83 or amino acids 31-35 (TYALH) of SEQ ID NO: 84. Similarly, in some embodiments , deamidation sites can be removed from anti-TREM-1 antibodies, particularly in CDRs.

[00130] In some embodiments, the VH and VL of the anti-TREM-1 antibody comprises a VH and VL sequences of the anti-TREM-1 antibodies described in International Publication No. WO 2017/152102 A2, which is incorporated herein document by reference in its entirety. In some embodiments, the VL of the anti-TREM-1 antibody comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 9-27 of WO 2017/152102, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 28-40 of WO 2017/152102, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 41-119 of WO 2017/152102. In one embodiment, the VH of the anti-TREM-1 antibody comprises a CDR1 sequence selected from the group consisting of SEQ ID NOs: 120-143 of WO 2017/152102, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 144 -172 of WO 2017/152102, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 173-247 of WO 2017/152102.

[00131] In some embodiments, the anti-TREM-1 antibodies of the present disclosure comprise CDR and/or variable region sequences that have at least 80% identity (e.g., at least 85%, at least 95%, at least 95%, or at least 99% identity) to the CDR and/or variable region sequences of the mAb 0318 antibody.

[00132] In some embodiments, anti-TREM-1 antibodies comprise a heavy chain variable region (VH) and a light chain variable region (VL) comprising SEQ ID NOs: 14 and 15, respectively. In some embodiments, the anti-TREM-1 antibodies comprise a heavy chain (HC) and a light chain (LC), wherein the HC comprises SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. In some embodiments, the LC comprises SEQ ID NO: 54.

[00133] In some embodiments, the anti-TREM-1 antibodies of the present disclosure comprise a heavy chain (VH) variable region selected from the group consisting of SEQ ID NOs: 396475 of WO 2017/152102 and/or a heavy chain variable region light (VL) selected from the group consisting of SEQ ID NOs: 316-395 of WO 2017/152102.

[00134] In some embodiments, the anti-TREM-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise amino acid sequences as shown in Table 7. In some embodiments, the anti-TREM-1 antibody TREM-1 comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence referred to as SEQ ID NO: 50 and a light chain comprises the amino acid sequence referred to as SEQ ID NO: 54. 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 mentioned as SEQ ID NO: 51 and a light chain comprises the amino acid sequence mentioned as SEQ ID NO: 54. 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 referred to as SEQ ID NO: 52 and a light chain comprises the amino acid sequence referred to as SEQ ID NO: 54. 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 referred to as SEQ ID NO: 53 and a light chain comprises the amino acid sequence referred to as SEQ ID NO: 54.

[00135] Light and Heavy Chains comprising an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to any of the light and heavy chains mentioned in present document, for example, SEQ ID NOs: 50 to 54 can be used to form anti-TREM-1 antibodies having the desired characteristics, for example, those also described herein.

[00136] In some embodiments, the anti-TREM-1 antibody of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence mentioned as SEQ ID NO: 50, 51, 52, or 53 and wherein the light chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence mentioned as SEQ ID NO: 54.

[00137] In some embodiments, the anti-TREM1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E , L358M, and any combination thereof by EU numbering. In some embodiments, the anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, L358M, and any combination thereof by EU numbering. In some embodiments, the anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, P238S, and any combination of them by EU numbering. In some embodiments, the anti-TREM-1 antibody comprises a heavy chain constant region, wherein the heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, P238S, and any combination thereof by EU numbering.

[00138] In one embodiment, the anti-TREM-1 antibody is capable of binding variants of TREM-1 (e.g., TREM-1 isoforms 2 and 3, SEQ ID NOs: 2 and 3, respectively), as determined using , for example, surface plasmon resonance. In another embodiment, the anti-TREM-1 antibody is capable of binding cynomolgus TREM-1 (SEQ ID NO: 7), as determined using, for example, surface plasmon resonance.

[00139] In some embodiments, anti-TREM-1 antibodies described herein bind to human TREM-1 with high affinity, for example, as determined by BIACORE™ (e.g., as described in the Examples), with a KD of 10-7 M or less, 10-8 M or less, 10-9 M (1 nM) or less, 10-10 M or less, 10-11 M or less, 10-12 M or less, 10-12 M at 10-7 M, 10-11 M at 10-7 M, 10-10 M at 10-7 M, or 10-9 M at 10-7 M. In some embodiments, anti-TREM-1 antibodies described herein document bind to the TREM-1 cyno, for example, as determined by BIACORE™ (e.g., as described in the Examples), with a KD 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 M to 10-7 M, 10-11 M to 10-7 M, 10-10 M to 10- 7 M, or 10-9 M to 10-7 M.

[00140] In one embodiment, the antibodies of the present invention bind anti-TREM-1 to one or more of the same epitopes as the mAb 0318 antibody. In some embodiments, the anti-TREM-1 antibody is capable of specifically binding (i) at least one amino acid residue selected from the group consisting of A21, T22, K23, L24, T25, E26, and any combination thereof and (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 of human TREM-1 (e.g., Isoform 1, SEQ ID NO: 1). See WO 2016/009086.

[00141] In one embodiment, the anti-TREM-1 antibody is capable of specifically binding to amino acids D38 to F48 of SEQ ID NO: 1 (human TREM-1), as determined using, for example, HX-MS or diffraction X-ray. In some embodiments, the anti-TREM-1 antibody has an epitope comprising one, two, three, four, five, six, seven, or all of the amino acid residues D38, V39, K40, C41, D42, Y43 , T44, and L45 of SEQ ID NO: 1 (human TREM-1) and one, two, or all of the amino acid residues selected from the group consisting of E46, K47, and F48 of SEQ ID NO: 1 (TREM-1 human), as determined using, for example, HX-MS or X-ray diffraction. In certain embodiments, the anti-TREM-1 antibody has an epitope comprising one, two, three, or all amino acid residues selected from the group that consists of D42, E46, D92, and H93 of SEQ ID NO: 1 (human TREM-1), as determined using TREM-1 variants and surface plasmon resonance.

[00142] In one embodiment, the anti-TREM-1 antibody of the present invention has an epitope comprising at least amino acid residues E46 and/or D92 of SEQ ID NO: 1 (human TREM-1), as determined using variants of TREM-1 and surface plasmon resonance. In another embodiment, the anti-TREM-1 antibody comprises one, two, or all amino acid residues selected from the group consisting of L31, 186 and V101 of SEQ ID NO: 1 (human TREM-1). In certain embodiments, the anti-TREM-1 antibody is capable of specifically binding to a polypeptide comprising amino acid residues E19 to L26 of cynomolgus monkey TREM-1 (SEQ ID NO: 7), as determined using, for example, HX -MS or X-ray diffraction.

[00143] In one embodiment, the anti-TREM-1 antibody is capable of specifically binding to human TREM-1, wherein the epitope of the antibody comprises one, two, three, four, five, six, seven, eight, nine , or all amino acid residues selected from the group consisting of V39, K40, C41, D42, Y43, L45, E46, K47, F48, and A49 of SEQ ID NO: 1.

[00144] In one embodiment, the anti-TREM-1 antibody is capable of specifically binding to human TREM-1, wherein the epitope of the antibody comprises D42 of SEQ ID NO: 1. In other embodiments, the anti-TREM-1 antibody TREM-1 is capable of specifically binding to human TREM-1, wherein the antibody epitope comprises E46 of SEQ ID NO: 1. In some embodiments, the antibody epitope may comprise V39, C41, D42, Y43, L45 of SEQ ID NO: 1. In other embodiments, the antibody epitope may comprise E46, K47 and A49 of SEQ ID NO: 1. In a specific embodiment, the anti-TREM-1 antibody epitope may also comprise F48 of SEQ ID NO: 1.

[00145] In some embodiments, the anti-TREM-1 antibody has a viscosity profile similar to that of the mAb 0318 antibody. In some embodiments, the anti-TREM-1 antibody of the present invention has a viscosity lower than 5 cP, lower than 4 cP, less than 3 cP, less than 2.5 cP, less than 2.4 cP, less than 2.3 cP, less than 2.2 cP, less than 2.1 cP, less than 2 cP, less than 1.9 cP, less than 1.8 cP, less than 1.7 cP, less than 1.6 cP, less than 1.5 cP, less than 1.4 cP, less than 1.3 cP, less than 1.2 cP, less than 1.1 cP, less than 1.0 cP, less than 0.9 cP, less than 0.8 cP, less than 0.7 cP, less than 0.6 cP, less than 0 .5 cP, less than 0.4 cP, less than 0.3 cP, less than 0.2 cP, or less than 0.1 cP at a concentration of 80 mg/mL. In some embodiments, the anti-TREM-1 antibody has a viscosity of less than 10 cP (e.g., 9 cP) at a concentration of 130 mg/mL.

[00146] In some embodiments, the anti-TREM-1 antibody of the present invention comprises mutations, in which one or more negatively charged residues in the light CDR1 and CDR3 regions of the antibody are replaced with uncharged residues. In some embodiments, the anti-TREM-1 antibody comprises a substitution in 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 "cargo patch" mutations.

[00147] In some embodiments, the anti-TREM-1 antibody of the present invention comprises mutations in the Fab-Fab interaction area of SEQ ID NO: 14 to reduce Fab-Fab dimerization. It was previously shown with the mAb 0318 antibody that because the antibodies comprise two Fabs, multimerization can impact viscosity. These mutations are referred to as "Fab-Fab interaction" mutations. In certain embodiments, the anti-TREM-1 antibody comprises a mutation in any of residues Y32, R52, S55, S56, N57, A59, M102, I104 and R106 of SEQ ID NO: 14 or F32, D33, Y34, Y53 , R54, and D98 of SEQ ID NO 15 with an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine.

[00148] In one embodiment, the anti-TREM-1 antibody as described herein comprises a mutation at position 32 of SEQ ID NO: 15, in which phenylalanine is mutated to an amino acid selected from amino acid residues glycine, serine, threonine , cysteine, alanine, valine, leucine, isoleucine and methionine. Such a mutation is based on the observation that an Ala substitution at position Y90 of SEQ ID NO: 1 improved the affinity of SEQ ID NO: 3 for TREM-1. Y90 was found to interact with a phenylalanine residue of SEQ ID NO: 15. Mutations of SEQ ID NO: 15 in order to improve the Fab-TREM-1 interaction are referred to as "Fab-TREM-1 interaction" mutations. Provided herein are anti-TREM-1 antibodies whose variable regions are linked (e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3 or IgG4 Fc, which may be of any allotype or isoallotype, for example, 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. (2009) mAbs 1:1). In some embodiments, the variable regions of the anti-TREM-1 antibodies described herein are linked to a less effector or primarily less effector Fc, e.g., IgG1. In some embodiments, the variable regions of the anti-TREM-1 antibodies are linked to an Fc that has reduced binding or is unable to bind to one or more FCYRS.

[00149] In one embodiment, a VH domain of the anti-TREM-1 antibody described herein can be fused to the constant domain of a human IgG (i.e., Fc), e.g., IgG1, IgG2, IgG3, or IgG4, which is naturally occurring or modified, for example, as also described herein. For example, a VH domain may comprise an amino acid sequence of any VH domain described herein fused to a human IgG, e.g., an IgG1, constant region, such as, the following human IgG1 constant domain amino acid sequence of wild type: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 9) or that of an allotypic variant of SEQ ID NO: 9 and ter The following amino acid sequences: VVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO : 77; allotype-specific amino acid residues are in bold and underlined).

[00150] In one embodiment, a VH domain of the anti-TREM-1 antibody described herein may comprise the amino acid sequence of any VH domain described herein fused to a less effector constant region, for example, the following sequences of constant domain amino acid of human IgG1 least effectors:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 78; "IgG1, 1f," comprising substitutions L234A, L235E, G237A, A330S and P331S, by EU numbering, which are underlined ) or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS V LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK(SEQ ID NO: 79; "IgG1,3f", comprising substitutions L234A, L235E and G237A, by EU numbering, which are underlined).

[00151] For example, an IgG1 allotypic variant comprises a K97R, D239E, and/or L241M (underlined and bolded above) and numbering according to that in SEQ ID NOs: 77-79. Within the full-length heavy region and according to EU numbering, these amino acid substitutions are numbered K214R, D356E and L358M. In some embodiments, the constant region of an anti-TREM-1 antibody also comprises one or more mutations or substitutions in the amino acids L117, A118, G120, A213 and P214 (underlined above) as numbered in SEQ ID NO: 77-79, or L234, A235, G237, A330 and P331, by EU numbering. In other embodiments, the constant region of the anti-TREM-1 antibody comprises one or more mutations or substitutions in the amino acids L117A, A118E, G120A, A213S, and P214S of SEQ ID NO: 77-79, or L234A, L235E, G237A, A330S and P331S, with EU numbering. The constant region of the anti-TREM-1 antibody may also comprise one or more mutations or substitutions L117A, A118E and G120A of SEQ ID NO: 9, or L234A, L235E and G237A, by EU numbering.

[00152] In some embodiments, a VH domain of the anti-TREM-1 antibodies described herein comprises the amino acid sequence of any VH domain described herein fused to an IgG1 constant domain comprising the following amino acid sequences:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 11; "IgG1-Aba", comprising substitutions K214R, C226S, C229S, and P238S, by EU numbering, which are underlined); or ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVEPKSCDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLH QDWLngKey R, G137E, G138S, Q196K, I199T, N203D, K214R,C226S, C229S, and P238S, by EU numbering , which are underlined).

[00153] A VL domain described herein can be fused to the constant domain of a human Kappa or Lambda light chain. For example, a VL domain of an anti-TREM-1 antibody may comprise the amino acid sequence of any VL domain described herein fused to the following human IgG1 kappa light chain amino acid sequence: SEQ ID NO: 13)

[00154] In certain embodiments, the heavy chain constant region comprises a lysine or other amino acid at the C terminus, for example, it comprises the following last 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.

[00155] In one embodiment, the variable region of the anti-TREM-1 antibody is linked to a less effector or mainly less effector Fc. In certain embodiments, 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, as described herein.

[00156] Generally, variable regions described herein can be linked to an Fc comprising one or more modifications, typically to alter one or more functional properties of the antibody, such as binding to the Fc receptor, release of inflammatory cytokines, serum half-life, complement fixation and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody described herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or be modified to alter its glycosylation, to alter one or more functional properties of the antibody. Each of these modalities is described in more detail below. The numbering of residues in the Fc region is that of Kabat's EU index.

[00157] The Fc region encompasses domains derived from the constant region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE and IgM), including a fragment, analogue, variant, mutant or derived from the constant region. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide homologous to the C-terminal region of the immunoglobulin, and may include a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately. or in combination.

[00158] Ig molecules interact with several classes of cellular receptors. For example, IgG molecules interact with three classes of FCY receptors (FCYR) specific to the IgG antibody class, namely FCYRI, FCYRII, and FCYRIII. It has been reported that sequences important for IgG binding to FCYR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by the ability of that antibody to bind to an Fc receptor (FcR).

[00159] In one embodiment, the Fc region of the anti-TREM-1 antibodies is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and/or insertion) with respect to a parental Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural characteristics and/or biological activity.

[00160] For example, it is possible to make modifications to the Fc region in order to generate an Fc variant that (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 an Fc receptor relative to the parental Fc. Such Fc region variants will generally comprise at least one amino acid modification in the Fc region. The combination of amino acid modifications is considered particularly desirable. For example, the variant Fc region may include two, three, four, five, etc. substitutions therein, for example, of the specific Fc region positions identified herein.

[00161] A variant Fc region may also comprise a sequence change in which the amino acids involved in the formation of the disulfide bond are removed or replaced by other amino acids. This removal may prevent reaction with other cysteine-containing proteins present in the host cell used to produce the anti-TREM-1 antibodies described herein. Even when cysteine residues are removed, single-chain Fc domains can still form a dimeric Fc domain that is held together noncovalently. In other embodiments, the Fc region can be modified to make it more compatible with a selected host cell. For example, one can remove the PA sequence near the N terminus of a typical native Fc region, which can be recognized by a digestive enzyme in E. coli, such as proline iminopeptidase. In other embodiments, one or more glycosylation sites within the Fc domain can be removed. Residues that are typically glycosylated (e.g., asparagine) can confer cytolytic response. These residues can be deleted or replaced with non-glycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement, such as the C1q binding site, can be removed from the Fc region. For example, it is possible to delete or replace the EKK sequence of human IgG1. In certain embodiments, sites that affect binding to Fc receptors can be removed, preferably sites other than salvage receptor binding sites. In other embodiments, an Fc region can be modified to remove an ADCC site. ADCC sites are known in the art; see, for example, Sarmay et al., Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. Specific examples of variant Fc domains are described, for example, in WO 97/34631 and WO 96/32478.

[00162] In one embodiment, the Fc hinge region is modified such that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. This method is described in more detail in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the Fc hinge region is altered to, for example, facilitate the assembly of light and heavy chains or increase or decrease antibody stability. In one embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fc hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to SpA binding of the Fc domain. native Fc articulation. This method is described in more detail in U.S. Patent No. 6,165,745 by Ward et al.

[00163] In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and/or 331 may be replaced by a different amino acid residue so that the antibody have an altered affinity for an effector ligand but retain 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 the C1 component of complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[00164] In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced by a different amino acid residue, so that the antibody has altered and/or reduced or abolished C1q binding. complement-dependent cytotoxicity (CDC). This method is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[00165] In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This method is also described in PCT Publication WO 94/29351 by Bodmer et al.

[00166] In yet another example, the Fc region can be modified to decrease antibody-dependent cellular cytotoxicity (ADCC) and/or to decrease affinity for an 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 3 25, 326, 327 4 35, 436, 437 , 438 or 439. Exemplary replacements include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D and 332E. Exemplary variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T and 267E/268F/324T. Other modifications to enhance FCYR and complement interactions include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051 , and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20: 685-691,

[00167] Other Fc modifications that can be made to Fcs are those to reduce or ablate binding to complement proteins and/or FCYR, reducing or ablating Fc-mediated effector functions, such as ADCC, ADCP and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330 and/or 331 (e.g., 330 and 331), wherein the Numbering is according to the EU index. Exemplary replacements include, but are not limited to, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S and 331S (e.g., 330S and 331S), where numbering is in accordance with the EU index. An Fc variant may comprise 236R/328R. Other modifications to reduce FCYR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as, removal of glycosylation at position 297 by mutational or enzymatic means or by production in organisms, such as bacteria, that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20: 685-691.

[00168] Optionally, the Fc region may comprise a non-naturally occurring amino acid residue in additional and/or alternative positions known to one skilled in the art (see, for example, U.S. Patents 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720; International Publication Nos. WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035 752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/020114).

[00169] The affinities and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assay methods (biochemically or immunologically based assays) known in the art including, but not limited to, equilibrium methods ( e.g., enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetic (e.g., BIACORE analysis), and other methods such as direct binding assays, competitive inhibition assays, resonance energy transfer fluorescence (FRET), gel electrophoresis and chromatography (e.g. gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or use a variety of detection methods including, but not limited to, chromogenic, fluorescent, luminescent or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[00170] In certain embodiments, the anti-TREM-1 antibodies of the present invention comprise an Fc that has reduced binding or is unable to bind to FCYRS. In some embodiments, the anti-TREM-1 antibody has a reduced binding affinity to FCYRI (CD64), FcYRIIA (CD32), FCYRIIB (CD32), FCYRIIIA (CD16a), FCYRIIIB (CD16b), or any combination thereof compared to with an antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 54. In some embodiments, the anti-TREM- 1 has a reduced binding affinity to FcyRI (CD64) by at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine times, or at least 10 times compared to an antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 54.

[00171] In some embodiments, the anti-TREM-1 antibodies comprise an Fc variant of IgG1 comprising: (a) one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, and any combination thereof, e.g. EU numbering; (b) one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, P331S, and any combination thereof by EU numbering; (c) one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, P238S, and any combination thereof by 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 by EU numbering.

[00172] In some embodiments, the anti-TREM-1 antibody, as described herein, has (a) an IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at the amino acid residue selected from the group consisting of: N297A , N297Q, D270A, D265A, L234A, L235A, C226S, C229S,P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A,L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, L328E, P238D , S267E, L328F, E233D, G237D, H268D, P271G, A330R, and any combination thereof, wherein the residue numbering is in accordance with EU or Kabat numbering, or comprises an amino acid deletion in the Fc region at a corresponding position glycine 236; (b) an IgG2 isotype and comprises one or more amino acid substitutions in the Fc region at an amino acid residue 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 is in accordance with the EU or Kabat numbering; or (c) an IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at an amino acid residue 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 is in accordance with the EU or Kabat numbering. In some embodiments, (a) the Fc region further comprises one or more additional amino acid substitutions at an amino acid residue selected from the group consisting of A330L, L234F; L235E, P331S and any combination thereof, where the residue numbering is in accordance with EU or Kabat numbering; (b) the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein the residue numbering is in accordance with EU or Kabat numbering; or (c) the Fc region further comprises an S228P amino acid substitution according to EU or Kabat numbering. See WO 2017/152102.

[00173] In certain embodiments, an Fc is chosen that has reduced complement fixation. An exemplary Fc, e.g., IgG1 Fc, with reduced complement fixation has the following two amino acid substitutions: A330S and P331S.

[00174] In certain embodiments, an Fc is chosen that essentially has no effector function, that is, that has reduced binding to FCYRS and reduced complement fixation. An exemplary Fc, e.g. IgG1 Fc, that is non-effector comprises the following five mutations: L234A, L235E, G237A, A330S and P331S.

II. Physical Properties of Antibody

[00175] Anti-TREM-1 antibodies, for example, those described herein, have some or all of the physical characteristics of the specific anti-TREM-1 antibodies described herein, such as the characteristics described in the Examples.

[00176] Glycosylation sites, particularly within the variable regions, can result in increased antibody immunogenicity or a change in antibody pK due to altered antigen binding (Marshall et al., (1972) Annu Rev Biochem 41: 673 -702; Gala and Morrison (2004) J. Immunol 172: 5489-94; Wallick et al., (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al. , (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37: 697-706). Glycosylation is known to occur in motifs containing an N-X-S/T sequence. In some embodiments, the anti-TREM-1 antibodies of the present invention do not contain or have reduced variable region glycosylation. This can be achieved by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues in the glycosylation region. Accordingly, in some embodiments, the anti-TREM-1 antibody as described herein is less immunogenic compared to an antibody comprising a heavy chain consisting of the amino acid sequence as mentioned in SEQ ID NO: 76 and a light chain consisting of in the amino acid sequence as set forth in SEQ ID NO: 54.

[00177] In some embodiments, anti-TREM-1 antibodies do not contain sites of asparagine isomerism. Deamidation of asparagine can occur in the N-G or D-G sequences and results in the creation of an isoaspartic acid residue that introduces a kink in the polypeptide chain and decreases its stability (isoaspartic acid effect).

[00178] Each antibody will have a unique isoelectric point (pi), which generally falls in the pH range between 6 and 9.5. The pI for an IgG1 antibody is typically within the pH range of 7-9.5 and the pI for an IgG4 antibody is typically within the pH range of 6-8. It is speculated that antibodies with a pI outside the normal range may have some misfolding and instability under in vivo conditions. Thus, anti-TREM-1 antibodies, as described herein, may contain a pI value that falls within the normal range (e.g., 89). This can be achieved by selecting antibodies with a pI in the normal range or by mutating charged surface residues.

[00179] Each antibody will have a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71). Generally, the TMi (the initial unfolding temperature) can be greater than 60 °C, greater than 65 °C, or greater than 70 °C. In some embodiments, the anti-TREM-1 antibodies of the present invention have a higher melting temperature compared to an antibody comprising a heavy chain consisting of the amino acid sequence as shown in SEQ ID NO: 76 and a light chain consisting of in the amino acid sequence as shown in SEQ ID NO: 54. Accordingly, in some embodiments, the anti-TREM-1 antibodies of the present invention are thermally stable compared to a reference antibody comprising a heavy chain consisting of the amino acid sequence as shown in SEQ ID NO: 76 and a light chain consisting of the amino acid sequence as shown in SEQ ID NO: 54, as measured, for example, by a Capillary Differential Scanning Calorimeter (CAP-DSC). The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al., (2003) Pharm Res 20:1952-60; Ghirlando et al., (1999) Immunol Lett 68:47-52) or dichroism circular (Murray et al., (2002) J. Chromatogr Sci 40: 343-9). In some embodiments, about 10% to 20%, about 20% to 30% (e.g., 24%), or about 30% to 40% of the antibody is reversible when it is heated to 77 °C.

[00180] In some embodiments, the anti-TREM-1 antibodies of the present invention do not degrade quickly. Degradation of an antibody can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem 67:362632).

[00181] In some embodiments, anti-TREM-1 antibodies, as described herein, present minimal aggregation effects, which may lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured by several techniques, including size exclusion column (SEC), high-performance liquid chromatography (HPLC), and dynamic light scattering (DLS). In some embodiments, the anti-TREM-1 antibodies of the present invention are monomeric as observed by size exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, anti-TREM-1 antibodies present minimal risk of fragmentation as observed by two-dimensional liquid chromatography tandem mass spectrometry (2D-LC/MS) or intact mass analysis using liquid chromatography tandem mass spectrometry (LC/MS). MS).

III. Nucleic Acids, Vectors and Cells

[00182] Another aspect described herein concerns the nucleic acid molecules that encode the anti-TREM-1 antibodies described herein. Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., the chromosomal DNA that is linked to the isolated DNA in nature) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl band, column chromatography, restriction enzymes, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., Ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid described 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.

[00183] Nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or techniques cDNA cloning. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), the nucleic acid encoding the antibody can be recovered from the library.

[00184] In some embodiments, the nucleic acids described herein are those that encode the VH and VL sequences of anti-TREM-1 antibodies of the present invention. Exemplary DNA sequences encoding VH and VL sequences are set forth in SEQ ID NOs: 58-61 and 62-65, respectively.

[00185] A method for preparing an anti-TREM-1 antibody as described herein may comprise expressing a heavy chain and light chains in a cell line comprising the nucleotide sequences encoding the light and heavy chains with a peptide signal, for example, SEQ ID NOs: 58-61 and 62-65, respectively. Host cells comprising these nucleotide sequences are covered herein.

[00186] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into chain genes. full-length antibody, on Fab fragment genes or on an scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in structure.

[00187] Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH encoding DNA to another DNA molecule encoding heavy chain constant regions (joint, CH1, CH2 and/or CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, Publication NIH No. 91-3242) and DNA fragments spanning these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for example, an IgG2 and/or IgG4 constant region. For a Fab fragment heavy chain gene, the DNA encoding VH can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[00188] 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 operatively linking the VL encoding DNA to another DNA molecule encoding the region light chain constant, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immuno-Logical Interest, fifth edition, US Department of Health and Human Services , NIH Publication No. 91-3242) and DNA fragments spanning these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[00189] Another aspect described herein refers to cells (e.g., host cells) that express (e.g., recombinantly) anti-TREM-1 antibodies described herein and related polynucleotides and expression vectors. Also provided herein are vectors comprising polynucleotides comprising nucleotide sequences encoding anti-TREM-1 antibodies or a fragment thereof. In some embodiments, vectors can be used to recombinantly express anti-TREM-1 antibodies described herein in host cells, for example, in mammalian cells. In some embodiments, the vectors can be used for gene therapy.

[00190] Suitable vectors for the invention include expression vectors, viral vectors and plasmid vectors. In some embodiments, the vector is a viral vector.

[00191] As used herein, an expression vector refers to any nucleic acid construct that contains the elements necessary for the transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the elements necessary for replication and translation when introduced into an appropriate host cell. Expression vectors may include plasmids, phagemids, viruses and derivatives thereof.

[00192] The expression vectors of the invention may include polynucleotides that encode the antibody or antigen-binding portion thereof described herein. In some embodiments, the coding sequences for the antibody or the antigen-binding portion thereof are operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked so as to allow each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operatively linked when they are covalently linked so as to place expression or transcription and/or translation of the coding sequence under the influence or control of the gene control sequence. gene expression. Two DNA sequences are said to be operably linked if the induction of a promoter in the 5' gene expression sequence results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired antibody. or antigen-binding portion thereof.

[00193] Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, Rous sarcoma virus ; lentivirus; adenovirus; adeno-associated virus; SV40 type virus; polyomavirus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; polio virus; and RNA viruses, such as a retrovirus. Other vectors well known in the art can readily be used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves the reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into the host's cellular DNA. Retroviruses have been approved for human gene therapy trials. More useful are those retroviruses deficient in replication (i.e., capable of directing the synthesis of desired proteins but unable to manufacture an infectious particle). Such genetically altered retroviral expression vectors have general utility for high-efficiency transduction of genes in vivo. Standard protocols for the production of replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with plasmid, producing recombinant retroviruses by the packaging cell line, collecting viral particles of tissue culture media, and infection of target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

[00194] In some embodiments, the virus is an adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It also has advantages, such as heat and lipid solvent stability; high transduction frequencies in cells of different lineages, including hematopoietic cells; and lack of inhibition of superinfection, thus allowing multiple series of transductions. Reportedly, adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thus minimizing the possibility of insertional mutagenesis and variability of expression of the inserted gene, characteristic of retroviral infection. Furthermore, wild-type adeno-associated virus infections have been followed in tissue culture for more than 100 passages in the absence of selective pressure, implying that adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function extrachromosomally.

IV. Immunoconjugates

[00195] The present invention also provides immunoconjugates comprising any of the anti-TREM-1 antibodies described herein. In some embodiments, the immunoconjugate comprises an antibody or antigen-binding moiety linked to an agent. In some embodiments, the immunoconjugate comprises a bispecific molecule described herein linked to an agent (e.g., as a therapeutic agent or a diagnostic agent).

[00196] For diagnostic purposes, appropriate agents are detectable markers that include radioisotopes, for whole-body imaging, and radioisotopes, enzymes, fluorescent markers and other antibody markers suitable for testing samples. Detectable markers that can be bound to any anti-TREM-1 antibody described herein can be any of several types currently used in the field of in vitro diagnostics, including particulate markers including metal solutions such as colloidal gold, isotopes such as I125 or Tc99 presented, for example, with a peptide chelating agent of the N2S2, N3S or N4 type, chromophores including fluorescent labels, luminescent labels, phosphorescent labels and the like, as well as enzyme labels that convert a given substrate into a detectable label, and labels of polynucleotide that are revealed after amplification, such as by polymerase chain reaction. Suitable enzyme markers include horseradish peroxidase, alkaline phosphatase and the like. For example, the marker may be the enzyme alkaline phosphatase, detected by measuring the presence or formation of chemiluminescence upon conversion of 1,2-dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl dioxane (AMPPD), 3-(4-(methoxyspiro{1 ,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3.7}decan}-4-yl)disodium phenyl phosphate (CSPD), as well as CDP and CDP-STAR® or other luminescent substrates well known to those in the art, for example, suitable lanthanide chelates such as Terbium(III) and Europium(III). The detection medium is determined by the chosen label. Appearance of the label or its reaction products it can be achieved with the naked eye, in the case where the marker is particulate and accumulates at appropriate levels, or by means of instruments such as spectrophotometer, luminometer, fluorimeter and similar, all in accordance with standard practice.

[00197] In some embodiments, the conjugation methods result in linkages that are substantially (or nearly) non-immunogenic, e.g., peptide- (i.e., amide-), sulfide-, (sterically hindered), disulfide-, hydrazone - , ether bonds. These bonds are almost non-immunogenic and show reasonable stability in serum (see, for example, Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009/059278; WO 95/17886).

[00198] Depending on the biochemical nature of the moiety and the antibody, different conjugation strategies can be used. In the event that the portion is naturally occurring or recombinant of between 50 to 500 amino acids, there are standard procedures in textbooks describing the chemistry for the synthesis of protein conjugates, which can be easily followed by the skilled artisan (see, for example, Hackenberger, C.P.R., and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, the reaction of a maleinimide moiety with a cysteine residue within the antibody or moiety is used. This is an especially suitable coupling chemistry if, for example, a Fab or Fab' fragment of an antibody is used. Alternatively, in some embodiments, coupling to the C-terminal end of the antibody or portion is performed. C-terminal modification of a protein, for example, of a Fab fragment, can be carried out as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[00199] In general, the site-specific reaction and covalent coupling are based on the transformation of a natural amino acid into an amino acid with a reactivity that is orthogonal to the reactivity of the other functional groups present. For example, a specific cysteine within a rare sequence context can be enzymatically converted to an aldehyde (see Frese, M. A., and Dierks, T., Chem Bio Chem. 10 (2009) 425-427). It is also possible to obtain a desired amino acid modification utilizing the specific enzymatic reactivity of certain enzymes with a natural amino acid in a given sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; and the protease-catalyzed formation of C - N bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry ( 2004) 389-403). Site-specific reaction and covalent coupling can also be achieved by selectively reacting terminal amino acids with appropriate modifying reagents.

[00200] The reactivity of an N-terminal cysteine with benzonitriles (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve specific covalent coupling to the site.

[00201] The native chemical bond may also rely on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[00202] US6437095 B1 describes a conjugation method that is based on the faster reaction of a cysteine within a stretch of negatively charged amino acids with a cysteine located in a stretch of positively charged amino acids.

[00203] The portion may also be a synthetic peptide or peptide mimic. In case a polypeptide is synthesized chemically, amino acids with orthogonal chemical reactivity can be incorporated during this synthesis (see, for example, de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since a wide variety of orthogonal functional groups are in play and can be introduced into a synthetic peptide, conjugation of such a peptide to a ligand is standard chemistry.

[00204] In order to obtain a monolabeled polypeptide, the conjugate with 1:1 stoichiometry can be separated by chromatography from other conjugation side products. This procedure can be facilitated by the use of a dye-labeled binding pair member and a charged ligand. By using this type of labeled and highly negatively charged binding pair member, monoconjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides that carry more than one ligand, since the difference in charge and molecular weight can be used for separation. Fluorescent dye can be useful for purifying the complex from unbound components, such as a labeled monovalent ligand.

[00205] In some embodiments, the portion bound to an anti-TREM-1 antibody is selected from the group consisting of a binding portion, a labeling portion, and a biologically active portion.

[00206] Anti-TREM-1 antibodies described 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 binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, protein inhibitors of heat shock, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In ADC, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. In some embodiments, the linker is a peptidyl linker, such as, Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 80), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO 08/103693; U.S. Patent No. Publications 20060024317; 20060004081; and 20060247295.

[00207] Anti-TREM-1 antibodies, e.g., those described herein, can also be used to detect TREM-1, such as human TREM-1, e.g., human TREM-1 in tissues or tissue samples . Antibodies can be used, for example, in ELISA assay or flow cytometry. In some embodiments, an anti-TREM-1 antibody is contacted with cells, e.g., cells in a tissue, for a time suitable for specific binding to occur, and then a reagent, e.g., an antibody that detects the antibody anti-TREM-1, is added. Exemplary essays are provided in the Examples. The anti-TREM-1 antibody may be a fully human antibody, or it may be a chimeric antibody, such as an antibody with human variable regions and murine constant regions or a portion thereof. Exemplary methods for detecting TREM-1, e.g., human TREM-1, in a sample (cell or tissue sample) comprise (i) contacting a sample with an anti-TREM-1 antibody for a time sufficient to allow specific binding of the anti-TREM-1 antibody to TREM-1 in the sample, and (2) contacting the sample with a detection reagent, e.g., an antibody, that specifically binds to the anti-TREM-1 antibody, such as, to the Fc region of the anti-TREM-1 antibody, in order to detect TREM-1 bound by the anti-TREM-1 antibody. Washing steps may be included after incubation with the antibody and/or detection reagent. Anti-TREM-1 antibodies for use in these methods do not need to be linked to a marker or detection agents, as a separate detection agent can be used.

[00208] Other uses for anti-TREM-1 antibodies, for example, as monotherapy or combination therapy, are provided elsewhere in this document, for example, in the section relating to combination treatments.

V. Bispecific molecules

[00209] Anti-TREM-1 antibodies described herein can be used to form bispecific molecules. An anti-TREM-1 antibody, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a molecule bispecific that binds to at least two different binding sites or target molecules. For example, an anti-TREM-1 antibody can be linked to an antibody or scFv that specifically binds to any protein that can be used as potential targets for combination treatments, such as the proteins described herein (e.g., antibodies to IP -10 or TNF-α). The antibody described herein can, in fact, be derived from or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule described herein, an antibody described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as other antibody, antibody fragment, peptide or binding mimetic, so that a bispecific molecule results.

[00210] Consequently, provided herein are bispecific molecules comprising at least a first binding specificity for TREM-1 and a second binding specificity for a second target epitope. In some embodiments described herein where the bispecific molecule is multispecific, the molecule may further include a third binding specificity.

[00211] In some embodiments, the bispecific molecules described herein comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, for example, a Fab, Fab', F(ab')2 , Fv, or a single-chain Fv (scFv). The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as an Fv or a single chain construct, as described in Ladner et al. US Patent No. 4,946,778.

[00212] Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules herein are murine, chimeric and humanized monoclonal antibodies.

[00213] The bispecific molecules herein can be prepared by conjugating the binding specificities of the constituents using methods known in the art. For example, each specific molecule's binding specificity can be generated separately and then conjugated together. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl -3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovskyet al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139: 23672375). Some conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[00214] When the binding specificities are antibodies, they can be conjugated through the sulfhydryl bond of the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[00215] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, mAb x (scFv)2, Fab x F(ab')2 or linker x Fab fusion protein. A bispecific antibody may comprise an antibody comprising one scFv at the C terminus of each heavy chain. A bispecific molecule described 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 can comprise at least two single-chain molecules. Methods for preparing the bispecific molecules are described, for example, in US Patent No. 5,260,203; US Patent No. 5,455,030; US Patent No. 4,881,175; US Patent No. 5,132,405; US Patent No. 5,091,513; US Patent No. 5,476,786; US Patent No. 5,013,653; US Patent No. 5,258,498; and U.S. Patent No. 5,482,858.

[00216] The binding of bispecific molecules to their specific targets can be confirmed using methods recognized in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay.

[00217] (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest using a labeled reagent (e.g., an antibody) specific to the complex of interest.

SAW. Kits

[00218] Kits comprising one or more anti-TREM-1 antibodies described herein, or antigen-binding portions thereof, bispecific molecules, or immunoconjugates thereof are provided herein. In some embodiments, there is herein provided a pharmaceutical package or kit comprising one or more containers loaded with one or more of the ingredients of the pharmaceutical compositions herein described, such as one or more antibodies herein provided or a drug-binding moiety. antigen of the same, an optional instruction for use. In some embodiments, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.

VII. Compositions and Formulations

[00219] Also provided herein are compositions (e.g., pharmaceutical compositions) and formulations comprising one or more of the anti-TREM-1 (including polynucleotides, vectors, and cells that encode and/or express the anti-TREM-1 antibodies ) described in this document. For example, in one embodiment, the present invention provides a pharmaceutical composition comprising one or more anti-TREM-1 antibodies as described herein, formulated together with a pharmaceutically acceptable carrier.

[00220] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[00221] Consequently, one of the objectives of the present invention is to provide a pharmaceutical formulation that improves the stability of anti-TREM-1 antibodies and, thus, allows their long-term storage. In some embodiments, the pharmaceutical formulation described herein comprises: (a) an anti-TREM-1 antibody; (b) a buffering agent; (c) a stabilizing agent; (d) a salt; (e) a bulking agent; and/or (f) a surfactant. In some embodiments, the pharmaceutical formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or more. In some embodiments, the formulation is stable when stored at 4°C, 25°C, or 40°C.

Buffering Agent

[00222] Buffering agents useful for the present invention can be a weak base or acid used to maintain the acidity (pH) of a solution close to a chosen value after the addition of another acid or base. Suitable buffering agents can maximize the stability of pharmaceutical formulations while maintaining control over the pH of the formulation. Suitable buffering agents can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties may also depend on the pH of the formulation. Common buffering agents include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, a buffering agent comprises histidine (e.g., L-histidine) with isotonicity and potentially pH-adjusting agents with an acid or base known in the art. In certain embodiments, the buffering agent is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

Stabilizing Agent

[00223] Stabilizing agents are added to a pharmaceutical product to stabilize that product. These agents can stabilize proteins in several different ways. Common stabilizing agents include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine, carbohydrates such as glucose, sucrose, trehalose, raphmose, or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrins or dextrans of any type and molecular weight, or PEG. In one aspect of the invention, the stabilizing agent is chosen in order to maximize the stability of the FIX polypeptide in lyophilized preparations. In certain embodiments, the stabilizing agent is sucrose and/or arginine. Bulking Agent

[00224] Bulking agents can be added to a pharmaceutical product to add volume and mass to the product, thereby facilitating accurate dosing and handling thereof. Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

Surfactant

[00225] Surfactants are amphipathic substances with lyophilic and lyophobic groups. A surfactant can be anionic, cationic, zuiterionic or non-ionic. 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 MEA, polysorbates or dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80,

[00226] In some embodiments, the pharmaceutical formulation of the present invention comprises: (a) about 0.25 mg/mL to 250 mg/mL (e.g., 10 to 200 mg/mL) of an anti-TREM-1 antibody ; (b) about 20 mM histidine; (c) about 150 mM sucrose; (d) about 25 mM arginine; and (e) about 50 mM NaCl.

[00227] The formulation may also comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[00228] In some embodiments, the pharmaceutical formulation is an aqueous formulation. Such a formulation is typically a solution or a suspension, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation comprising at least 50% w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50% w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w/w water.

[00229] In some embodiments, the pharmaceutical formulation is a lyophilized formulation, to which the doctor or patient adds solvents and/or diluents before use.

[00230] Pharmaceutical compositions described herein can also be administered in combination therapy, that is, combined with other agents. For example, the combination therapy may include an anti-TREM-1 antibody herein combined 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 (e.g., an inflammatory disorder). Such compounds, drugs, and/or agents may include, for example, anti-inflammatory drugs or antibodies that block or reduce the production of inflammatory cytokines. In some embodiments, the therapeutic agents may include an anti-IP-10 antibody, an anti-TNF-α antibody (e.g., adalimumab (HUMIRA®), golimumab (SIMPONI®), infliximab (REMICADE®), Certolizumab pegol (CIMZIA ®)), interferon beta-1a (e.g. AVONEX®, REBIF®), interferon beta-1b (e.g. BETASERON®, EXTAVIA®), glatiramer acetate (e.g. COPAXONE®, GLATOPA®), mitoxantrone ( for example, NOVANTRONE®), non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids, and combinations thereof.

[00231] The pharmaceutical compounds mentioned herein may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not confer any undesirable toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phorphorous and the like, as well as from non-toxic organic acids, such as mono- and dicarboxylic aliphatic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[00232] A pharmaceutical composition described 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) oil-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.

[00233] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[00234] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures, above, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption such as aluminum monostearate and gelatin.

[00235] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except to the extent that any conventional medium or agent is incompatible with the active compound, the use thereof in the pharmaceutical compositions described herein is contemplated. A pharmaceutical composition may comprise a preservative or may be devoid of a preservative. Supplementary active compounds can be incorporated into the compositions.

[00236] Therapeutic compositions typically must be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. In many cases, the compositions may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[00237] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, some preparation methods are vacuum drying and freeze drying (lyophilization) which produce a powder of the active ingredient plus any desired additional ingredient from a sterile previously filtered solution.

[00238] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the individual being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Generally, from 100%, this amount will range from about 0.01% to about 99% active ingredient, from about 0.1% to about 70%, or from about 1% to about 30% active ingredient. active in combination with a pharmaceutically acceptable carrier.

[00239] Dosage regimens are adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionately reduced or increased as indicated by the demands of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. The dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the individuals to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms described herein are dictated by, and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the compounding technique. of such an active compound for the treatment of sensitivity in individuals.

[00240] For administration of an anti-TREM-1 antibody, for example, described herein, the dosage varies from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 or 10 mg/kg , the host's body weight. For example, dosages may be 0.3 mg/kg of body weight, 1 mg/kg of body weight, 3 mg/kg of body weight, 5 mg/kg of body weight or 10 mg/kg of body weight or within the range of 1 to 10 mg/kg. An exemplary treatment regimen involves 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 to 6 months. Exemplary dosage regimens for an anti-TREM-1 antibody herein include 1 mg/kg body weight or 3 mg/kg body weight intravenously of administration, with the antibody being given using one of the following dosage schedules. : (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

[00241] In some embodiments, the anti-TREM-1 antibody is administered in a fixed dose (fixed dose regimen). In other embodiments, the anti-TREM-1 antibody is administered in a fixed dose with another antibody. In certain embodiments, the anti-TREM-1 antibody is administered in a dose based on body weight.

[00242] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each administered antibody falls within the indicated ranges. Antibody is usually administered on multiple occasions. Intervals between single dosages may be, for example, weekly, monthly, every three months or annually. Intervals may also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1 to 1000 μg/ml and in some methods about 25 to 300 μg/ml.

[00243] An antibody can be administered as an extended-release formulation, in which case less frequent administration is necessary. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes necessary until disease progression is slowed or terminated, and until the patient shows complete or partial improvement in disease symptoms. After that, the patient can be administered a prophylactic regimen.

[00244] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient that is sufficient to achieve the desired therapeutic response for a particular patient, composition, and mode of administration , without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the particular compositions described herein used, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular compositions used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in medical techniques.

[00245] A composition described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending on the results desired. Routes of administration for the anti-TREM-1 antibodies described herein may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, generally by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal.

[00246] Alternatively, an antibody described herein can potentially be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[00247] Active compounds can be prepared with vehicles that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated release systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to one skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[00248] Therapeutic compositions can be administered with medical devices known in the art. For example, in a particular embodiment, a therapeutic composition described herein can be administered with a needleless hypodermic injection device, such as devices described in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants or modules for use with anti-TREM-1 antibodies described herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for delivering medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medications through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable-flow implantable infusion apparatus for continuous drug release; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chambered compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to one skilled in the art.

[00249] In some embodiments, the anti-TREM-1 antibodies described herein can be formulated to ensure appropriate distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if desired, for example, for brain cancers), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may comprise one or more moieties that are selectively transported to specific cells or organs, thereby improving targeted drug release (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawaet al., (1988) Biochem. Biophys. Res. Commun.153: 1038); antibodies (P.G. Bloemanet al. (1995) FEBS Lett. 357: 140; M. Owaiset al. (1995) Antimicrob. AgentsChemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pl20 (Schreieret al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

VIII. Uses and Methods

[00250] The anti-TREM-1 antibodies of the present invention and compositions comprising such antibodies (e.g., pharmaceutical composition, formulations, polynucleotides, vectors, and cells) can be used for the treatment of an inflammatory disease (e.g., inhibiting TREM-1 activity).

[00251] Accordingly, in one aspect, the present invention provides methods for treating an inflammatory disease in an individual in need thereof, comprising administering a therapeutically effective dose of the anti-TREM-1 antibody to the individual. Examples of inflammatory diseases that can be treated with the present anti-TREM-1 antibodies 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 nephritis, type I diabetes, Grave's disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, lung disease chronic obstructive disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft versus host disease, Sjogrens Syndrome, autoimmune nephritis, Goodpasture Syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma and others autoimmune diseases that are a result of acute or chronic inflammation.

[00252] In one embodiment, anti-TREM-1 antibodies are suitable for use in treating 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 mainly causes abdominal pain, diarrhea (which may be bloody), vomiting or weight loss, but it can also cause complications outside the gastrointestinal tract, such as skin rashes, arthritis, eye inflammation, fatigue and poor concentration. Patients with IBD can be divided into two main classes, those with ulcerative colitis (UC) and those with Crohn's disease (CD). CD usually involves the ileum and colon, it can affect any region of the intestine, but is often discontinuous (focused areas of disease spread throughout the intestine). UC always involves the rectum (colonic) and is more continuous. In CD, inflammation is transmural, resulting in abscesses, fistulas, and strictures, whereas in UC, inflammation is typically confined to the mucosa. There is no known pharmaceutical or surgical cure for Crohn's disease, while some UC patients can be cured by surgical removal of the colon. Treatment options are restricted to controlling symptoms, maintaining remission, and preventing relapse. Effectiveness in inflammatory bowel disease in the clinic can be measured as a reduction in the Crohn's Disease Activity Index (CDAI) score for CD which is a scoring scale based on laboratory tests and a quality of life questionnaire. In animal models, efficacy is primarily measured by increase in weight and also a disease activity index (DAI), which is a combination of stool consistency and weight, and blood in the stool.

[00253] In one embodiment, the anti-TREM-1 antibodies of the present invention are suitable for use in treating individuals with rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease that affects almost if not the entire body and is one of the most common forms of arthritis. It is characterized by inflammation of the joint, which causes pain, stiffness, heat, redness and swelling. This inflammation is a consequence of inflammatory cells invading the joints, and these inflammatory cells release enzymes that can digest bone and cartilage. As a result, this inflammation can lead to severe damage to bones and cartilage and joint deterioration and intense pain, among other physiological effects. The involved joint may lose its shape and alignment, resulting in pain and loss of movement. There are several animal models for rheumatoid arthritis known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop an inflammatory arthritis that resembles human rheumatoid arthritis. Since CIA shares similar immunological and pathological features with RA, this makes it a suitable model for screening potential human anti-inflammatory compounds. Efficacy in this model is measured by decreased joint swelling. Efficacy in RA in the clinic is measured by the ability to reduce symptoms in patients, which is measured as a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein levels, and levels of serum factors such as anti-citrullinated protein antibodies. .

[00254] In one embodiment, anti-TREM-1 antibodies as described herein are suitable for use in treating individuals with psoriasis. Psoriasis is a T cell-mediated inflammatory disorder of the skin that can cause considerable discomfort. It is a disease for which there is currently no cure and which affects people of all ages. Although individuals with mild psoriasis can often control their disease with topical agents, more than a million patients worldwide require ultraviolet light treatments or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of ultraviolet radiation and the toxicities of many therapies limit their long-term use. Furthermore, patients often have psoriasis recurrence, and in some cases rebound, shortly after discontinuing immunosuppressive therapy. A recently developed model of psoriasis based on the transfer of CD4+ T cells mimics many aspects of human psoriasis and therefore can be used to identify suitable compounds for use in the treatment of psoriasis (Davenport et al., Internat. Immunopharmacol 2: 653- 672, 2002). Effectiveness in this model is measured by reduction in skin pathology using a scoring system. Similarly, effectiveness in patients is measured by a reduction in skin pathology.

[00255] In one embodiment, anti-TREM-1 antibodies are suitable for use in treating individuals with psoriatic arthritis. Psoriatic arthritis (PA) is a type of inflammatory arthritis that occurs in a subset of patients with psoriasis. In these patients, the skin pathology/symptoms are accompanied by joint swelling similar to that seen in rheumatoid arthritis. It presents with uneven, enlarged and red areas of inflamed skin with scaling. Psoriasis often affects the tips of the elbows and knees, the scalp, the belly button, and around the genital areas or anus. Approximately 10% of patients who have psoriasis also develop associated inflammation of their joints.

[00256] Pursuant to the present invention, prophylactic, palliative, symptomatic and/or curative treatments may represent separate aspects of the disclosure. An antibody of the invention may be administered parenterally, such as intravenously, such as intramuscularly, such as subcutaneously. Alternatively, an antibody of the invention may be administered by a non-parenteral route, such as perorally or topically. An antibody of the invention can be administered prophylactically. An antibody of the invention can be administered therapeutically (on demand).

[00257] The following examples are offered by way of illustration and not by way of limitation. The contents of all references cited throughout this application are expressly incorporated herein by reference.

EXAMPLES Example 1: Analysis of Interaction Kinetics for the 318 Antibody Variants for Both Human TREM-1 and Cynomolgus by Surface Plasma Resonance

[00258] The binding kinetics of mAb 0318 variants towards human TREM-l-Fc (hTREM-1) and cynomolgus TREM-l-Fc (cTREM-1) were determined. Binding studies were performed on a ProteOn Analyzer (BioRad) that measures molecular interactions in real time via 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) reported by ProteOn is directly correlated to the mass on the individual sensor chip surfaces in the six parallel flow cells. Anti-human Fc monoclonal antibody or anti-murine Fc polyclonal antibody from Biacore human or mouse Fc capture kits were immobilized in the horizontal direction on flow cells of a GLM sensor chip according to the manufacturer's instructions. The level of capture antibody immobilization was approximately 2600 to 6000 RU in each experiment. Capture of purified mouse monoclonal or recombinantly expressed anti-hTREM-1 antibodies was conducted by diluting the antibodies to 5 to 10 nM in running buffer (10 mM Hepes, 0.15 M NaCl, 5 mM EDTA, 0.05 % surfactant P20, pH 7.4) followed by injection in the vertical direction at 30 μl/min for 60 sec, creating intermediate reference points adjacent to all flow cells with only anti-Fc antibody immobilized. This typically resulted in final test antibody capture levels of approximately 100 to 300 RU and analyte Rmax values of 30 to 90 RU. Binding of hTREM-1 or cTREM-1 proteins was conducted by injecting analyte (antigen) into all flow cells in the horizontal direction to allow comparative analyzes of binding to different captured anti-TREM-1 antibodies with respect to binding to the reference midpoint. . hTREM-1 or cTREM-1 proteins were serially diluted 1:3 to 1.2 to 100 nM or in running buffer, injected at 100 μl/min for 250 s, and allowed to dissociate for 600 s. The GLM surface was regenerated after each analyte injection cycle by two 18-s injections of 10 mM Glycine, pH 1.7, and 50 mM NaOH at 100 μl/min. This regeneration step removed the anti-TREM-1 antibody and any bound TREM-1 protein from the immobilized capture antibody surface and allowed subsequent binding of the next interacting sample pair. The regeneration procedure did not remove the directly immobilized anti-Fc capture antibody from the chip surface.

[00259] Binding affinity between antibodies and antigen was quantified by determining the equilibrium dissociation constant (KD) determined by measuring the kinetics of complex formation and dissociation. The rate constants corresponding to the association and dissociation of a monovalent complex, such as ka (association rate) and kd (dissociation rate) were recovered by fitting the data to the 1:1 Langmuir model using ProteOn evaluation software for analysis. of data. KD is related to ka and kd through the equation KD = kd / ka.

[00260] Binding curves were processed by double reference (subtraction of reference surface signals as well as blank buffer injections over captured anti-TREM-1 antibodies) prior to data analysis. This allowed correction for instrument noise, mass shifting and movement during sample injections.

[00261] As shown in FIG. 1A and 1B, the mAb 0318 variants (i.e., 318-IgG1.1f, 318-IgG1.3f, 318-IgG4-Aba, and 318-IgG1-Aba) were all found to have similar affinity toward TREM-1. -Human Fc as mAb 0318-IgG4. The 0318-IgG1.3f variant also binds to cynomolgus TREM-1, although with slightly reduced affinity compared to human TREM-1. See FIG. 1A.

Example 2: Internalization Analysis of mAb 0318-IgG1.3f after Binding to TREM-1 Receptor

[00262] The mAb 0318-IgG1.3f variant antibody was tested for its internalization in primary human monocytes using confocal laser scanning microscopy (data not shown) and Amnis ImageStream® Imaging Flow Cytometry Analysis. As shown in FIG. 2A, at 0 hours, TREM-1 is expressed mainly on the surface of monocytes. However, by 24 hours after antibody binding, a significant percentage (~36%) of the TREM-1 receptor (as evidenced by 0318-IgG1.3f staining) was internalized, suggesting that after binding of mAb 0318-IgG1 antibody. 3f, the entire antibody-receptor complex is internalized within the cell.

[00263] Next, to determine the fate of the TREM-1 receptor once it was internalized, the TREM26 antibody (cat. No. 314902, Biolegend; See US20150274825 in paragraph [0005]) was used for comparison. The TREM26 antibody does not compete with the 0318 antibody variants described in the present invention. As shown in FIG. 2B, compared to 0 hours, there was a significant reduction in TREM26+ expression (~51%) at the 20 hour time point. There was also a significant (4-fold) reduction in TREM26+ MFI (mean fluorescence index) at the 20 hour time point (after treatment with mAb 0318-IgG1.3f), suggesting that the TREM-1 receptor was degraded upon internalization. This loss of TREM-1 receptor expression following antibody exposure is reversible, however, once the antibody is removed (data not shown).

Example 3: Analysis of mAb 0318 Variants to Efficiently Block TREM-1 Activation Using the BWZ/hTREM-1 Reporter Cell Assay

[00264] The ability of the anti-TREM-1 mAb 0318 variants to inhibit human TREM-1 signaling was determined using the BWZ.36/hTREM-1DAP12:NFAT-LacZ cell line assay (herein also referred to as " BWZ/hTREM-1 reporter cell") as described in, for example, U.S. Patent No. 9,550,830 B2 and International Publication No. WO 2016/009086 A1. Briefly, approximately 40,000 hTREM-1/BWZ.36 cells/well were plated in a clear bottom, black 96-well plate in the presence of 75 ng/ml PGLYRP1 (SEQ ID NO: 8) with 2.5 μg/ml of PGN-ECndi (Cat. no. tlrl-kipgn, Invivogen San Diego, Calif., USA) to provide a submaximal positive signal, or alternatively in the presence of a submaximal level (1 μg/ml) of anti-TREM-1 monoclonal antibody absorbed onto plastic (Cat. no. MAB1278, R&D Systems, Minneapolis, Minn., USA) to provide a positive signal.

[00265] mAb 0318 variants (e.g., 0318-IgG1.3f; 0318-IgG1.1f; 0318-IgG1-Aba; and 0318-IgG4-Aba) were titrated into the assay starting at 10 μg/ml, with 5 2-fold serial dilutions. The assay was incubated overnight at 37°C, subsequently developed with Beta Glo (Cat. no. E4740, Promega Madison, Wis., USA) as per the Beta Glo protocol, and luminescence was recorded. Data were plotted showing luminescent units relative to Beta Glo vs test antibody concentration. non-neutralizing negative control mIgG1 (Cat. no. MAB002, R&D Systems Minneapolis, Minn., USA) and neutralizing positive control polyclonal goat anti hPGLYRP1 antibody (Cat. no. AF2590, R &D Systems, Minneapolis, Minn., USA ) were run on each assay plate. The MAB1278 antibody (cat. no. MAB1278, R&D Systems; see US20150274825 in paragraph [0005]), a known agonist of TREM-1 signaling, was also used as a positive control (see inset box).

[00266] As shown in FIG. 3, all mAb 0318 variants were potent in inhibiting human TREM-1 signaling as previously observed with mAb 0318 IgG4 antibody in International Publication No. WO 2016/009086 A1.

Example 4: In vitro analysis of the potency of mAb 0318 antibody variants in inhibiting TREM-1-mediated production of inflammatory cytokines by different primary human cells

[00267] To also evaluate the antagonistic properties of the anti-TREM-1 mAb 0318 variants, their potency to block the release of various inflammatory cytokines (e.g., TNF-α, IL-6, or IL-8) from primary cells activated human cells were evaluated. Primary monocytes, neurotrophils, and peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood and stimulated with plate-bound PGRP1 and soluble peptidoglycan (PGN-ECndss; a form of peptidoglycan without TLR2 activity).

[00268] As shown in FIG. 4, all variants of mAb 0318 (i.e., IgG1.3f, IgG1.1f, IgG1-Aba, and IgG4-Aba) were potent (IC50 values in the range of ~10 to 20 pM) in inhibiting MAb-mediated release. TREM -1 of TNF-α from PBMC and monocytes. The potency of these mAb 0318 variants was similar to that observed with the mAb 0318-IgG4 antibody. The mAb 0318-IgG1.3f antibody was also potent in inhibiting IL-6 production (IC50 value of ~32 pM). For neutrophils, the mAb 0318-IgG1.3f variant appeared to be better compared to the mAb 0318-IgG4 antibody in blocking TREM-1-mediated IL-8 production from neutrophils. (See FIG. 4).

[00269] As a further demonstration of the antagonistic properties of the anti-TREM-1 mAb 0318 antibody variants, a monocyte-neutrophil co-culture assay was also used. Neutrophil-associated PGRP1, when co-cultured with monocytes, can bind TREM-1 receptors, resulting in the production of monocyte-derived TNF-α. As shown in FIG. 4, all mAb 0318 antibody variants effectively blocked this endogenous activation (IC50 values ranging from 19 to 44 pM). Similar results were observed with RBC-sedimented whole blood. (See FIG. 4).

Example 5: In vitro analysis of the potency of mAb 0318-IgG1.3f to block IL-8 production from stimulated whole blood

[00270] One of the main challenges for developing a whole blood (PD) pharmacodynamics assay to measure the antagonistic properties of anti-TREM-1 antibody variants is the high base resulting from PGN stimulation. To help address this question, whole blood was stimulated with pre-complexed PGRP1 + PGN in the presence of NOD2 inhibitor (to block background cytokines produced by NOD2 stimulation by PGN). IL-8 levels were measured using the standard pharmacodynamic ligand binding assay (HTRF®) (FIG. 5A) or an intracellular cytokine staining (ICS) assay (FIG. 5B).

[00271] As shown in FIGs. 5A and 5B, the 0318-IgG1.3f antibody effectively blocked TREM-1-mediated IL-8 production with percentage inhibition ranging from about 60 to 90% (see FIG. 5A). The IC50 values observed (mean value of 12 pM with HTRF and 19.6 pM with ICS) were similar to those observed with other functional assays (e.g., see Example 4).

Example 6: In vitro analysis of the potency of mAb 0318-IgG1.3f to block mRNA expression of different inflammatory mediators in stimulated whole blood

[00272] To further demonstrate the antagonistic properties of mAb 0318-IgG1.3f, the expression levels of selected inflammatory mediators (i.e., chitinase 3-like protein 1 ("CHI3L1"), IL1β, and IL6) were measured by PCR in real-time (qPCR). Briefly, human whole blood collected in EDTA tubes from three normal healthy volunteers (donor #126, 290, and 322) was stimulated with pre-complexed hPGRPI (50 μg/ml) and PGN-ECndss (10 μg/ml) (Invivogentlrl - ksspgn) overnight in the presence of varying concentrations of mAb 0318-IgG1.3f (0 to 1 nM). After stimulation, plasma was removed and frozen for cytokine measurements. mRNA was isolated from samples using the MagMax-96 Blood Isolation Kit (ThermoFisherAM1837) according to the manufacturer's protocol. Isolated mRNA was then converted to cDNA using SuperScriptVILO Master Mix (Thermo Fisher 11755250). Then, qPCR was performed using the following probes: HPRT1 (Hs99999909_m1) (Thermo Fisher 4351370), CHI3L1 (Hs01072228_m1) (Thermo Fisher 4331182), IL1β (Hs00174097_m1) (Thermo Fisher 4331182), and IL6 (Hs00985 639_m1) (Thermo Fisher 4331182 ) and TaqMan Fast Universal Master Mix (2x) (Thermo Fisher 4366072). Gene expression values were normalized to HPRT1 and ΔΔCT values were generated. The results were then plotted and IC50 values were determined.

[00273] As shown in FIGs. 6A-6C and in accordance with the previous examples, mAb 0318-IgG1.3f was able to inhibit TREM-1-mediated expression of different inflammatory mediators in human whole blood. Inhibition appears to be dose dependent. IC50 values are shown in Table 1 below.

[00274] Collectively, the above results demonstrate that the mAb 0318 antibody variants described in the present invention are antagonistic and can effectively block TREM-1-mediated inflammatory cytokine production from various human cells. Table 1.

Example 7: Viscosity of mAb 0318 Variants

[00275] The sample was buffer exchanged and dialyzed in the formulation of choice (20 mM histidine, 150 mM sucrose, 25 mM arginine, 50 mM sodium chloride, pH 6.0), followed by concentration using filters. Amicon Ultra centrifugal molecular weight cutoff. The aggregation state of the samples was measured by size exclusion chromatography to control for potential changes in monomericity upon concentration, which were not observed. Concentration-dependent viscosity was determined using a RheoSense m-VROC solution viscometer using a 3-point upward shear sweep for each measured concentration. Concentration was determined by measuring absorbance at 280 nm by nanoDrop using dilution and extrapolation series.

[00276] As shown in FIG. 7, both variants 318-IgG1.1f and 318-IgG1.3f had a similar viscosity profile at a concentration of about 130 mg/mL, the 0318-IgG1.3f variant showed a viscosity value of around 9 cP. Such a viscosity profile closely mirrored that previously observed with mAb 0318-IgG4 (see International publication No. WO 2016/009086).

Example 8: Immunogenicity potential of mAb 0318 variants

[00277] As shown in FIG. 8 and Table 2 (below), the 318-IgG1.1f and 318-IgG1.3f variants had low to medium risk of immunogenicity in human patients. Only about 22 to 30% of the donor had immunogenic responses (as measured by in vitro CD4+ T cell proliferation) to these antibodies. Immunogenicity for the 0318-IgG1-Aba and 0318-IgG4-Aba variants was 10% and 42.5%, respectively. See Table 2 (below). In contrast, mAb 0318-IgG4 was much more immunogenic (55%) in human patients. KLH (keyhole limpet hemokinin) and VL6 (IL-21RmAb), which were used as positive controls, were highly immunogenic in human patients (100% and 40%, respectively). Table 2.

Example 9: Binding analysis of mAb 0318 variants to FcRn using surface plasmon resonance (SPR)

[00278] To determine whether the different variants of mAb 0318 were capable of binding to FcRn, FcRn receptors (mouse, human, and cyno) were immobilized on a BIAcoreCM5 biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) via amine coupling at a level of 400 Response Units (RU). The assay was performed at room temperature with PBS, 0.05% Tween-20™ pH 6.0 (GE Healthcare Bioscience) as running and dilution buffer. The different variants of mAb 0318 (200 nM) were injected at a flow rate of 50 μL/min at room temperature at pH 6.0. The association time was 180 seconds, the dissociation phase (also at pH 6.0) took 360 seconds. Regeneration of the chip surface back to baseline was achieved by a short injection of 50 mM Tris, pH 8.0, and 150 mM NaCl. Evaluation of SPR data was performed by comparing the height of the biological response signal at 180 seconds post-injection and 300 seconds post-injection. The corresponding parameters are the maximum RU level (180 seconds after the injection) and the late stability (300 seconds after the end of the injection).

[00279] As shown in FIGs. 9A and 9B, the mAb 0318 antibody variants (IgG1-Aba mod, IgG4-Aba mod, IgG1.1f, and IgG1.3f) were all capable of binding to human, mouse, and cyno FcRn in a pH-dependent manner. This was also true for the mAb 0318 (IgG4) antibody.

Example 10: Binding analysis of mAb 0318 variants to one or more FCYRS

[00280] As previously discussed, in vivo administration of antibodies to certain cell surface immune receptors has the potential to induce the release of cytokines, which may result in the induction of a common and toxic clinical complication known as cytokine release syndrome ( RSC). Due to concern that FCYR engagement could lead to potential TREM-1 agonistic activity through cross-linking, mAb 0318-IgG4 was redesigned into one of the variant formats described in the present invention (i.e., IgG1.1f, IgG1.3f , IgG1-Aba, or IgG4-Aba). Then, the ability of the 0318 antibody variants to bind to different FcyRs was evaluated.

[00281] As shown in FIGs. 10A and 10B, variants 318-IgG1.1f and 318-IgG1.3f had minimal binding to all FcyRs (i.e., FcyRI (CD64), FcyRIIA (variants CD32a-H131 and CD32a-R131), FcyRIIB (CD32b), FcyRIIIA (CD16a-V158 variant), and FcyRIIIB (CD16b-NA2 variant)). The 318-IgG1-Aba and 318-IgG4-Aba variants did not bind to FcyRIIA, FcyRIIB, FcyRIIIA, and FcyRIIIB, but did bind to FcyRI. In contrast, mAb 0318 (IgG4) showed significant binding to all FcyRs.

Example 11: Analysis of the induction of inflammatory cytokines by mAb 0318 variants

[00282] To determine whether in vivo treatment with mAb 0318 variants poses a risk of cytokine release syndrome, whole blood was collected from 8 human donors and monocytes were isolated. Then, monocytes were differentiated into immature dendritic cells by plating the monocytes (4x10 cells/well) and culturing them in a differentiation medium containing IL-4 and GM-CSF (100 ng/mL). By days 2 to 3 post-plating, 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 flow cytometer. Next, immature dendritic cells were seeded in a flat-bottom plate (0.8x105 cells/well) and mAb 0318 variants were added to the respective wells (with or without CHO-CD32a). 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 were harvested from the wells and the amount of TNF-α, IL-6, and IL-12 produced was assessed using an ELISA assay.

[00283] Results from representative donors are shown in FIGs. 11A to 11I. Addition of the different mAb 0318 variants (0318-IgG1.1f, 0318-IgG1.3f, and 0318-IgG1.1 Aba) resulted in minimal IL-6 (FIGS. 11A, 11B, and 11C), TNF-α ( FIGS. 11D, 11E, and 11F), and IL-12 production (FIGS. 11G, 11H, and 11I) by immature dendritic cells. These results, together with those of Example 9, demonstrate that the anti-TREM-1 antibodies described herein have a low risk of inducing a cytokine release syndrome when administered to patients in vivo.

Example 12: Additional characteristics of mAb 0318 variants

[00284] The biophysical characteristics of the 0318-IgG1.3f variant are provided in Table 3 (below). Table 3. Biophysical characteristics of the anti-TREM-1 antibody 0318-IgG1.3f

[00285] The biophysical properties of the mAb 0318-IgG1.3f variant are favorable for clinical development. Antibody identity was confirmed by mass spectrometry analysis (Intact Mass Analysis & Peptide Mapping). The antibody was >96% monomeric as tested by size exclusion chromatography. A single N-glycosylation site was confirmed at N301 in a heavy chain glycan profile with a corresponding glycan profile of CHO-expressed monoclonal antibodies (G0F, G1F, and G2F). The thermal stability (Tm1 = 66.2°C; Tm2 = 78.4°C; Tm3 = 83.2°C) and thermal reversibility (24% at 77°C) of mAb 0318-IgG1.3f was within the range of a typical human IgG1.3 monoclonal antibody.

[00286] The stability characteristics of the mAb 0318- IgG1.3f variant are provided in Table 4 (below). Table 4. Stability of the anti-TREM-1 antibody 0318-IgG1.3f

[00287] No physical stability problems were observed during freeze-thaw stress (3 cycles) at 150 mg/mL in the described formulation (20 mM histidine, 150 mM sucrose, 50 mM sodium chloride, 25 mM arginine mM, pH 6.0). Forced degradation studies on the studied formulation at 150 mg/mL were set up at 4, 25, and 40 °C (up to 3 months). CDR chemical modifications remained low across all temperature conditions and did not affect activity as determined by SPR. Deamidation of VSNK was below expectations compared to other monoclonal antibodies in the IgG1.3f structure (increase of 3%/month at 40 °C storage), changes were time and temperature dependent. All other chemical modifications (oxidation, deamidation, isomerization) also remained low as monitored during stability studies. Of note, storage at 40 °C indicates a formation of HMW and LMW variants (demonstrating 1.2%/month and 2.4%/month, respectively). The observed changes depended on time and temperature, such that HMW increased by 0.25%/month, which LMW remained unchanged under 4 °C storage during the study period. Low LMW formation under 40 °C storage was characterized by 2D-LC/MS with high-resolution accurate mass measurements as the cumulative Fab-1 loss species, as well as the Fab arm, putatively in a conserved sequence in the region upper articulation.

Example 13: PK/TK /PD study for mAb variant 0318- IgG1.3f in Cynomolgus monkeys

[00288] A single dose pharmacokinetic (PK), toxicokinetic (TK) and pharmacodynamic (PD) study was conducted in Cynomolgus monkeys for the anti-TREM-1 antibody 0318-IgG1.3f. Some of the animals received 2 mg/kg of the 0318-IgG1.3f antibody intravenously (n=3). Other animals received one of the following doses of the 0318-IgG1.3f antibody subcutaneously: (i) 0 mg/kg (i.e. control) (n = 4), (ii) 0.1 mg/ kg (n = 4 ), (iii) 0.5 mg/kg (n=4), (iv) 2 mg/kg (n=3), or (v) 10 mg/kg (n=4). Following antibody administration, pharmacokinetics, anti-drug antibodies (ADA), TREM-1 receptor (RO) occupancy, and ex vivo pharmacodynamic responses were examined at predetermined time points.

Pharmacokinetics (PK)

[00289] To evaluate pharmacokinetics, serum concentrations of the 0318-IgG1.3f antibody were evaluated in the animals with a ligand binding assay using a biotinylated recombinant TREM-1 protein as the capture reagent and a polyclonal goat anti- Commercial TREM-1 as detection reagent.

[00290] As shown in FIG. 12, the pharmacokinetics of the 0318-IgG1.3f antibody were determined to be nonlinear between 0.1 and 10 mg/kg, predominantly due to target-mediated clearance (i.e., internalization and degradation of the antibody upon binding to the TREM-1 receptor; see Example 2).

[00291] Pharmacokinetic parameters based on non-compartmental analysis are presented in Tables 5 (intravenous administration) and 6 (subcutaneous administration). Briefly, a 100-fold increase in dose resulted in an 830-fold increase in serum exposures (AUCs). See Table 6. Clearance following the 2 mg/kg IV dose was 0.1 ± 0.02 mL/h/kg and similar to other IgG1-based mAbs in monkeys. See Table 4. Vss at 36 ± 5 mL/kg was similar to plasma volume, indicating limited extravascular distribution. The half-life of the 0318-IgG1.3f antibody increased from 2 days at the 0.1 mg/kg dose to 10 days at 2 and 10 mg/kg (single subcutaneous dose administration). See Table 6. Bioavailability of antibody 0318-IgG1.3f after subcutaneous administration (2 mg/kg) was high at 84%. Table 5. PK Parameters of mAb 0318-IgG1.3f in Monkey * Number of monkeys examined that showed persistent ADA. Table 6. PK parameters of mAb 0318-IgG1.3f in monkeys *Number of monkeys examined that showed persistent ADA.

Antidrug Antibodies (ADA)

[00292] Although serum ADA was detected in the majority of monkeys (16 of 18) after the single dose (regardless of the route of administration), exposure of the 0318-IgG1.3f antibody and occupancy of the TREM-1 receptor (RO ) was not compromised in the majority of ADA-positive animals. See Tables 4 and 5. The accelerated decline in terminal exposure of the 0318-IgG1.3f antibody that was accompanied with ADA formation was observed only in two monkeys (one in the 0.1 mg/kg dose group on day 7 and one in 0.5 mg/kg dose group on day 21). Since ADA affected exposure in these two monkeys in the terminal phase, the corresponding data points were eliminated for PK analysis.

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

[00293] Next, the occupancy of TREM-1 receptors expressed on peripheral blood monocytes and granulocytes was evaluated. As shown in FIGS. 15A and 15B, for all doses tested, the percentage of occupied TREM-1 receptors (i.e., bound to the anti-TREM-1 antibody) was similar between monocytes and granulocytes. Furthermore, the duration of receptor occupancy appeared to be dependent on the dose of the 0318-IgG1.3f antibody administered to the animals. At 0.5 mg/kg, there was >85% RO observed for up to 2 weeks after antibody administration. In contrast, at 2 and 10 mg/kg, >85% of TREM-1 receptors remained occupied for at least 1 month after administration.

[00294] After measuring the 0318-IgG1.3f antibody, total levels of the TREM-1 receptor were reduced in monocytes and granulocytes. See FIGS. 14A and 14B. As discussed previously, this reduction is presumably due to increased receptor turnover following antibody binding. The decrease in surface TREM-1 receptor expression was reversible and the duration of surface receptor loss was correlated with the duration of receptor occupancy at least at the doses examined.

[00295] Levels of soluble TREM-1 (sTREM-1) appeared to increase 10 to 50-fold after a single dose of the mAb 0318 mAb-IgG1.3f variant in all dose groups tested. (Data not shown). It is unclear whether the increase in sTREM-1 levels was related to the administration of the anti-TREM-1 antibody or the general handling of the animals during the course of the study. In any case, since drug concentrations greatly exceeded (>1000-fold) sTREM-1 levels, the soluble target should not effect binding of the mAb 0318 mAb-IgG1.3f variant to the TREM-1 cell surface.

Example 14: PK/TK/PD study for the mAb 0318-IgG1.3f variant in Cynomolgus monkeys, described by a 2-compartment PK model with TMDD in the central compartment

[00296] Monkey PK, RO, total receptor levels, and PD data were described using a 2-compartment PK model with saturable target-mediated drug disposition (TMDD) in the central compartment to accommodate the observed nonlinear PK ( see Example 12 and FIG. 13). A direct effect inhibitory model was used to describe the PD response. This model was able to effectively capture the PK/RO/PD time course observed in monkeys. The observed (open circle) and model-predicted (solid line) endpoints are provided in FIGs. 16A (subcutaneous dosage of 0.1 mg/kg) and 16B (subcutaneous dosage of 10 mg/kg). Table 7 provides the estimated PK/PD parameters. When serum exposures of variant mAb 0318- IgG1.3f and TREM-1 RO data were pooled from all monkeys, a concentration-dependent increase in RO was observed. See FIGS. 16A and 16B. An Emax model used to describe the concentration-RO relationship, estimated in vivo RO EC50 to be 1.6 ± 0.2 nM. Table 7. Parameter estimates from the PK/PD model used to describe data from Monkey PK/TK/PD and projected human parameter estimates Table 8.

[00297] This PCT application claims the priority benefit of North American Provisional Application No. 62/651,605, filed on April 2, 2018, which is incorporated herein by reference in its entirety.

Claims (25)

1. Heavy chain characterized by comprising SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 85. 2. Heavy chain according to claim 1, characterized in that it comprises SEQ ID NO: 50. 3. Heavy chain according to claim 1, characterized in that it comprises SEQ ID NO: 85. 4. Isolated antibody that specifically binds to TREM-1, characterized by comprising a heavy chain and a light chain, where the heavy chain comprises SEQ ID NO: 85 and where the light chain comprises SEQ ID NO: 54. 5. Isolated antibody that specifically binds to TREM-1, characterized by comprising a heavy chain and a light chain, where the heavy chain comprises SEQ ID NO: 50 and where the light chain comprises SEQ ID NO: 54. 6. Isolated antibody that specifically binds to TREM-1, characterized by comprising a heavy chain and a light chain, where the heavy chain comprises SEQ ID NO: 51 and where the light chain comprises SEQ ID NO: 54. 7. Isolated antibody that specifically binds to TREM-1, characterized by comprising a heavy chain and a light chain, where the heavy chain comprises SEQ ID NO: 52 and where the light chain comprises SEQ ID NO: 54. 8. Isolated antibody that specifically binds to TREM-1, characterized by comprising CDR1, CDR2, CDR3 of a heavy chain; CDR1, CDR2, CDR3 of a light chain; and an IgG1 heavy chain constant region, wherein CDR1, CDR2 and CDR3 of the heavy chain comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively ; wherein the CDR1, CDR2 and CDR3 of the light chain comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively; wherein the heavy chain constant region of IgG1 comprises three or more amino acid substitutions comprising L234A, L235E and G237A, by EU numbering and in which the heavy chain lacks a C-terminal lysine. 9. Antibody according to claim 8, characterized in that it comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 14 and the VL comprises the SEQ ID NO: 15. 10. Antibody according to claim 9, characterized in that the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 85 and wherein the light chain comprises SEQ ID NO :54. 11. Isolated antibody that specifically binds to TREM-1, characterized by comprising CDR1, CDR2, CDR3 of a heavy chain; CDR1, CDR2, CDR3 of a light chain; and an IgG1 heavy chain constant region, wherein the CDR1, CDR2 and CDR3 of the heavy chain comprise TYAMH (SEQ ID NO: 24), RIRTKSSNYATYYAASVKG (SEQ ID NO: 25) and DMGIRRQFAY (SEQ ID NO: 26), respectively ; wherein the CDR1, CDR2 and CDR3 of the light chain comprise RASQSVDTFDYSFLH (SEQ ID NO: 27), RASNLES (SEQ ID NO: 28) and QQSNQDPYT (SEQ ID NO: 29), respectively; wherein the IgG1 heavy chain constant region comprises three or more amino acid substitutions comprising L234A, L235E and G237A, by EU numbering, or wherein the IgG1 heavy chain constant region comprises three or more amino acid substitutions and one or more amino acid deletions, where the substitutions comprise L234A, L235E and G237A, by EU numbering. 12. Antibody according to claim 11, characterized in that it comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 14 and the VL comprises the SEQ ID NO: 15. 13. Antibody according to claim 12, characterized in that it comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 85; and wherein the light chain comprises SEQ ID NO: 54. 14. Antibody according to claim 12, characterized by the fact that the constant region of the IgG1 heavy chain has three substitutions, where the substitutions are L234A, L235E and G237A, by EU numbering. 15. Antibody according to claim 12, characterized by the fact that the IgG1 heavy chain constant region has three substitutions, where the substitutions are L234A, L235E and G237A, by EU numbering, and a C-lysine residue terminal of the IgG1 heavy chain constant region is present. 16. Antibody according to claim 12, characterized by the fact that the IgG1 heavy chain constant region has three substitutions, in which the substitutions are L234A, L235E and G237A, by EU numbering, and in which the heavy chain does not has a C-terminal lysine residue. 17. Antibody according to claim 11, characterized in that it comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 85 and wherein the light chain comprises SEQ ID NO: 54. 18. Antibody according to claim 11, characterized in that it comprises a heavy chain, wherein the heavy chain does not have a C-terminal lysine residue. 19. Antibody according to claim 11, characterized by the fact that the constant region of the IgG1 heavy chain has three substitutions, where the substitutions are L234A, L235E and G237A, by EU numbering. 20. Antibody according to claim 11, characterized by the fact that the IgG1 heavy chain constant region has three substitutions, where the substitutions are L234A, L235E and G237A, by EU numbering, and a C-lysine residue terminal of the IgG1 heavy chain constant region is present. 21. Antibody according to claim 11, characterized by the fact that the IgG1 heavy chain constant region has three substitutions, in which the substitutions are L234A, L235E and G237A, by EU numbering, and in which the heavy chain does not has a C-terminal lysine residue. 22. Bispecific molecule characterized by the fact that it comprises the antibody as defined in claim 11 linked to a molecule having a second binding specificity. 23. Immunoconjugate characterized by the fact that it comprises an antibody as defined in claim 11, linked to an agent. 24. Composition characterized by the fact that it comprises antibody as defined in claim 11, and a vehicle. 25. Kit characterized by the fact that it comprises the antibody as defined in claim 11, and an instruction for use.