Antibodies against human TREM-1 and their use

Abstract

Provided herein are antibodies or antigen-binding portions thereof that specifically bind to TREM-1 and inhibit TREM-1 signaling, as well as uses of the antibodies or antigen-binding portions thereof in therapeutic applications, such as for the treatment of autoimmune diseases.

Description

Background Art

[0001] Cross - reference to related applications This PCT application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 874,316, filed on July 15, 2019, the content of which is incorporated herein by reference.

[0002] Reference to a sequence listing electronically submitted via EFS - WEB The content of the sequence listing of the ASCII text file (name: 3338_0960000_SeqListing_ST25.txt; size: 451,353 bytes; creation date: July 14, 2019), sent together with this application, is incorporated herein by reference in its entirety.

[0003] TREM - 1 is an activating receptor expressed on monocytes, macrophages and neutrophils. These cells play a central role in chronic inflammatory diseases by releasing cytokines and other mediators that induce inflammation. The expression of TREM - 1 mRNA and protein is up - regulated in patients with rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and TREM - 1 - positive cells accumulate at the site of inflammation and correlate with disease severity. See Bouchon et al., Nature 410:1103 - 1107 (2001); Schenk et al., Clin Invest 117:3097 - 3106 (2007); and Kuai et al., Rheumatology 48:1352 - 1358 (2009). Peptidoglycan - recognition - protein 1 (PGLYRP1), mainly expressed by activated neutrophils, is a ligand of TREM - 1 and mediates TREM - 1 signaling upon binding.

[0004] In vitro, TREM-1 engagement induces the secretion of pro-inflammatory cytokines, including TNF, IL-8, and monocyte chemotactic protein-1. In addition, TREM-1 signaling synergistically interacts with multiple Toll-like receptors (TLRs), further boosting pro-inflammatory signals. This, in turn, upregulates TREM-1 expression, creating a dangerous cycle that amplifies inflammation. (See Bouchon et al., J Immunol 164:4991-4995 (2000)). There is growing evidence, for example, that TLRs contribute to the development and progression of chronic inflammatory diseases such as rheumatoid arthritis (RA) and inflamed heart disease (IBD).

[0005] Humanized anti-TREM-1 mAbs that inhibit the function of TREM-1 in both humans and cynomolgus monkeys have been disclosed separately. See WO2013 / 120553A1 and WO2016 / 009086A1. However, such antibodies have viscosity profiles that can interfere with the manufacturing process or have other problems (e.g., cytokine storms or ADCC) that may limit their therapeutic capacity. See Shire et al., J.Pharm.Sci.93:1390-1402(2004); and Warnkke et al., J Immunol.188:4405-11(2012). Therefore, there is a need for another anti-TREM-1 antibody that can specifically bind to TREM-1 and inhibit its function without the challenges of previous anti-TREM-1 antibodies. [Overview of the Initiative]

[0006] This specification provides isolated antibodies, such as monoclonal antibodies (e.g., human monoclonal antibodies), which specifically bind to triggering receptor expressed on myeloid cells-1 (TREM-1) and possess desirable functional properties. In some embodiments, the antibodies of this disclosure include a heavy chain variable region (VH) and a light chain variable region (VL), in which case the antibodies bind to TREM-1 with an epitope containing amino acids E27-L37 (EKYELKEGQTL, SEQ ID NO: 9), E88-M100 (EDYHDHGLLRVRM, SEQ ID NO: 10), and / or K120-R128 (KEPHMLFDR, SEQ ID NO: 11). In certain embodiments, the antibodies of this disclosure bind to TREM-1 with an epitope containing amino acids E27-L37 (EKYELKEGQTL, SEQ ID NO: 9). In other embodiments, the antibody of the Disclosure binds to TREM-1 via an epitope containing amino acids E88-M100 (EDYHDHGLLRVRM, SEQ ID NO: 10). In further embodiments, the antibody of the Disclosure binds to TREM-1 via an epitope containing amino acids K120-R128 (KEPHMLFDR, SEQ ID NO: 11).

[0007] In some embodiments, the disclosure provides an isolated antibody that specifically binds to TREM-1 and includes VH and VL, in which case the antibody binds to TREM-1 at epitopes other than D38-F48 of SEQ ID NO: 1.

[0008] In some embodiments, the disclosure provides an isolated antibody that specifically binds to TREM-1 and includes VH and VL, in which case the antibody binds to TREM-1 at an epitope different from that of mAb 0170.

[0009] In some embodiments, the disclosure further provides an isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-1 (TREM-1) and comprises VH and VL, wherein the antibody cross-competes with a reference antibody for binding to TREM-1, wherein the reference antibody comprises a heavy chain variable region (VH) including SEQ ID NOs. 13, 15, 23, 25, or 130, and / or a light chain variable region (VL) including SEQ ID NOs. 14, 16, 17, 24, 131, or 132.

[0010] In some embodiments, the antibodies disclosed herein include heavy chain CDR1, CDR2, and CDR3 in VH, and light chain CDR1, CDR2, and CDR3 in VL, in which case the heavy chain CDR3 includes EGYDILTGYEYYGMDV (SEQ ID NO: 28), GVLWFGELLPLLDY (SEQ ID NO: 34), MVRGNYFYFYGMDV (SEQ ID NO: 47), DGRHYYGSTSYFGMDV (SEQ ID NO: 52), and TYYDILTYHYHYGMDV (SEQ ID NO: 138).

[0011] In some embodiments, the heavy chain CDR1 of the antibody disclosed herein comprises X1, X2, X3, X4, and X5, where X1 is S or N, X2 is S, Y, or E, X3 is YG, or A, X4 is W, M, or I, and X5 is S, T, H, or N.

[0012] In some embodiments, the heavy chain CDR1 of the antibody disclosed herein includes NSEAIN (SEQ ID NO: 136).

[0013] In some embodiments, the heavy chain CDR2 of the antibody disclosed herein includes X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, and X17, where X1 is Y, V, or G; X2 is T or I; X3 is W, I, or none; X4 is H, Y, or P; X5 is Y, D, or I; X6 X7 is S, G, or F; X8 is I, Y, N, or T; X9 is S, T, or K; X10 is N or Y; X11 is Y or G; X12 is N or A; X13 is P, D, or Q; X14 is S or K; X15 is L, V, or F; X16 is K or Q; and X17 is S or G.

[0014] In some embodiments, the light chain CDR1 of the antibody disclosed herein comprises R, A, S, Q, X1, X2, X3, S, S, X4, L, and A, where X1 is S or G, X2 is V or I, X3 is S or none, and X4 is Y or A. In some embodiments, the light chain CDR2 of the antibody disclosed herein comprises X1, A, S, S, X2, X3, and X4, where X1 is G, D, or A, X2 is R or L, X3 is A, E, or Q, and X4 is T or S.

[0015] In some embodiments, the light chain CDR3 of the antibody disclosed herein comprises Q, Q, X1, X2, S, X3, P, X4, and T, where X1 is Y or F, X2 is G or N, X4 is S or Y, and X5 is L, Y, or none.

[0016] In some embodiments, the heavy chain CDR2 of the antibodies disclosed herein includes YTHYSGISNYNPSLKS (SEQ ID NO: 27), YIYDSGYTNYNPSLKS (SEQ ID NO: 33), GIIPIFGTTNGAQKFQG (SEQ ID NO: 46), VIWYDGSNKYYADSVKG (SEQ ID NO: 51), or GIIPIFDITNYAQKFQG (SEQ ID NO: 137).

[0017] In some embodiments, the heavy chain CDR1 of the antibodies disclosed herein includes SSYWS (SEQ ID NO: 26), NYYWT (SEQ ID NO: 32), SSAIS (SEQ ID NO: 45), or NYGMH (SEQ ID NO: 50).

[0018] In some embodiments, the light chain CDR1 of the antibodies disclosed herein includes RASQSVSSSYLA (SEQ ID NO: 29) or RASQGISSALA (SEQ ID NO: 35).

[0019] In some embodiments, the light chain CDR2 of the antibodies disclosed herein includes GASSRAT (SEQ ID NO: 30), DASSLES (SEQ ID NO: 36), or AASSLQS (SEQ ID NO: 48).

[0020] In some embodiments, the light chain CDR3 of the antibodies disclosed herein includes QQYGSSPT (SEQ ID NO: 31), QQFNSYPYT (SEQ ID NO: 37), QQYGSSPLT (SEQ ID NO: 38), QQYNSYPLT (SEQ ID NO: 49), or QQYNSYPIT (SEQ ID NO: 103).

[0021] In some embodiments, the antibodies of this disclosure comprise heavy chains CDR1, CDR2, and CDR3 in VH, and light chains CDR1, CDR2, and CDR3 in VL. (a) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 26, 27, and 28, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 29, 30, and 31, respectively. (b) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 32, 33, and 34, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 37, respectively. (c) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 32, 33, and 34, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 29, 30, and 38, respectively. (d) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 45, 46, and 47, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 48, and 49, respectively. (e) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 50, 51, and 52, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 37, respectively. (f) The heavy chain CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 136, 137, and 138, respectively, and the light chain CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 139, respectively, or (g) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 136, 137, and 138, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 103, respectively.

[0022] In some embodiments, the heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 32, 33, and 34, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 37, respectively.

[0023] In some embodiments, the heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 32, 33, and 34, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 29, 30, and 38, respectively.

[0024] In some embodiments, the heavy chains CDR1, CDR2, and CDR3 include the amino acid sequences described in SEQ ID NOs. 45, 46, and 47, respectively, and the light chains CDR1, CDR2, and CDR3 include the amino acid sequences described in SEQ ID NOs. 35, 48, and 49, respectively.

[0025] In some embodiments, the heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 50, 51, and 52, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 37, respectively.

[0026] In some embodiments, the heavy chains CDR1, CDR2, and CDR3 include the amino acid sequences described in SEQ ID NOs. 136, 137, and 138, respectively, and the light chains CDR1, CDR2, and CDR3 include the amino acid sequences described in SEQ ID NOs. 35, 36, and 139, respectively.

[0027] In some embodiments, the heavy chains CDR1, CDR2, and CDR3 include the amino acid sequences described in SEQ ID NOs. 136, 137, and 138, respectively, and the light chains CDR1, CDR2, and CDR3 include the amino acid sequences described in SEQ ID NOs. 35, 36, and 103, respectively.

[0028] In some embodiments, VH includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence described as SEQ ID NO: 13, 15, 23, 25, or 130. In certain embodiments, VL includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence described as SEQ ID NO: 14, 16, 17, 24, 131, or 132.

[0029] In some embodiments, the antibodies of this disclosure include VH and VL, in which case, (a) The VH includes sequence number 13, and the VL includes sequence number 14, (b) The VH includes sequence number 15, and the VL includes sequence number 16, (c) The VH includes sequence number 15, and the VL includes sequence number 17. (d) The VH includes sequence number 23, and the VL includes sequence number 24. (e) The VH includes sequence number 25, and the VL includes sequence number 16, (f) The VH includes sequence number 130, and the VL includes sequence number 131, or (g) The VH includes sequence number 130, and the VL includes sequence number 132.

[0030] In some embodiments, the antibodies disclosed herein further comprise a heavy chain (HC) constant region and a light chain (LC) constant region, wherein the HC constant region comprises an amino acid sequence that is at least about 70%, 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 SEQ ID NO: 123, SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID NO: 125. In some embodiments, the LC constant region comprises an amino acid sequence that is at least about 70%, 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 SEQ ID NO: 126.

[0031] Furthermore, this specification provides a bispecific molecule comprising the antibody of this disclosure linked to a molecule having a second binding specificity.

[0032] This disclosure further provides nucleic acids encoding antibodies disclosed herein, vectors containing said nucleic acids, and cells containing said vectors.

[0033] In this specification, immunoconjugates comprising an antibody or a bispecific molecule linked to an agent as disclosed herein are also provided.

[0034] This disclosure provides compositions comprising antibodies, bispecific molecules, nucleic acids, vectors, cells, or immunoconjugates, and carriers as disclosed herein.

[0035] Furthermore, this disclosure also provides kits comprising antibodies, bispecific molecules, nucleic acids, vectors, cells, or immunoconjugates disclosed herein, and instructions for use.

[0036] This specification provides a method for inhibiting TREM-1 activity in a target subject where such inhibition is necessary, comprising administering an antibody, bispecific molecule, nucleic acid, vector, cell, or immunoconjugate disclosed herein to the target subject.

[0037] This specification provides a method for treating an inflammatory or autoimmune disease in a target subject where such treatment is necessary, comprising administering an antibody, bispecific molecule, nucleic acid, vector, cell, or immunoconjugate disclosed herein to the target subject.

[0038] In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, vasculitis, sepsis, systemic inflammatory response syndrome (SIRS), type 1 diabetes mellitus, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma, other autoimmune diseases resulting from either acute or chronic inflammation, and any combination thereof.

[0039] In some embodiments, the methods disclosed herein further include administering one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent is an anti-IP-10 antibody or an anti-TNFα antibody. [Brief explanation of the drawing]

[0040] [Figure 1] Figure 1 shows the sequence alignment of the heavy chain variable regions (VHs) of different epitope-steered anti-TREM-1 antibodies disclosed herein. The antibodies shown include (i) P1-047248, (ii) P1-047246, (iii) P1-047247, (iv) P1-047239, (v) P1-047334, (vi) P1-047323, and (vii) P1-047328. The heavy chain CDR1, CDR2, and CDR3 regions are enclosed in boxes.

[0041] [Figure 2] Figure 2 shows the sequence alignment of the light chain variable regions (VLs) of different epitope-steered anti-TREM-1 antibodies disclosed herein. The antibodies shown are the same antibodies shown in Figure 1. The light chain CDR1, CDR2, and CDR3 regions are shown (enclosed in boxes).

[0042] [Figure 3] Figure 3 shows a comparison of epitope competition analysis (y-axis) and THP1 inhibition assay results (x-axis) for different anti-TREM-1 antibodies. Epitope competition analysis data are provided as the inhibition rate of mAb170 binding to TREM-1. Diamonds represent different anti-TREM-1 antibodies produced from non-epitope-manipulated clones. Circles represent different anti-TREM-1 antibodies produced from epitope-manipulated clones.

[0043] [Figure 4] Figure 4 shows how bins of steered and non-steered epitopes correlate with antibodies grouped by heavy chain CDR3 (HCDR3) amino acid sequences. Each HCDR3 sequence provided represents an individual group, each group consisting of one or more antibodies sharing the same HCDR3 sequence. Based on the distribution of IL-1 beta protein signals measured with the Cisbio HTRF kit, the different HCDR3 groups were further grouped into low nM (0–50, shaded), medium nM (51–500, gray), and high nM (501–900, black) IC50 categories. The bars shown on the left side of the figure correspond to anti-TREM-1 antibodies produced from non-steered clones. The bars shown on the right side of the figure correspond to anti-TREM-1 antibodies produced from epitope-steered clones.

[0044] [Figure 5A]Figure 5A shows a comparison of binding analyses of various anti-TREM-1 antibodies. The y-axis shows the ability of various anti-TREM-1 antibodies to compete with mAb170 for binding to TREM-1. The data is shown as the percentage of inhibition of mAb170 binding. The x-axis shows the ability of various anti-TREM-1 antibodies to compete with PGRP for binding to TREM-1. The data is shown as the percentage of inhibition of PGRP binding. The different anti-TREM-1 antibodies shown were produced from either non-epitope-manipulated clones (black diamonds) or epitope-manipulated clones (gray circles). The antibodies enclosed in circles in Figure 5A (lower right quadrant) correspond to the epitope-manipulated anti-TREM-1 antibodies that most effectively inhibited the binding of PGRP to TREM-1. The antibodies enclosed in boxes (upper right quadrant) correspond to the non-epitope-manipulated antibodies that most effectively inhibited the binding of both PGRP and mAb170 to TREM-1. HMEP (High Throughput Mammalian Expression and Purification) buffer (i.e., no antibody) was used as the negative control (white square). mAb 0170 was used as the positive control (white circle).

[0045] [Figure 5B] Figure 5B shows both the results of the THP1 inhibition assay (y-axis) and the human germline genes corresponding to the heavy chain variable region (VH) of the various anti-TREM-1 antibodies shown in Figure 5A (x-axis). Human germline genes corresponding to the light chain variable region are also presented, with each shape representing a different germline gene. The THP1 inhibition assay results are shown as inhibition percentages. The various anti-TREM-1 antibodies shown were produced from either un-epitope-manipulated clones (black / gray) or epitope-manipulated clones (white). In Figure 5A, antibodies enclosed in circles and boxes are indicated by black outlines and black shading, respectively. [Modes for carrying out the invention]

[0046] To facilitate understanding of this specification, certain terms are defined first. Additional definitions are provided throughout the detailed description.

[0047] It should be noted that the term "a" or "an" entity refers to one or more of those entities. For example, "a nucleoride sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0048] Furthermore, when used herein, “and / or” is understood to mean a specific disclosure of each of the two specified characteristics or components, whether accompanied by “other.” Thus, when used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0049] Where an embodiment is described using the word “including,” it should be understood that other similar embodiments described as “consisting of” and / or “essentially consisting of” are also provided.

[0050] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those universally understood by those skilled in the art in the field to which this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *The Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide dictionaries for many of the terms used herein.

[0051] Units, prefixes, and symbols are shown in the format permitted by the Systeme International de Unites (SI). Numerical ranges include the digit defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right in the 5' to 3' direction. Amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not limitations on various aspects of this disclosure, but rather may be derived by reference to this disclosure as a whole. Thus, the terms defined below are more fully defined by referring to this disclosure as a whole.

[0052] The term “approximately” is used herein to mean roughly, roughly, or about a range. When the term “approximately” is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the stated numerical value. Generally, the term “approximately” may modify the numerical values ​​above and below the stated value by, for example, a 10% upper and lower (up and down) variance.

[0053] The term “triggering receptor expressed on myeloid cells 1” (also known as TREM1, TREM-1, and CD354) refers to a receptor expressed on monocytes, macrophages, and neutrophils. A major ligand for TREM-1 is peptidoglycan-recognition-protein 1 (PGLYRP1), which belongs to the family of peptidoglycan (PGN)-binding proteins (PGRPs). Upon activation, TREM-1 associates with DAP12, an ITAM-containing signaling adapter protein. Downstream signaling may involve activation of NFAT transcription factors, which upregulates the production of pro-inflammatory cytokines. The term “TREM-1” includes any variant or isoform of TREM-1 spontaneously expressed by cells. Therefore, in some embodiments, the antibodies described herein may cross-react with TREM-1 from non-human species (e.g., cynomolgus monkey TREM-1).

[0054] 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 is the standard sequence. Isoform 2 (accession number NP_001229518.1, SEQ ID NO: 2) consists of 225 amino acids and differs from the standard sequence at amino acid residues 201-234. These amino acid residues encode part of the transmembrane domain and the cytoplasmic domain. Isoform 3 (accession number NP_001229519, SEQ ID NO: 3) consists of 150 amino acids and is soluble. It lacks amino acid residues 151-234, and this portion encodes part of the transmembrane domain, cytoplasmic domain, and extracellular domain. Amino acid residues 138-150 also differ from the standard sequence described above.

[0055] The following are the amino acid sequences of three known human TREM-1 isoforms. (A) Human TREM-1 isoform 1 (encoded by the nucleotide sequences of accession number NP_061113.1, SEQ ID NO: 1, accession number NM_018643, SEQ ID NO: 4): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP (Underlined part is the signal sequence); (B) Human TREM-1 isoform 2 (encoded by the nucleotide sequences of accession number NP_001229518.1, SEQ ID NO: 2, accession number NM_001242589, SEQ ID NO: 5): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRYSFQVPGPLVWTLSPLFPSLCAERM (Underlined part is the signal sequence); (C) Human TREM-1 isoform 3 (encoded by the nucleotide sequences of accession number NP_001229519, SEQ ID NO: 3, accession number NM_001242590, SEQ ID NO: 6): MRKTRLWGLLWMLFVSELRA ATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFRCSTLSFSWLVDS (The underlined part is the signal sequence).

[0056] The cynomolgus monkey TREM-1 protein (accession number XP_001082517, SEQ ID NO: 7) is predicted to have the following amino acid sequence: MRKTRLWGLLWMLFVSELRA TTELTEEKYEYKEGQTLEVKCDYALEKYANSRKAWQKMEGKMPKILAKTERPSENSHPVQVGRITLEDYPDHGLLQVQMTNLQVEDSGLYQCVIYQHPKESHVLFNPICLVVTKGSSGTPGSSENSTQNVYRTPSTTAKALGPRYTSPRTVTQAPPESTVVVSTPGSEINLTNVTDIIRVPVFNIVIIVAGGFLSKSLVFSVLFAVTLRSFGP (The underlined part is the signal sequence).

[0057] This disclosure relates to an antibody that specifically binds to and inhibits the function of TREM-1. The antibody interferes with the function of TREM-1 by reducing / blocking TREM-1 activation and downstream signaling.

[0058] The anti-TREM-1 antibodies of this disclosure block TREM-1 signaling by one or a combination of several different mechanisms that directly or indirectly interfere with TREM-1. In one embodiment, the antibody prevents peptideidoglycan recognition protein 1 (PGLYRP1), the natural ligand of TREM-1, from forming a functional complex with TREM-1. In another embodiment, the antibody inhibits 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 an anti-TREM-1 antibody that has the ability to bind to a portion of TREM-1, which otherwise resides at the interface of the TREM-1 dimer, thus preventing individual TREM-1 molecules from associating with each other. In other embodiments, dimerization or multimerization of TREM-1 is reduced or prevented by an anti-TREM-1 antibody that interferes with the interaction of TREM-1 with its ligand.

[0059] In some embodiments, anti-TREM-1 antibodies can interfere with PGLYRP1-induced TREM-1 activation. PGLYRP1 is highly conserved, a 196-amino acid protein consisting of a signal peptide and a peptidoglycan-binding domain, expressed in neutrophils and released upon neutrophil activation. The amino acid sequence of PGLYRP1 (accession number NP_005082.1, SEQ ID NO: 8) is shown below: MSRRSMLLAWALPSLLRLGAA QETEDPACCSPIVPRNEWKALASECAQHLSLPLRYVVVSHTAGSSCNTPASCQQQARNVQHYHMKTLGWCDVGYNFLIGEDGLVYEGRGWNFTGAHSGHLWNPMSIGISFMGNYMDRVPTPQAIRAAQGLLACGVAQGALRSNYVLKGHRDVQRTLSPGNQLYHLIQNWPHYRSP (The underlined part is the signal sequence).

[0060] Accordingly, in some embodiments, the anti-TREM-1 antibodies of this disclosure downregulate or block the release of pro-inflammatory cytokines from myeloid cells such as dendritic cells and monocytes (e.g., THP-1 cells). In some embodiments, the anti-TREM-1 antibodies block the release of TNF-α, MIP-1 beta, MCP-1, IL-1 beta, GM-CSF, IL-6, and / or IL-8 from macrophages, neutrophils, synovial tissue cells, and / or reporter cells, as disclosed herein.

[0061] In some embodiments, the anti-TREM-1 antibodies of this disclosure bind to both human TREM-1 and other species of TREM-1. Therefore, as used herein, the term “TREM-1” encompasses any native form of TREM-1 that 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 derived from primates (e.g., humans, chimpanzees, cynomolgus macaques, or rhesus macaques), rodents (e.g., mice or rats), lagomorphs (e.g., rabbits), or artiodactyls (e.g., cattle, sheep, pigs, or camels). In certain embodiments, TREM-1 is SEQ ID NO: 1 (human TREM-1, isoform 1). TREM-1 may also be a mature form of TREM-1, such as a TREM-1 protein that has undergone post-translational processing in a suitable cell. Such a mature TREM-1 protein may be glycosylated, for example. TREM-1 may also be the full-length TREM-1 protein.

[0062] In some embodiments, the anti-TREM-1 antibodies of this disclosure are monoclonal antibodies, meaning they are derived directly or indirectly from a single clone of B lymphocytes. In some embodiments, the anti-TREM-1 antibodies are prepared, screened, and purified using, for example, the method described in International Patent Application Publication WO2013 / 120553. Briefly, suitable mice, such as TREM-1 or TREM-1 / TREM-3 knockout (KO) mice, are immunized with TREM-1, TREM-1 expressing cells, or a combination of both. In other embodiments, the anti-TREM-1 antibodies are polyclonal antibodies, meaning they are a mixture of the monoclonal antibodies disclosed herein.

[0063] In some embodiments, the anti-TREM-1 antibodies of this disclosure are recombinantly expressed in prokaryotic or eukaryotic cells. In some embodiments, the prokaryotic cells are Escherichia coli. In certain embodiments, the eukaryotes are yeast, insect, or mammalian cells, and are cells derived from organisms such as primates (e.g., humans, chimpanzees, cynomolgus macaques, or rhesus macaques), rodents (e.g., mice or rats), lagomorphs (e.g., rabbits), or artiodactyls (e.g., cattle, sheep, pigs, or camels). Suitable mammalian cell lines, but not limited to, include HEK293 cells, CHO cells, and HELA cells. The anti-TREM-1 antibodies disclosed herein can also be prepared by other methods known to those skilled in the art, such as phage display or yeast display. Once prepared, the antibodies can be screened for binding to full-length TREM-1 or its variants using, for example, the methods described in the examples of International Patent Application Publication 2013 / 120553.

[0064] In some embodiments, the anti-TREM-1 antibody of this disclosure is operated away from the epitope on human TREM-1 recognized by a reference antibody (e.g., mAb 0170). Therefore, in some embodiments, the anti-TREM-1 antibody disclosed herein does not compete with the reference antibody (e.g., mAb 0170) for binding to human TREM-1. In some embodiments, the anti-TREM-1 antibody of this disclosure does not bind to amino acids D38-F48 of human TREM-1 (SEQ ID NO: 1). In certain embodiments, the anti-TREM-1 antibody disclosed herein does not bind to amino acids D38-L45, E46-Q56, and / or Y90-L96 of human TREM-1 (SEQ ID NO: 1). The binding epitope of the reference antibody mAb 0170 is known in the art; see, for example, U.S. Patent No. 9,000,127.

[0065] As used herein, the term “epitope-engineered” refers to an anti-TREM-1 antibody selected to bind to epitopes other than D38–L45, E46–Q56, and / or Y90–L96 of human TREM-1 (SEQ ID NO: 1). In some embodiments, the epitope-engineered anti-TREM-1 antibody is (1) of human TREM-1 (e.g., isoform 1, SEQ ID NO: 1). 27 EKYELKEGQTL 37 (Sequence ID 9), (2) 88 EDYHDHGLLRVRM 100 (Sequence ID 10), (3) 120 KEPHMLFDR 128 It binds to one or more epitopes selected from the group consisting of (Sequence ID 11) and any combination thereof.

[0066] The epitope-modified anti-TREM-1 antibodies described herein can be prepared by any method known in the art, such as the methods described in the examples. In some embodiments, the epitope-modified anti-TREM-1 antibody can be produced by immunizing an animal (e.g., a mouse) with a human TREM-1 polypeptide containing a mutation in one of the above epitopes (e.g., amino acid residues 38-48 of SEQ ID NO: 1). After immunization, the produced antibody may be further characterized for binding to human TREM-1. In some embodiments, a synthetic peptide containing the target epitope may be synthesized and used to immunize an animal (e.g., a mouse). In some embodiments, an alternative scaffold containing the target epitope (e.g., a 10th human fibronectin type III domain) may be used. 10 Fn3 or α3D (a highly thermally stable 3-helix bundle protein) may be used.

[0067] In some embodiments, the anti-TREM-1 antibody of this disclosure is not epitope-modified and is therefore capable of binding to the same epitope as a reference antibody (e.g., mAb170).

[0068] As used herein, the term “antibody” refers to a protein derived from a germline immunoglobulin sequence that has the ability to specifically bind to an antigen (TREM-1) or a portion thereof. The term includes full-length antibodies of any class or isotype (i.e., IgA, IgE, IgG, IgM, and / or IgY), and any single-chain or fragment thereof. Antibodies that specifically bind to an antigen or a portion thereof may bind exclusively to that antigen or a portion thereof, or to a limited number of homologous antigens or portions thereof. Full-length antibodies typically contain at least four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. One subclass of immunoglobulins of particular pharmaceutically acceptable use is the IgG family. In humans, the IgG class can be subdivided into four subclasses: IgG1, IgG2, IgG3, and IgG4. These are based on the sequence of their heavy chain constant region. Light chains can be divided into two types, kappa and lambda, based on differences in sequence composition. An IgG molecule consists of two heavy chains linked by two or more disulfide bonds, and two light chains, each attached to the heavy chains by disulfide bonds. The heavy chains may contain a heavy chain variable region (VH) and up to three heavy chain constant (CH) regions, CH1, CH2, and CH3. The light chains may contain a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into a more conserved region called the framework region (FR) and a hypervariable region called the complementarity-determining region (CDR) located between them. The VH and VL regions typically consist of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy and light chains form binding domains that can interact with the antigen. On the other hand, the constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (effector cells), Fc receptors, and host tissues or factors, including the first component of the classical complement system (C1q), though this is not limited to these. The antibody of the present invention can be isolated.The term "isolated antibody" refers to an antibody that has been isolated and / or recovered from other components in the environment in which it was produced, and / or an antibody that has been purified from a mixture of components present in the environment in which it was produced. Since it has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody, a particular antigen-binding fragment of an antibody may be suitable in the context of the present invention.

[0069] The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen, such as TREM-1, as described herein. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)S, Fv (typically the VL and VH domains of one arm of an antibody), single-stranded Fv (scFv, see, e.g., Bird et al., Science 242:42S-426 (1988); Huston et al., PNAS 85:5879-5883 (1988)), dsFv, Fd (typically the VH and CH1 domains), and dAb (typically the VH domain) fragments; VH, VL, VhH, and V-NAR domains; monovalent molecules containing one VH and one VL; mini-bodies, dia-bodies, tria-bodies, tetra-bodies, and kappa-bodies (see, e.g., Ill et al., Protein Eng See 10:949-57 (1997): Examples include camelid IgG; IgNAR; and one or more isolated CDRs or functional paratopes, where the isolated CDRs or antigen-binding residues or polypeptides can be associated with or linked to each other to form functional antibody fragments. Various types of antibody fragments have been described and reviewed, for example, in Holliger and Hudson, Nat Biotechnol 2S:1126-1136 (2005); International Patent Application Publication WO2005 / 040219, and U.S. Patent Publications 2005 / 0238646 and 2002 / 0161201. These antibody fragments may be obtained using prior art known to those skilled in the art, and the fragments may be screened for utility in the same manner as complete antibodies.

[0070] A “human” antibody (HuMAb) refers to an antibody having a variable region in which both the framework region and the CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Anti-TREM-1 antibodies described herein may contain amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or mutations introduced by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted onto a human framework sequence. The terms “human” antibody and “fully human” antibody are used synonymously.

[0071] A "humanized" antibody refers to a human / non-human chimeric antibody containing one or more sequences (CDR regions or parts thereof) derived from non-human immunoglobulins. That is, a humanized antibody is a human immunoglobulin (recipient antibody) in which at least several residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of an antibody derived from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing desired specificity, affinity, sequence composition, and functionality. In some cases, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. One example of such modification is the introduction of one or more so-called reverse mutations, which are typically amino acid residues derived from the donor antibody. Antibody humanization can be carried out using recombinant techniques known to those skilled in the art (see, e.g., Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny KCLo). A human recipient framework suitable for both light-chain and heavy-chain variable domains may be identified, for example, by sequence homology or structural homology. Alternatively, a fixed recipient framework may be used, for example, based on knowledge of its structure, biophysical, and biochemical properties. The recipient framework may be germline-derived or derived from a mature antibody sequence. The CDR region derived from the donor antibody may be transferred by CDR transplantation. The CDR-transplanted humanized antibody can be further optimized, for example, in terms of affinity, functionality, and biophysical properties, by identifying key framework locations where the reintroduction (reverse mutation) of amino acid residues derived from the donor antibody has a beneficial effect on the properties of the humanized antibody. In addition to reverse mutations derived from the donor antibody, the humanized antibody can be manipulated by introducing germline residues into the CDR or framework region, removing immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0072] Furthermore, humanized antibodies may contain residues not present in recipient or donor antibodies. These modifications are made to further improve antibody performance. Generally, humanized antibodies contain at least one, typically two, variable domains, in which all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR residues are the FR region of a human immunoglobulin sequence. Humanized antibodies also optionally, typically, contain at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. The term "humanized antibody derivative" refers to any modification of a humanized antibody, such as a conjugate of an antibody with another agent or another antibody.

[0073] The term “recombinant human antibody,” as used herein, includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as: (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal with respect to human immunoglobulin genes, or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express antibodies, such as transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur during antibody maturation, for example. As is well known in this field (see, for example, Lonberg Nature Biotech. 23(9):1117-1125 (2005)), the variable region contains an antigen-binding domain, which is encoded by various genes that are reconstituted to form antibodies specific to exogenous antigens. In addition to reconstitution, the variable region may be further modified by multiple single amino acid changes (referred to as somatic mutations or hypermutations) to increase the affinity of antibodies against exogenous antigens. The constant region changes (i.e., isotype switches) in further responses to the antigen. Thus, nucleic acid molecules that are reconstituted in response to an antigen and those that are somatically mutated, encoding light chain immunoglobulin polypeptides and heavy chain immunoglobulin polypeptides, may not have sequence identity with the original nucleic acid molecule, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0074] A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region originates from another species. For example, an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0075] In some embodiments, the anti-TREM-1 antibody of this disclosure is an IgG antibody. As used herein, “IgG antibody,” such as human IgG1, has, in certain embodiments, the structure of a natural IgG antibody. That is, the IgG antibody has the same number of heavy and light chains and disulfide bonds as a natural IgG antibody of the same subclass. For example, a TREM-1 IgG1 antibody consists of two heavy chains (HC) and two light chains (LC), in which case the heavy and light chains are linked in the same number and positions as the disulfide crosslinks present in a natural IgG1 antibody (unless the antibody has undergone a mutation that alters the disulfide crosslinks).

[0076] As used herein, “isotype” refers to an antibody class encoded by a heavy chain constant region gene (e.g., antibodies of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE).

[0077] An "allotype" refers to a variant that occurs spontaneously within a particular group of isotypes, and such variants differ in several amino acids (see, for example, Jefferis et al., mAbs 1:1 (2009)). The anti-TREM-1 antibodies described herein may be any allotype. In some embodiments, the anti-TREM-1 antibody is an antibody of the "IgG1.3f" allotype, and the antibody contains one or more amino acid substitutions selected from the group consisting of L234A, L235E, and G237A in EU numbering, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 12). In other embodiments, the anti-TREM-1 is an antibody of the "IgG1.1f" allotype, and the antibody contains one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S, and P331S in EU numbering, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 12). In certain embodiments, the anti-TREM-1 antibody is an "IgG1-Aba" allotype antibody, which, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 12), contains one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S in EU numbering. In further embodiments, the anti-TREM-1 antibody is an "IgG4-Aba" allotype antibody, which, compared to the wild-type IgG1 isotype (e.g., SEQ ID NO: 12), contains one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S in EU numbering.

[0078] The terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are interchangeable in this specification with the term "antibody that specifically binds to an antigen."

[0079] As used herein, “isolated antibody” refers to an antibody that has been isolated and / or recovered from other components in the environment in which it was produced, and / or an antibody that has been purified from a mixture of components present in the environment in which it was produced.

[0080] "Effector function" refers to the interaction between an antibody Fc region and an Fc receptor or ligand, or the resulting biochemical events. Examples of effector functions include C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, Fcγ-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptors, BCRs). Such effector functions generally require the Fc region to bind to a binding domain (e.g., an antibody variable domain). In one embodiment, the anti-TREM-1 antibody of this disclosure includes an Fc region that does not bind to one or more FcγRs and therefore lacks effector function (i.e., effectorless).

[0081] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include the FcγR family of receptors, including allele variants and alternative splicing forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Various properties of human FcγR are known in this field. Most innate effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB. Natural killer (NK) cells, on the other hand, selectively express one activating Fc receptor (FcγRIII in mice, FcγRIIIA in humans), but do not express the inhibitory FcγRIIB in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the types of activated Fc receptors it binds to.

[0082] The "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1q) of the classical complement system. Therefore, the Fc region includes the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL).

[0083] In IgG, the Fc region includes the CH2 and CH3 domains of the immunoglobulin domain, as well as the hinge between the CH1 and CH2 domains. While the definition of the boundary of the Fc region of the immunoglobulin heavy chain may vary as defined herein, the Fc region of the human IgG heavy chain is defined as the range from amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxyl terminus of the heavy chain, in which case the numbering follows the Kabat EU index. The CH2 domain of the human IgG Fc region spans amino acids 237 to 340, and the CH3 domain is located on the C-terminal side of the CH2 domain within the Fc region, that is, from amino acids 341 to 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of IgG. As used herein, the Fc region may be a native sequence Fc containing any allotype variant, or a variant Fc (e.g., a non-native Fc). Fc may further refer to the isolated region, or to the region in the background of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein" also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesion).

[0084] "Natural sequence Fc region" or "natural sequence Fc" refers to a sequence of amino acids identical to that of a naturally occurring Fc region. Natural sequence human Fc regions include the natural sequence human IgG1 Fc region, the natural sequence human IgG2 Fc region, the natural sequence human IgG3 Fc region, and the natural sequence human IgG4 Fc region, as well as their natural variants. Natural sequence Fc includes various allotypes of Fc (see, for example, Jefferis et al., mAbs 1:1 (2009)).

[0085] A “variant sequence Fc region” or “non-natural Fc” involves a modification, typically altering one or more of its functional properties, such as serum half-life, complement binding response, Fc receptor binding, protein stability, and / or antigen-dependent cell-mediated cytotoxicity, or particularly their absence. In some embodiments, the anti-TREM-1 antibody of this disclosure may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, again altering one or more of the functional properties of the antibody. In one embodiment, the anti-TREM-1 antibody is an IgG1 isotype and carries a modified Fc domain containing one or more, possibly all, of the following mutations: reducing affinity to specific Fc receptors (L234A, L235E, and G237A), and reducing C1q-mediated complement binding response (A330S and P331S) (residues are numbered according to the EU index).

[0086] The terms “hinge,” “hinge domain,” “hinge region,” and “antibody hinge region” refer to the domain of the heavy chain constant region that binds the CH1 domain to the CH2 domain, and include the upper, middle, and lower parts of the hinge (Roux et al., J Immunol 161:4083 (1998)). The hinge provides varying levels of flexibility between the antibody binding region and the effector region, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, the hinge begins at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., J Immunol 161:4083 (1998)). The sequences of the wild-type IgG1, IgG2, IgG3, and IgG4 hinges are publicly known in the art (e.g., WO 2017 / 087678, International PCT Publication). In one embodiment, the hinge region of CH1 in the anti-TREM-1 antibody is modified so that the number of cysteine ​​residues within the hinge region changes, for example, by increasing or decreasing it. This method is further described, for example, in U.S. Patent No. 5,677,425.

[0087] The constant region may be modified to stabilize the antibody and reduce the risk, for example, that a bivalent antibody separates into two monovalent VH-VL fragments. For example, in the IgG4 constant region, residue S228 (residue numbering by EU index) may be mutated to a proline (P) residue to stabilize inter-heavy-chain disulfide crosslinking at the hinge (see, e.g., Angal et al., Mol Immunol. 30:105-8 (1995)). The antibody or fragment may also be defined in terms of its complementarity-determining region (CDR). As used herein, the terms “complementarity-determining region” or “hypervariable region” refer to the region of the antibody where amino acid residues involved in antigen binding are located. The hypervariable region or CDR region can be identified as the region with the highest variability in the amino acid alignment of the antibody variable domain. For example, databases such as the Kabat database can be used to identify CDRs, such as a CDR defined as containing amino acid residues 24-34 (CDR1), 50-59 (CDR2), and 89-97 (CDR3) of the light chain variable domain, and amino acid residues 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) of the heavy chain variable domain (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, the CDR can be defined as residues derived from the "hypervariable loop" (residues 26-33 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain (Chothia and Lesk, J. Mol. Biol 196: 901-917 (1987)). Typically, the numbering of amino acid residues in this region is performed using the method described by Kabat et al. above.For example, the terms “Kabat position,” “Kabat residue,” and “according to Kabat” refer, in this specification, to the numbering system relating to the heavy chain variable domain or light chain variable domain. By using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, which correspond to abbreviations or insertions of the variable domain framework (FR) or CDR. For example, a heavy chain variable domain may contain an amino acid insertion after residue 52 of the CDR H2 (residues 52a, 52b, and 52c according to Kabat) and an inserted residue after residue 82 of the heavy chain FR (residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the homologous region of the antibody sequence with a “standard” Kabat numbering sequence.

[0088] The terms “epitope” or “antigenic determinant” refer to a site on an antigen (e.g., TREM-1) to which an immunoglobulin or antibody specifically binds, and are defined, for example, by specific methods used to identify epitopes. Epitopes can be formed from a sequence of amino acids (usually linear epitopes) or from discontinuous amino acids juxtaposed by the three-dimensional folding of a protein (usually structural epitopes). Epitopes formed from a sequence of amino acids are typically retained upon exposure to denaturing solvents, though not always, whereas epitopes formed by three-dimensional folding are typically lost upon processing with denaturing solvents. Typically, an epitope contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial structure. Methods for determining which epitopes bind to a given antibody (i.e., epitope mapping) are known in the art, and include, for example, immunoblotting and immunoprecipitation assays. In this case, the duplicated or continuous peptide (e.g., derived from TREM-1) is tested for reactivity with a given antibody (e.g., an anti-TREM-1 antibody). Methods for determining the spatial structure of the epitope include techniques in this art and those described herein, such as X-ray crystallography, antigen mutation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0089] The term “binding to the same epitope” when referring to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the “same epitope on TREM-1” as the antibodies described herein include, for example, epitope mapping methods, such as X-ray analysis of the antigen:antibody complex crystals, which provides atomic dissolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods involve monitoring the binding of antibodies to antigen fragments or variants of the antigen, where loss of binding due to modification of amino acid residues in the antigen sequence is often considered an indicator of epitope components. Furthermore, computer combinatorial methods for epitope mapping may also be used. These methods depend on the ability of the target antibody to affinity isolate specific short peptides derived from a peptide library of combinatorial phage displays. Antibodies having the same VH and VL, or the same CDR1, 2, and 3 sequences, are predicted to bind to the same epitope.

[0090] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits the binding of the other antibody to the target and to what extent, can be determined using known competition experiments such as BIACORE® surface plasmon resonance (SPR) analysis. In certain embodiments, the antibody competes with another antibody for binding to the target and inhibits by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). The competition assay can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc ;2006;doi: 10.1101 / pdb.prot4277, or Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if they inhibit each other by at least 50% in both directions, i.e., regardless of whether one antibody or the other antibody was first contacted with the antigen in the competition experiment.

[0091] As used herein, the terms "specific binding", "selective binding", "selectively binds", and "specifically binds" refer to an antibody that binds to an epitope on a given antigen. Typically, an antibody binds with an affinity of about 10 -7 M or less, such as approximately 10 -8 M or less, 10 -9 M or less or 10-10 Equilibrium dissociation constant (K) less than or even lower than M D (ii) binds to the specified antigen with an affinity at least twice as high as the affinity for binding to nonspecific antigens other than the specified antigen or closely related antigens (e.g., BSA, casein, etc.). Therefore, an antibody that "specifically binds to human TREM-1" is defined as 10 -7 M or less, for example, approximately 10 -8 Less than M, 10 -9 Less than M or 10 -10 K less than M or even lower D This refers to antibodies that bind to soluble or cell-bound human TREM-1. Antibodies that "cross-react with cynomolgus monkey TREM-1" are 10 -7 M or less, for example, approximately 10 -8 Less than M, 10 -9 Less than M, or 10 -10 K less than M or even lower D This refers to antibodies that bind to cynomolgus monkey TREM-1. In certain embodiments, antibodies that do not cross-react with TREM-1 from non-human species exhibit essentially undetectable binding to these proteins in standard binding assays.

[0092] In this specification, the term "binding specificity" refers to the interaction between a molecule, such as an antibody or a fragment thereof, and a single exclusive antigen, or with a limited number of highly homologous antigens (or epitopes). In contrast, an antibody that can specifically bind to TREM-1 cannot bind to dissimilar molecules. Antibodies according to the present invention may not have the ability to bind to Nkp44, a natural killer cell p44-related protein.

[0093] The specificity of the interaction and the value of the equilibrium binding constant can be directly determined by known methods. Standard assays for evaluating the ability of a ligand (e.g., an antibody) to bind to its target are known in the art and include, for example, ELISA, Western blotting, RIA, and flow cytometry. Antibody binding kinetics and binding affinity can also be evaluated by standard assays known in the art, such as SPR.

[0094] A competitive binding assay to determine whether two antibodies compete for or cross-compete with each other in terms of binding is, for example, a flow cytometry analysis of competition for binding to TREM-1 expressing myeloid cells, as described in the examples. Other methods include: SPR (e.g., BIACORE®), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)), solid-phase direct biotin-avidin EIA (see Kirkland et al., J.Immunol. 137:3614 (1986)), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), solid-phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol.Immunol. 25(1):7 (1988)), solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546(1990)), and directly labeled RIA. (See Moldenhauer et al., Scand. J. Immunol. 32:77(1990)).

[0095] As used herein, the term “bin” is defined using a reference antibody. If a second antibody cannot bind to the antigen simultaneously with the reference antibody, the second antibody is said to belong to the same “bin” as the reference antibody. In this case, the reference antibody and the second antibody competitively bind to the same portion of the antigen, and the term “competitive antibody” is coined. If a second antibody can bind to the antigen simultaneously with the reference antibody, the second antibody is said to belong to a separate “bin.” In this case, the reference antibody and the second antibody do not competitively bind to the same portion of the antigen, and the term “non-competitive antibody” is coined.

[0096] Antibody "binning" does not provide direct information about epitopes. Competitive antibodies, i.e., antibodies belonging to the same "bin," may have the same epitope, overlapping epitopes, or even distinct epitopes. The latter occurs when a reference antibody bound to its own epitope on the antigen occupies space necessary for a second antibody to contact its own epitope on the antigen (steric hindrance). Non-competitive antibodies generally have distinct epitopes.

[0097] In this specification, the term "binding affinity" refers to a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or a fragment of an antigen. The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity).

[0098] For example, the binding affinity between two molecules via monovalent interactions, such as an antibody or a fragment of an antigen, is determined by the equilibrium dissociation constant (K). D It can be quantified by the determination of K. Similarly, K D This can be determined, for example, by measuring the dynamics of complex formation and dissociation using the SPR method. The rate constants corresponding to the binding and dissociation of the monovalent complex are the binding rate constant k, respectively. a (or k on ) and the dissociation rate constant k d (or k 0ff ) is referred to as K D is, formula K D =k d / ka Through, k a and k d It is associated with K for individual antibody / antigen complexes. Following the above definition, binding affinity associated with different molecular interactions, such as comparing the binding affinity of different antibodies to a given antigen, is related to K for individual antibody / antigen complexes. D They can be compared by comparing their values.

[0099] As used herein, the term “high affinity” for IgG antibodies means 10% or higher against the target antigen. -8 M or less, 10 -9 M or less, or 10 -10 K below M D This refers to antibodies that possess high affinity. However, for other antibody isotypes, the binding of "high affinity" may change. For example, the binding of "high affinity" for IgM isotypes is 10 -10 M or less or 10 -8 K below M D This refers to antibodies that possess [a certain characteristic].

[0100] Under the circumstances of an in vitro or in vivo assay using an antibody or its antigen-binding fragment, "EC 50 The term "maximum response" refers to the concentration of an antibody or its antigen-binding moiety that induces a response that is 50% of the maximum response, i.e., an intermediate response between the maximum response and the baseline.

[0101] As used herein, the term “natural” when applied to an object means the fact that the object may exist in nature. For example, a polypeptide or polynucleotide sequence present in a living organism (including a virus) that can be isolated from its source in nature and has not been intentionally modified by a human in a laboratory is natural.

[0102] A "polypeptide" refers to a chain containing at least two consecutive linked amino acid residues, with no upper limit on chain length. One or more amino acid residues in a protein may undergo modifications such as glycosylation, phosphorylation, or disulfide bond formation, for example, but are not limited to these. A "protein" may contain one or more polypeptides.

[0103] As used herein, the term “nucleic acid molecule” is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0104] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in this art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, predicted non-essential amino acid residues in an anti-TREM-1 antibody are substituted with other amino acid residues derived from the same side chain family. Methods for identifying conserved nucleotide and amino acid substitutions that do not quench antigen binding are well known in this field (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0105] With respect to nucleic acids, the term "substantial homology" indicates that when two nucleic acids or their specified sequences are optimally aligned and compared, they are identical in at least approximately 80% of nucleotides, at least approximately 90% to 95% of nucleotides, or at least approximately 98% to 99.5% of nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when a segment hybridizes to the complementary strand of that strand under selective hybridization conditions.

[0106] For polypeptides, the term "substantial homology" indicates that, when two polypeptides or their specified sequences are optimally aligned and compared, they are identical in at least approximately 80% of amino acids, at least approximately 90% to 95% of amino acids, or at least approximately 98% to 99.5% of amino acids, with appropriate amino acid insertions or deletions.

[0107] The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology % = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap. The comparison of sequences and the determination of the percentage of identity between the two sequences may be performed using mathematical algorithms such as those described in the non-restrictive examples below.

[0108] The percentage of identity between two nucleotide sequences may be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide sequences or between amino acid sequences may also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)), incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences may be determined using the algorithm of Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)) incorporated into the GAP program of the GCG software package (available at worldwideweb.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0109] The nucleic acid and protein sequences described herein may further be used as “query sequences” for performing searches against public databases, for example, to identify related sequences. Such searches may be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990), J.Mol.Biol.215:403-10. A BLAST nucleotide search may be performed using the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search may be performed using the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignment for comparative purposes, Gapped BLAST may be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and Gapped BLAST programs, you may use the default parameters for each program (e.g., XBLAST and NBLAST). See worldwideweb.ncbi.nlm.nih.gov.

[0110] Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. When purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods known in the art, nucleic acids are “isolated” or “substantially pure.” See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0111] For example, nucleic acids such as cDNA may be mutated according to standard techniques for providing gene sequences. With respect to coding sequences, these mutations may have desirable effects on amino acid sequences. In particular, DNA sequences that are substantially homologous to or derived from natural V, D, J, constant, switch sequences, and other such sequences described herein are expected (in this case, "derived" means that the sequence is identical to or modified from another sequence).

[0112] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another ligated nucleic acid. Certain types of vectors are “plasmids,” which refer to a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is a viral vector, in which an additional DNA segment can be ligated within the viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), once introduced into a host cell, can be integrated into the host cell’s genome, thereby replicating alongside the host genome. Furthermore, certain vectors have the ability to induce the expression of a gene into which they are manipulably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA methods are often in plasmid form. Hereinafter, “plasmid” and “vector” may be used interchangeably because plasmids are the most commonly used form of vector. However, this also includes other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0113] As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell that may contain nucleic acids not naturally present in the cell and into which a recombinant expression vector has been introduced. It should be understood that the term is intended to refer not only to a specific target cell but also to its progeny. Such progeny cells may not be identical to the parent cell in practice, as certain modifications may occur in subsequent generations due to either mutation or environmental influences, but as used herein, they are still included within the scope of the term “host cell.”

[0114] As used herein, the term “bound” refers to the association of two or more molecules. The bond may be covalent or non-covalent. The bond may also be genetic (i.e., recombinant fusion). Such bonds may be achieved using a wide range of techniques recognized in the art, such as chemical bonding or the creation of recombinant proteins.

[0115] As used herein, “administer” means the physical introduction of a composition containing a therapeutic agent into a target using any of the various methods and delivery systems known to those skilled in the art. Various routes of administration for the anti-TREM-1 antibodies described herein include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration by injection or infusion. As used herein, the term “parenteral administration” means a mode of administration other than intestinal and topical administration, and is usually performed by injection, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, lymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and substernal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via, for example, topical, epidermal, or mucosal routes of administration, or parenteral routes such as intranasal, oral, vaginal, rectal, sublingual, or topical. Furthermore, administration may be carried out, for example, once, multiple times, and / or over one or more extension periods.

[0116] As used herein, the terms “inhibit” or “block” (for example, referring to inhibition / blocking of the binding of TREM-1 ligand to TREM-1 on cells) are used interchangeably and encompass both partial and complete inhibition / blocking. In some embodiments, an anti-TREM-1 antibody is determined to inhibit the binding of TREM-1 ligand to TREM-1 by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as further described herein, for example. In some embodiments, an anti-TREM-1 antibody is determined to inhibit the binding of TREM-1 ligand to TREM-1 by 50% or less, e.g., about 40%, 30%, 20%, 10%, 5%, or 1%, as further described herein, for example.

[0117] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, improving, inhibiting, delaying, or preventing the progression, manifestation, severity, or recurrence of any disease-related symptoms, complications, conditions, or biochemical signs. Treatment may be the treatment of a subject with the disease, or the treatment of a subject without the disease (e.g., for preventive purposes).

[0118] The term “effective dose” or “effective dosage” is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The “therapeutic effective dose” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug that promotes disease regression, demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or the prevention of functional or physical impairment resulting from disease onset, when used alone or in combination with another therapeutic agent. The therapeutic effective dose or therapeutic effective dosage of a drug includes the “preventive effective dose” or “preventive effective dosage,” which is any amount of the drug that inhibits the onset or recurrence of disease when administered alone or in combination with another therapeutic agent to subjects at risk of developing the disease or at risk of disease relapse. The ability of a therapeutic agent to promote disease regression or inhibit the onset or recurrence of disease can be evaluated using various methods known to those skilled in the art, for example, by evaluating the activity of the agent in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or in in vitro assays.

[0119] The term "patient" includes human and other mammalian subjects receiving either preventive or therapeutic treatment.

[0120] As used herein, the term “subject” includes any human or non-human animal. For example, a subject having cancer may be treated using the methods and compositions described herein. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0121] As used herein, "ug" and "uM" are interchangeable with "μg" and "μM," respectively.

[0122] The various embodiments described herein are described in further detail in the following subsections.

[0123] I. Anti-TREM-1 antibody This specification describes antibodies, such as fully human antibodies, characterized by specific functional features or properties. For example, the antibodies of this disclosure specifically bind to human TREM-1, more specifically to a specific domain (e.g., a functional domain) within the extracellular domain of human TREM-1. In some embodiments, the antibody specifically binds to a site on the TREM-1 ligand to which the TREM-1 ligand (e.g., PGLYRP1) binds. In some embodiments, the antibody is an antagonist antibody; that is, the antibody inhibits or suppresses the activity of TREM-1 on cells such as monocytes, macrophages, and neutrophils (i.e., does not agonize upon binding). In some embodiments, the anti-TREM-1 antibody cross-reacts with TREM-1 derived from one or more non-human primates, such as cynomolgus monkey 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 activated cells (e.g., macrophages, dendritic cells, neutrophils). In some embodiments, the specific anti-TREM-1 antibodies described herein are antibodies that bind to human TREM-1 at a different epitope than the reference antibody (e.g., mAb170) (i.e., epitope-engineered), such as monoclonal antibodies, recombinant antibodies, and / or human antibodies. Thus, in some embodiments, the anti-TREM-1 antibodies do not cross-compete with the reference antibody (e.g., mAb170) with respect to binding to human TREM-1. In other words, in some embodiments, the anti-TREM-1 antibodies of this disclosure belong to a different "bin" than the reference antibody (e.g., mAb170).

[0124] In some embodiments, the epitope-operated anti-TREM-1 antibody of this disclosure comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown in Table 1. In certain embodiments, VH comprises an amino acid sequence described as SEQ ID NOs. 13, 15, 23, 25, or 130. In certain embodiments, VL comprises an amino acid sequence described as SEQ ID NOs. 14, 16, 17, 24, 131, or 132.

[0125] In some embodiments, the epitope-operated anti-TREM-1 antibodies of the present disclosure comprise VH and VL, in which case, (a) VH and VL contain the amino acid sequences described as SEQ ID NOs: 13 and 14, respectively. (b) VH and VL contain the amino acid sequences described as SEQ ID NOs: 15 and 16, respectively. (c) VH and VL contain the amino acid sequences described as SEQ ID NOs. 15 and 17, respectively. (d) VH and VL contain the amino acid sequences described as SEQ ID NOs. 23 and 24, respectively. (e) VH and VL contain the amino acid sequences described as SEQ ID NOs. 25 and 16, respectively. VH and VL contain the amino acid sequences described as SEQ ID NOs. 130 and 131, respectively, or (f) VH and VL contain the amino acid sequences described as SEQ ID NOs. 130 and 132, respectively.

[0126] In some embodiments, the epitope-operated anti-TREM-1 antibody disclosed herein includes a CDR of a heavy chain variable region selected from the group consisting of SEQ ID NOs: 13, 15, 23, 25, and 130. In some embodiments, the epitope-operated anti-TREM-1 antibody disclosed herein includes a CDR of a light chain variable region selected from the group consisting of SEQ ID NOs: 14, 16, 17, 24, 131, and 132.

[0127] In some embodiments, the epitope-operated anti-TREM-1 antibody of the present disclosure comprises CDR1, CDR2, and CDR3 of the heavy chain variable region (VH), and CDR1, CDR2, and CDR3 of the light chain variable region (VL), in which case, (a) VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 32, 45, 50, and 136, (b) VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 33, 46, 51, and 137, (c) VH CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 34, 47, 52, and 138. (d) VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 29 and 35, (e) VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 36, and 48, and / or (f) VL CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 37, 38, 39, 103, and 139.

[0128] In some embodiments, the epitope-operated anti-TREM-1 antibody disclosed herein comprises heavy chain variable regions (VH) CDR1, CDR2, and CDR3, and light chain variable regions (VL) CDR1, CDR2, and CDR3, in which case, (a) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 26, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 27, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 28, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 29, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 30, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 31. (b) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 32, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 33, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 34, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 35, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 36, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 37. (c) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 32, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 33, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 34, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 29, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 30, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 38. (d) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 45, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 46, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 47, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 35, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 49. (e) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 50, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 51, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 52, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 35, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 36, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 37. (f) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 136, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 137, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 138, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 35, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 36, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 139, or (g) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 136, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 137, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 138, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 35, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 36, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 103.

[0129] In some embodiments, the epitope-engineered anti-TREM-1 antibody disclosed herein comprises CDR1, CDR2, and CDR3 of the heavy chain variable region (VH), and CDR1, CDR2, and CDR3 of the light chain variable region (VL), in which case one or more of the CDRs contain one or more amino acid mutations (e.g., substitutions or deletions) compared to the anti-TREM-1 antibody disclosed herein. Thus, in certain embodiments, the epitope-engineered anti-TREM-1 antibody comprises VH CDR1 comprising X1, X2, X3, X4, and X5, in which case X1 is S or N, X2 is S, Y, or E, X3 is YG, or A, X4 is W, M, or I, and X5 is S, T, H, or N. In some embodiments, the epitope-modified anti-TREM-1 antibody includes VH antibodies X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, and X17. Including CDR2, in which case X1 is YV or G, X2 is T or I, X3 is W, I or none, X4 is H, Y, or P, X5 is Y, D, or I, X6 is S, G, or F, X7 is G, S, or D, X8 is I, Y, N, or T, X9 is S, T, or K, X10 is N or Y, X11 is Y or G, X12 is N or A, X13 is P, D, or Q, X14 is S or K, X15 is L, V, or F, X16 is K or Q, and X17 is S or G.In some embodiments, the epitope-modified anti-TREM-1 antibody is VH, including X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, G, X13, X14, X15, X16, X17, X18, D, and X19. Including CDR3, in this case X1 is E, D, M, T, or none; X2 is G, V, or Y; X3 is Y, R, or none; X4 is D, H, G, or none; X5 is I, Y, or none; X6 is L, Y, or none; X7 is T, G, N, or none; X8 is G, S, Y, or none; X9 is Y, V, T, F, or H; X10 is E, L, S, or Y; X11 is Y, W, F, or H; X12 is Y or F; X13 is E or none; X14 is L or none; X15 is L or none; X16 is P or none; X17 is L or none; X18 is M or L; and X19 is V or Y. In certain embodiments, the epitope-operated anti-TREM-1 antibody comprises a VL CDR1 containing R, A, S, Q, X1, X2, X3, S, S, X4, L, and A, where X1 is S or G, X2 is V or I, X3 is S or none, and X4 is Y or A. In some embodiments, the epitope-operated anti-TREM-1 antibody disclosed herein comprises a VL CDR2 containing X1, A, S, S, X2, X3, and X4, where X1 is G, D, or A, X2 is R or L, X3 is A, E, or Q, and X4 is T or S. In certain embodiments, the epitope-engineered anti-TREM-1 antibody comprises a VL CDR3 containing Q, Q, X1, X2, S, X3, P, X4, and T, where X1 is Y or F, X2 is G or N, X3 is S or Y, and X4 is L, Y, I, or none.

[0130] Furthermore, in this specification, an anti-TREM-1 antibody that binds to human TREM-1 at the same epitope as a reference antibody (e.g., mAb170) (i.e., not epitope-modified) is used, but the anti-TREM-1 antibody is not mAb170. Therefore, in some embodiments, these unepitope-modified anti-TREM-1 antibodies cross-compete with the reference antibody (e.g., mAb170) for binding to human TREM-1. In other words, in some embodiments, the anti-TREM-1 antibodies of this disclosure belong to the same "bin" as the reference antibody (e.g., mAb170), in which case the anti-TREM-1 antibody is not mAb170. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the reference antibody mAb 0170 are as follows: (A) Heavy chain variable region: EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMHWVRQASGKGLEWVGRIRTKSSNYATYYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRDMGIRRQFAYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 193); (B) Light chain variable region: DIVLTQSPDSLAVSLGERATINCRASESVDTFDYSFLHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 194). See International Patent Application Publication WO2016 / 009086A1.

[0131] In some embodiments, the unepitoped anti-TREM-1 antibodies disclosed herein include a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown in Table 2. In certain embodiments, VH includes an amino acid sequence described as SEQ ID NOs. 53, 55, 57, 59, 62, 64, 66, 68, 73, 74, 75, 76, 78, 80, 81, 83, or 133. In certain embodiments, VL includes an amino acid sequence described as SEQ ID NOs. 54, 56, 58, 60, 61, 63, 65, 67, 69, 70, 71, 72, 77, 79, 82, 134, or 135.

[0132] In some embodiments, the unepitoped anti-TREM-1 antibodies disclosed herein include VH and VL, in which case, (a) VH contains the amino acid sequence described in SEQ ID NO: 53, and VL contains the amino acid sequence described in SEQ ID NO: 54. (b) VH contains the amino acid sequence described in SEQ ID NO: 55, and VL contains the amino acid sequence described in SEQ ID NO: 56. (c) VH contains the amino acid sequence described in SEQ ID NO: 57, and VL contains the amino acid sequence described in SEQ ID NO: 58. (d) VH contains the amino acid sequence described in SEQ ID NO: 59, and VL contains the amino acid sequence described in SEQ ID NO: 60. (e) VH contains the amino acid sequence described in SEQ ID NO: 59, and VL contains the amino acid sequence described in SEQ ID NO: 61. (f) VH contains the amino acid sequence described in SEQ ID NO: 59, and VL contains the amino acid sequence described in SEQ ID NO: 54. (g)VH contains the amino acid sequence described in SEQ ID NO: 62, and VL contains the amino acid sequence described in SEQ ID NO: 61. (h)VH contains the amino acid sequence described in SEQ ID NO: 59, and VL contains the amino acid sequence described in SEQ ID NO: 63. (i) VH contains the amino acid sequence described in SEQ ID NO: 64, and VL contains the amino acid sequence described in SEQ ID NO: 65. (j)VH contains the amino acid sequence described in SEQ ID NO: 66, and VL contains the amino acid sequence described in SEQ ID NO: 67. (k)VH contains the amino acid sequence described in SEQ ID NO: 68, and VL contains the amino acid sequence described in SEQ ID NO: 54. (l) VH contains the amino acid sequence described in SEQ ID NO: 68, and VL contains the amino acid sequence described in SEQ ID NO: 69. (m)VH contains the amino acid sequence described in SEQ ID NO: 68, and VL contains the amino acid sequence described in SEQ ID NO: 70. (n)VH contains the amino acid sequence described in SEQ ID NO: 68, and VL contains the amino acid sequence described in SEQ ID NO: 71. (o)VH contains the amino acid sequence described in SEQ ID NO: 68, and VL contains the amino acid sequence described in SEQ ID NO: 72. (p)VH contains the amino acid sequence described in SEQ ID NO: 68, and VL contains the amino acid sequence described in SEQ ID NO: 60. (q)VH contains the amino acid sequence described in SEQ ID NO: 73, and VL contains the amino acid sequence described in SEQ ID NO: 54. (r)VH contains the amino acid sequence described in SEQ ID NO: 73, and VL contains the amino acid sequence described in SEQ ID NO: 63. (s)VH contains the amino acid sequence described in SEQ ID NO: 74, and VL contains the amino acid sequence described in SEQ ID NO: 54. (t)VH contains the amino acid sequence described in SEQ ID NO: 75, and VL contains the amino acid sequence described in SEQ ID NO: 54. (u)VH contains the amino acid sequence described in SEQ ID NO: 76, and VL contains the amino acid sequence described in SEQ ID NO: 77. (v)VH contains the amino acid sequence described in SEQ ID NO: 78, and VL contains the amino acid sequence described in SEQ ID NO: 79. (w)VH contains the amino acid sequence described in SEQ ID NO: 80, and VL contains the amino acid sequence described in SEQ ID NO: 54. (x)VH contains the amino acid sequence described in SEQ ID NO: 81, and VL contains the amino acid sequence described in SEQ ID NO: 82. (y)VH contains the amino acid sequence described in SEQ ID NO: 83, and VL contains the amino acid sequence described in SEQ ID NO: 60. (z)VH contains the amino acid sequence described in SEQ ID NO: 133, and VL contains the amino acid sequence described in SEQ ID NO: 134. (aa)VH contains the amino acid sequence described in SEQ ID NO: 133, and VL contains the amino acid sequence described in SEQ ID NO: 54, or (bb)VH contains the amino acid sequence described in SEQ ID NO: 59, and VL contains the amino acid sequence described in SEQ ID NO: 135.

[0133] In some embodiments, the unepitoped anti-TREM-1 antibody includes a CDR of a heavy chain variable region selected from the group consisting of 53, 55, 57, 59, 62, 64, 66, 68, 73, 74, 75, 76, 78, 80, 81, 83, and 133. In some embodiments, the unepitoped anti-TREM-1 antibody includes a CDR of a light chain variable region selected from the group consisting of 54, 56, 58, 60, 61, 63, 65, 67, 69, 70, 71, 72, 77, 79, 82, 134, and 135.

[0134] In some embodiments, the unepitoped anti-TREM-1 antibody of the present disclosure comprises CDR1, CDR2, and CDR3 of the heavy chain variable region (VH), and CDR1, CDR2, and CDR3 of the light chain variable region (VL), in which case, (a) VH CDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 84, 89, 93, 99, 106, 109, 112, and 140. (b) VH CDR2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 90, 94, 97, 98, 100, 102, 104, 107, 110, 113, 116, 119, and 141, (c)VH CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 91, 95, 101, 105, 108, 111, 114, 115, 117, 120, and 142. (d) VL CDR1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 87 and 42, (e) VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and 30, and / or (f) VL CDR3 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 49, 88, 92, 96, 103, 118, and 143.

[0135] In some embodiments, the unepitoped anti-TREM-1 antibodies disclosed herein include VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, in which case, (a) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 84, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 85, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 86, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88. (b) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 89, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 90, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 91, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 92. (c) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 93, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 94, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 95, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 96. (d) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 93, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 97, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 95, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 49. (e) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 93, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 97, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 95, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88. (f) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 93, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 98, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 95, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 49. (g) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 99, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 100, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 101, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 49. (h)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 99, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 102, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 101, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88. (i) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 99, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 102, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 101, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 103. (j) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 99, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 102, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 101, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 49. (k)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 93, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 102, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 95, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88. (l) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 45, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 104, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 105, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88, or (m)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 106, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 107, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 108, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88. (n) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 109, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 110, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 111, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 49. (o) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 112, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 113, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 114, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 96. (p)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 112, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 113, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 115, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88. (q) VH CDR1 contains the amino acid sequence described as SEQ ID NO: 45, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 116, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 117, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 118. (r)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 45, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 119, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 120, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 49. (s)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 140, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 141, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 142, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 143. (t)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 140, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 141, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 142, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 87, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 48, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 88, or (t)VH CDR1 contains the amino acid sequence described as SEQ ID NO: 93, VH CDR2 contains the amino acid sequence described as SEQ ID NO: 97, and VH CDR3 contains the amino acid sequence described as SEQ ID NO: 95, VL CDR1 contains the amino acid sequence described as SEQ ID NO: 42, VL CDR2 contains the amino acid sequence described as SEQ ID NO: 30, and VL CDR3 contains the amino acid sequence described as SEQ ID NO: 38.

[0136] In some embodiments, the anti-TREM-1 antibody comprises a CDR and / or variable region sequence having at least 80% identity (e.g., at least 85%, at least 95%, at least 95%, or at least 99%) to the CDR and / or variable region sequences described herein (e.g., Tables 1, 2, 5, and 6).

[0137] In some embodiments, the anti-TREM-1 antibody disclosed herein comprises a heavy chain and a light chain, wherein the heavy chain comprises a VH domain disclosed herein (e.g., those shown in Tables 1 and 2) fused to a heavy chain constant region disclosed herein (e.g., SEQ ID NO: 122, 123, 124, or 125). In some embodiments, the anti-TREM-1 antibody disclosed herein comprises a heavy chain and a light chain, wherein the light chain comprises a VL domain disclosed herein (e.g., those shown in Tables 1 and 2) fused to a light chain constant region disclosed herein (e.g., SEQ ID NO: 126).

[0138] In some embodiments, the anti-TREM-1 antibody of the present disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 197-207 and 209-232, and / or the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 233-243 and 245-268.

[0139] Heavy and light chains having amino acid sequences that are at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to either the heavy or light chain described herein may be used to form anti-TREM-1 antibodies having desired features, such as those detailed herein.

[0140] In some embodiments, the anti-TREM-1 antibody has the ability to bind to variants of human TREM-1 (e.g., isoforms 2 and 3 of TREM-1, SEQ ID NOs. 2 and 3, respectively) as determined, for example, using surface plasmon resonance. In some embodiments, the anti-TREM-1 antibody has the ability to bind to cynomolgus monkey TREM-1 (SEQ ID NO: 7) as determined, for example, using surface plasmon resonance.

[0141] In some embodiments, the anti-TREM-1 antibodies described herein bind with high affinity to human TREM-1, for example, as determined by BIACORE® (as described in the examples), 10 -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, or 10 -9 M~10 -7 M's K D It binds to the cynomolgus monkey TREM-1, for example, as determined by BIACORE® (as described in the examples). -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, or 10 -9 M~10 -7 M's K D They are joined together.

[0142] In some embodiments, the anti-TREM-1 antibody binds to TREM-1 at an epitope different from that of the reference antibody (e.g., mAb170) (i.e., epitope-operated), thereby the anti-TREM-1 antibody of this disclosure does not compete with the reference antibody for binding to human TREM-1. Therefore, in certain embodiments, the anti-TREM-1 antibody disclosed herein does not bind to amino acids D38-L45, E46-Q56, and / or Y90-L96 of human TREM-1 (SEQ ID NO: 1). In some embodiments, the anti-TREM-1 antibody binds to (1) human TREM-1 (e.g., isoform 1, SEQ ID NO: 1). 27 EKYELKEGQTL 37 (Sequence ID 9), (2) 88 EDYHDHGLLRVRM 100 (Sequence ID 10), and (3) 120 KEPHMLFDR 128 The antibody binds to one or more epitopes selected from the group consisting of (SEQ ID NO: 11). In some embodiments, the anti-TREM-1 antibody has the ability to specifically bind to at least one amino acid residue selected from the group consisting of (1) E27, K28, Y29, E30, L31, K32, E33, G34, Q35, T36, L37, and any combination thereof of human TREM-1 (e.g., isoform 1, SEQ ID NO: 1), (2) E88, D89, Y90, H100, D101, H102, G103, L104, L105, R106, V107, R108, M109, and any combination thereof of human TREM-1 (e.g., isoform 1, SEQ ID NO: 1).

[0143] In some embodiments, the antibody of this disclosure binds to TREM-1 at the same epitope as the reference antibody (e.g., mAb170) (i.e., it is not epitope-modified). In some embodiments, the anti-TREM-1 antibody is (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) A49, S50, S51, Q52, K53, A54, W55, Q56, 157, 158, R59, D60, G61, E62, M63, P64, K65, T66, L67, A68, C6 9) It has the ability to specifically bind to at least one amino acid residue selected from the group consisting of T70, E71, R72, P73, S74, K75, N76, S77, H78, P79, V80, Q81, V82, G83, R84, 185 and any combination thereof, and (iii) at least one amino acid residue selected from the group consisting of C113, V114, 1115, Y116, Q117, P118, P119 and any combination thereof. See WO2016 / 009086.

[0144] In some embodiments, the anti-TREM-1 antibody has the ability to specifically bind to amino acids D38-F48 of SEQ ID NO: 1 (human TREM-1), as determined, for example, by HDX-MS or X-ray diffraction. 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 (human TREM-1), as determined, for example, by HDX-MS or X-ray diffraction. In certain embodiments, the anti-TREM-1 antibody has an epitope comprising one, two, three, or all of the amino acid residues selected from the group consisting of D42, E46, D92, and H93 of SEQ ID NO: 1 (human TREM-1), as determined using TREM-1 variants and surface plasmon resonance.

[0145] In some embodiments, the anti-TREM-1 antibody of this disclosure has an epitope comprising at least amino acid residues E46 and / or D92 of SEQ ID NO: 1 (human TREM-1), as determined using TREM-1 variants and surface plasmon resonance. In other embodiments, the anti-TREM-1 antibody comprises one, two, or all amino acid residues selected from the group consisting of L31, I86, and V101 of SEQ ID NO: 1 (human TREM-1). In certain embodiments, the anti-TREM-1 antibody has the ability to specifically bind to a polypeptide comprising amino acid residues E19-L26 of cynomolgus monkey TREM-1 (SEQ ID NO: 7), as determined, for example, using HDX-MS or X-ray diffraction.

[0146] In some embodiments, the anti-TREM-1 antibody has the ability to specifically bind to human TREM-1, in which case the epitope of the antibody includes one, two, three, four, five, six, seven, eight, nine, or all of the amino acid residues selected from the group consisting of V39, K40, C41, D42, Y43, L45, E46, K47, F48, and A49 of SEQ ID NO: 1.

[0147] In some embodiments, the anti-TREM-1 antibody has the ability to specifically bind to human TREM-1, in which case the epitope of the antibody includes D42 of SEQ ID NO: 1. In other embodiments, the anti-TREM-1 antibody has the ability to specifically bind to human TREM-1, in which case the epitope of the antibody includes E46 of SEQ ID NO: 1. In some embodiments, the epitopes of the antibody may include V39, C41, D42, Y43, and L45 of SEQ ID NO: 1. In further embodiments, the epitopes of the antibody may include E46, K47, and A49 of SEQ ID NO: 1. In certain embodiments, the epitope of the anti-TREM-1 antibody may further include F48 of SEQ ID NO: 1.

[0148] The variable region of the anti-TREM-1 antibody described herein may be bound to (for example, covalently or fused to) Fc such as that of IgG1, IgG2, IgG3, or IgG4. The Fc may be 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 (for example, Jeffries et al. (See al. (2009) mAbs 1:1). In some embodiments, the variable region of the anti-TREM-1 antibody disclosed herein binds to Fc cells that have no effectors, or few effectors, such as IgG1. In some embodiments, the variable region of the anti-TREM-1 antibody binds to Fc cells with reduced or no binding to one or more FcγR cells.

[0149] In some embodiments, the VH domain of the anti-TREM-1 antibody described herein may be fused to a constant domain (i.e., Fc) of human IgG, such as IgG1, IgG2, IgG3, or IgG4, which may be natural or modified, as detailed herein. For example, the VH domain may be, for example, the following wild-type human IgG1 constant domain amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12), or the sequence of an allotype variant of SEQ ID NO: 12, may include any VH domain amino acid sequence described herein fused to a constant region of human IgG such as IgG1, and may have the following amino acid sequences: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 121, allotype-specific amino acid residues are the underlined bold parts).

[0150] In some embodiments, the VH domain of the anti-TREM-1 antibody described herein is, for example, the following human IgG1 constant domain amino acid sequence without effectors: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPE AE G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP SS IEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 122, "IgG1.1f", including substitutions of L234A, L235E, G237A, A330S, and P331S according to EU numbering, underlined portion) or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPE AE G A PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP AP IEKTISKAKGQPREPQVYTLPPSR E E MThe amino acid sequence of any VH domain described herein, fused to a constant region without effectors such as TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 123, "IgG1.3f", including the underlined portion, with substitutions of L234A, L235E, and G237A according to EU numbering).

[0151] For example, allotype variants of IgG1 include K97R, D239E, and / or L241M (the underlined bold portion above), and numbering according to SEQ ID NOs. 121-123. Within the full-length heavy chain region, and according to EU numbering, these amino acid substitutions are numbered K214R, D356E, and L358M. In some embodiments, the constant region of the anti-TREM-1 antibody further includes one or more mutations or substitutions in amino acids L117, A118, G120, A213, and P214 (the underlined portion above) as numbered in SEQ ID NOs. 121-123, or in amino acids L234, A235, G237, A330, and P331 according to EU numbering. In further embodiments, the constant region of the anti-TREM-1 antibody may include one or more mutations or substitutions in the amino acids L117A, A118E, G120A, A213S, and P214S of SEQ ID NO: 12, or in the amino acids L234A, L235E, G237A, A330S, and P331S according to EU numbering. The constant region of the anti-TREM-1 antibody may also include one or more mutations or substitutions in the L117A, A118E, and G120A of SEQ ID NO: 12, or in the L234A, L235E, and G237A according to EU numbering.

[0152] In some embodiments, the VH domain of the anti-TREM-1 antibody described herein is, for example, the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHT S PP S PAPELLGG S SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 124, "IgG1-Aba", underlined portion, including substitutions of K214R, C226S, C229S, and P238S according to EU numbering), or ASTKGPSVFPLAP C S R STS ES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT K TY T CNV D HKPSNTKVDK R VEPKSCDKTHT S PP S PAPELLGG S The amino acid sequence of any VH domain described herein, fused to an IgG1 constant domain including SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 125, "IgG4-Aba", underlined portion, including substitutions S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, P238S according to EU numbering).

[0153] The VL domains described herein may be fused to the constant domain of a human kappa or lambda light chain. For example, the VL domain of an anti-TREM-1 antibody may be fused to the following human IgG1 kappa light chain amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 126) may contain any VL domain amino acid sequence described herein, fused to the specified sequence number.

[0154] In certain embodiments, the heavy chain constant region contains lysine or another amino acid at the C-terminus. For example, the heavy chain contains the following final amino acid: LSPGK (SEQ ID NO: 127). In certain embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus and has, for example, LSPG (SEQ ID NO: 128) or LSP in the C-terminal sequence.

[0155] In general, the variable regions described herein can be conjugated to Fc, typically including one or more modifications that alter one or more functional properties of the antibody, such as Fc receptor binding, inflammatory cytokine release, serum half-life, complement binding, and / or antigen-dependent cytotoxic activity. Furthermore, the antibodies described herein may be chemically modified (for example, one or more chemical moieties may be conjugated to the antibody) or modified to alter their glycosylation and alter one or more functional properties of the antibody. Each of these embodiments is described in detail below. The numbering of residues in the Fc region is the numbering of the Kabat EU index.

[0156] The Fc region is derived from the constant region of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM), and includes fragments, analogs, variants, mutants, or derivatives of said constant region. The constant region of an immunoglobulin is defined as a native or synthetic polypeptide homologous to the C-terminal region of the immunoglobulin, and may include the CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain separately or in combination.

[0157] Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specific to the IgG class of an antibody: FcγRI, FcγRII, and FcγRIII. Sequences crucial for IgG binding to FcγR receptors have been reported to be located within the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to Fc receptors (FcR).

[0158] In one embodiment, the Fc region of the anti-TREM-1 antibody is a variant Fc region, which is modified (e.g., by amino acid substitution, deletion, and / or insertion) compared to the parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate the variant), thereby providing desired structural properties and / or biological activity.

[0159] For example, modifications can be made in the Fc region to produce Fc variants with increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), increased or decreased complement-mediated cytotoxicity (CDC), increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for the Fc receptor compared to the parent Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications are considered particularly desirable. For example, a variant Fc region may contain two, three, four, five, or more substitutions in the region, such as at the location of a particular Fc region as specified herein.

[0160] The variant Fc region may also include sequence changes, in which amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the anti-TREM-1 antibody described herein. Even if cysteine ​​residues are removed, the single-stranded Fc domain can still form a dimeric Fc domain, which is held non-covalently. In other embodiments, the Fc region may be modified to improve compatibility with selected host cells. For example, a PA sequence near the N-terminus of a typical native Fc region may be removed. The PA sequence can be recognized by digestive enzymes in E. coli, such as proline iminopeptidase. In other embodiments, one or more glycosylation sites within the Fc domain may be removed. Typically glycosylated residues (e.g., asparagine) can lead to cytolytic reactions. Such residues may be deleted or replaced with non-glycosylated residues (e.g., alanine). In other embodiments, sites involved in complement interaction, such as the C1q binding site, may be removed from the Fc region. For example, the EKK sequence of human IgG1 may be deleted or substituted. In certain embodiments, sites affecting binding to the Fc receptor, preferably sites other than the salvage receptor binding site, may be removed. In other embodiments, the Fc region may be modified to remove the ADCC site. The ADCC site is known in the art; for example, regarding the ADCC site in IgG1, see Sarmay et al., Molec.Immunol.29(5):633-9(1992). Specific examples of variant Fc domains are disclosed, for example, in WO97 / 34631 and WO96 / 32478.

[0161] In one embodiment, the hinge region of Fc is modified so that the number of cysteine ​​residues in the hinge region changes, for example, by increasing or decreasing it. This method is detailed in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of Fc is modified, for example, to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fc-hinge fragment, thereby attenuating the binding of Staphylococcus protein A (SpA) to the antibody compared to the SpA binding of the natural Fc-hinge domain. This method is detailed in U.S. Patent No. 6,165,745 by Ward et al.

[0162] In yet another embodiment, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and / or 331 are substituted with a different amino acid residue, thereby changing the antibody's affinity for the effector ligand, while retaining the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered may be, for example, the Fc receptor or the C1 component of complement. This method is detailed in U.S. Patents 5,624,821 and 5,648,260 by Winter et al.

[0163] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 are substituted with different amino acid residues, thereby altering the C1q binding of the antibody and / or reducing or neutralizing its complement-dependent cytotoxic activity (CDC). This method is detailed in U.S. Patent No. 6,194,551 by Idusogie et al.

[0164] In another example, one or more amino acid residues within positions 231 and 239 of the amino acid spectrum are altered, thereby changing the complement-binding ability of the antibody. This method is described in detail in the international patent application publication WO94 / 29351 by Bodmer et al.

[0165] In yet another embodiment, the Fc region may be modified by altering one or more amino acids at the following positions to reduce antibody-dependent cytotoxic activity (ADCC) and / or decrease affinity to the Fcγ receptor: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280 ,283,285,286,289,290,292,293,294,295,296,298,299,301,303,305,307,309,312,313,315,320,322,324,325,326,327,329,330,331,332,333,334,335,337,338,340,360,373,376,378,382,388,389,398,414,416,419,430,433,434,435,436,437,438, or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. Other modifications that enhance the interaction between FcγR and complement include, but are not limited to, 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are summarized in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0166] Other Fc modifications that can be made to Fc are modifications to reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating the Fc-mediated effector function of, for example, ADCC, ADCP, and CDC. Exemplary modifications, but not limited to, include substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and / or 331 (e.g., 330 and 331). Numbering follows the EU index. Exemplary substitutions, but not limited to, include 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S). Numbering follows the EU index. Fc variants may include 236R / 328R. Other modifications to reduce the interaction between FcγR and complement include substitutions of 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 mutagenesis, enzymatic means, or by production in organisms such as non-glycosylating bacteria. These and other modifications are summarized in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0167] Optionally, the Fc region may include non-natural amino acid residues at additional and / or alternative positions known in the art (e.g., U.S. Patents No. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720, International Patent Application Publication WO00 / See 42072, WO01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925, and WO06 / 0201 14.

[0168] The affinity and binding characteristics of the Fc region to its ligand can be determined by various in vitro assay methods known in the art (biochemical or immunological assays), including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labeling of one or more of the components being tested, and / or may employ various detection methods including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0169] In some embodiments, the anti-TREM-1 antibody disclosed herein is (a) an IgG1 isotype containing one or more amino acid substitutions in the Fc region, selected from the group consisting of: N297A, N297Q, D270A, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254 (b) IgG2 isotypes, which are T, T256E, L328E, P238D, S267E, L328F, E233D, G237D, H268D, P271G, A330R and any combination thereof, in which case the numbering of the residue follows EU numbering or Kabat numbering, or contain an amino acid deletion in the Fc region at the position corresponding to glycine 236, and contain one or more amino acid substitutions in the Fc region, selected from the group consisting of: P238S (c) IgG4 isotypes selected from the group consisting of (c) IgG4 isotypes, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, in which case the numbering of the residues follows EU numbering or Kabat numbering, or (c) IgG4 isotypes selected from the group consisting of the following The following are no-acid residues containing one or more amino acid substitutions in the Fc region: E233P, F234V, L234A / F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, in which case the numbering of the residues follows EU numbering or Kabat numbering.In some embodiments, (a) the Fc region further comprises one or more additional amino acid substitutions at amino acid residues selected from the group consisting of A330L, L234F, L235E, P331S and any combination thereof, with residue numbering following EU or Kabat numbering; (b) the Fc region further comprises one or more additional amino acid substitutions at positions selected from the group consisting of M252Y, S254T, T256E and any combination thereof, with residue numbering following EU or Kabat numbering; or (c) the Fc region further comprises the amino acid substitution of S228P, with residue numbering following EU or Kabat numbering. See WO2017 / 152102.

[0170] In certain embodiments, Fc with reduced complement binding is selected. An exemplary Fc with reduced complement binding, such as IgG1 Fc, has the following two amino acid substitutions: A330S and P331S.

[0171] In certain embodiments, Fc molecules that are essentially devoid of effector function are selected. That is, they exhibit reduced binding to FcγR and reduced complement binding. Exemplary Fc molecules without effector function, such as IgG1 Fc, include the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0172] II. Nucleic acids, vectors, and cells Another embodiment described herein relates to a nucleic acid molecule encoding the anti-TREM-1 antibody described herein. The nucleic acid may be present in whole cells, in cell lysates, or in a partially purified or substantially pure form. The nucleic acid is “isolated” or “substantially pure” when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA bound to the isolated DNA in nature) or proteins by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis and other methods known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid is a cDNA molecule.

[0173] The nucleic acids described herein can be obtained using standard molecular biological techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as detailed below), the cDNA encoding the light and heavy chains of the antibodies produced from the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display), the nucleic acids encoding the antibodies can be collected from the library.

[0174] In some embodiments, the nucleic acids described herein are nucleic acids encoding the VH and VL sequences of the anti-TREM-1 antibody of this disclosure. Exemplary DNA sequences encoding the VH sequence are given as SEQ ID NOs: 144-168. Exemplary DNA sequences encoding the VL sequence are given as SEQ ID NOs: 169-192, 195, and 196. Sequences are also provided in Tables 3 and 4.

[0175] The method for producing an anti-TREM-1 antibody disclosed herein involves a signal peptide along with nucleotide sequences encoding the heavy chain and light chain, for example, SEQ ID NOs: 269 and 305, SEQ ID NOs: 270 and 306, SEQ ID NOs: 271 and 307, SEQ ID NOs: 272 and 308, SEQ ID NOs: 273 and 309, SEQ ID NOs: 274 and 310, SEQ ID NOs: 275 and 311, SEQ ID NOs: 276 and 312, SEQ ID NOs: 277 and 313, SEQ ID NOs: 278 and 314, SEQ ID NOs: 279 and 315, SEQ ID NOs: 281 and 317, SEQ ID NOs: 282 and 318, SEQ ID NOs: 283 and 319, SEQ ID NOs: 284 and 320, SEQ ID NOs: 285 and 321, and sequence number In cell lines including numbers 286 and 322, SEQ ID NOs. 287 and 323, SEQ ID NOs. 288 and 324, SEQ ID NOs. 289 and 325, SEQ ID NOs. 290 and 326, SEQ ID NOs. 291 and 327, SEQ ID NOs. 292 and 328, SEQ ID NOs. 293 and 329, SEQ ID NOs. 294 and 330, SEQ ID NOs. 295 and 331, SEQ ID NOs. 296 and 332, SEQ ID NOs. 297 and 333, SEQ ID NOs. 298 and 334, SEQ ID NOs. 299 and 335, SEQ ID NOs. 300 and 336, SEQ ID NOs. 301 and 337, SEQ ID NOs. 302 and 338, SEQ ID NOs. 303 and 339, SEQ ID NOs. 304 and 340, etc., the heavy and light chains may be expressed. Host cells containing these nucleotide sequences are included herein.

[0176] Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments may be further manipulated using standard recombinant DNA techniques to transform, for example, the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operably ligated to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. When used in this context, the term "operably ligated" is intended to mean that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0177] The isolated DNA encoding the VH region may be converted to a full-length heavy chain gene by operably ligating the VH-coding DNA to another DNA molecule encoding the heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are publicly known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, for example, the IgG2 and / or IgG4 constant region. For heavy chain genes in Fab fragments, the DNA encoding VH may be operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0178] The isolated DNA encoding the VL region may be converted to a full-length light chain gene (and Fab light chain gene) by operably ligating the VL-encoding DNA to another DNA molecule encoding the CL of the light chain constant region. The sequences of human light chain constant region genes are publicly known in this field (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

[0179] Another embodiment described herein relates to cells (e.g., host cells) that express (recombinantly) the anti-TREM-1 antibody and associated polynucleotides and expression vectors described herein. Vectors comprising polynucleotides containing a nucleotide sequence encoding the anti-TREM-1 antibody or a fragment thereof are also provided herein. In some embodiments, the vector may be used to recombinantly express the anti-TREM-1 antibody described herein in host cells, such as mammalian cells. Non-limiting examples of cells that may be used to express the anti-TREM-1 antibody disclosed herein include human embryonic kidney (HEK) cell lines (e.g., HEK293), Chinese hamster ovary (CHO) cell lines, baby hamster kidney (BHK) cell lines, COS cell lines, Maidin Derby canine kidney (MDCK) cell lines, and HeLa cell lines. In some embodiments, the vector may be used in gene therapy.

[0180] Preferred vectors of this disclosure include expression vectors, viral vectors, and plasmid vectors. In some embodiments, the vector is a viral vector.

[0181] As used herein, an expression vector refers to any nucleic acid construct containing the factors necessary for the transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, any nucleic acid construct containing the factors necessary for replication and translation when introduced into a suitable host cell. Expression vectors may include plasmids, phagemids, viruses, and their derivatives.

[0182] Expression vectors in this disclosure may comprise polynucleotides encoding an antibody or its antigen-binding moiety as described herein. In some embodiments, the coding sequence of an antibody or its antigen-binding moiety is operably ligated to an expression regulatory sequence. As used herein, two nucleic acid sequences are operably ligated if they are covalently bonded in such a manner that the nucleic acid sequences of each component are able to retain their functionality. A coding sequence and a gene expression regulatory sequence are said to be operably ligated when they are covalently bonded in such a manner that the expression or transcription and / or translation of the coding sequence is under the influence or control of the gene expression regulatory sequence. Two DNA sequences are said to be operably ligated if the induction of a promoter in the 5' gene expression sequence results in the transcription of the coding sequence, and the nature of the ligation between the two DNA sequences is such that (1) it does not result in the introduction of a frameshift mutation, (2) it does not interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) it does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Therefore, the gene expression sequence is operably ligated to the coding nucleic acid sequence if it can transcribe the coding nucleic acid sequence and the resulting transcript is translated into a desired antibody or its antigen-binding portion.

[0183] Examples of viral vectors, though not limited to them, include nucleic acid sequences derived from the following viruses: retroviruses such as Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40 virus; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses; and RNA viruses such as retroviruses. Other vectors well known in this field can also be readily employed. Certain viral vectors are based on non-cytotoxic eukaryotic viruses in which non-essential genes are replaced with the target gene. Examples of non-cytotoxic viruses include retroviruses, whose life cycle involves the reverse transcription of genomic viral RNA into DNA and subsequent integration of the provirus into host cell DNA. Retroviruses are approved for human gene therapy trials. Most useful are replication-deficient retroviruses (i.e., those that can induce the synthesis of the desired protein but cannot produce infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient in vivo gene transfer. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfection of a packaging cell line containing the plasmid, production of recombinant retrovirus by the packaging cell line, recovery of viral particles from tissue culture medium, and infection of target cells with viral particles) are presented in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990), and Murry, EJ, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0184] In some embodiments, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated viruses may be engineered to have replication defects and possess the ability to infect a wide range of cell types and cell species. They further offer advantages such as thermal stability, lipid solvent stability, high transduction frequency in diverse cell lineages including hematopoietic cells, and lack of duplication inhibition, thereby enabling multiple transduction. Adeno-associated viruses can integrate into human cell DNA in a site-specific manner, which has been reported to minimize the possibility of insertional mutation induction and the variability of inserted gene expression characteristic of retroviral infections. Furthermore, wild-type adeno-associated virus infection has been tracked in tissue cultures for more than 100 passages in the absence of selective pressure, suggesting that adeno-associated virus genomic integration is a relatively stable event. Adeno-associated viruses can also function in extrachromosomal ways.

[0185] III. Immunoconjugate This disclosure also provides immunoconjugates comprising any of the anti-TREM-1 antibodies disclosed herein. In some embodiments, the immunoconjugate comprises an antibody or its antigen-binding portion conjugated to an agent. In some embodiments, the immunoconjugate comprises a bispecific molecule disclosed herein conjugated to an agent (e.g., as a therapeutic or diagnostic agent).

[0186] For diagnostic purposes, appropriate agents are detectable labels, including radioisotopes for whole-body imaging, and radioisotopes, enzymes, fluorescent labels, and other appropriate antibody tags for sample testing. Detectable labels that can be conjugated to any anti-TREM-1 antibody described herein may be of any of the various types currently used in the field of in vitro diagnostics, such as particle labels containing metal sols, such as gold colloid, or, for example, N2S2, N3S, or N4 type peptide chelating agents. 125 or Tc 99Examples include chromophores containing isotopes, fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzymatic labels that convert a given substrate into a detectable marker, and polynucleotide tags that become apparent after amplification, such as by polymerase chain reaction. Suitable enzymatic labels include horseradish peroxidase and alkaline phosphatase. For example, the label may be the alkaline phosphatase enzyme, such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD) or 3-(4-(methoxyspiro{l,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl)phenyl phosphate disodium (CSPD). The presence or formation of chemiluminescence after conversion of 1,2-dioxetane substrates such as phosphate, and CDP and CDP-STAR® or other luminescent substrates known in the art, such as suitable lanthanide chelating agents like Terbium(III) and Europium(III), is detected by measuring the presence or formation of chemiluminescence. The detection means is determined by the selected label. The appearance of the label or its reaction products can be observed with the naked eye if the label is particulate and accumulates at an appropriate level, or can be observed using instruments such as spectrophotometers, luminometers, or fluorometers. All of these follow standard practice.

[0187] In some embodiments, the binding method results in bindings that are substantially (or nearly) non-immunogenic. For example, peptide-bindings (i.e., amide-bindings), sulfide-bindings, (sterically hindered), disulfide-bindings, hydrazone-bindings, and ether-bindings are formed. These bindings are nearly non-immunogenic and exhibit reasonable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO95 / 17886).

[0188] Depending on the biochemical properties of the moiety and antibody, various coupling strategies can be employed. If the moiety is a natural or recombinant substance of 50-500 amino acids, standard procedures exist in textbooks describing chemical techniques for the synthesis of protein conjugates, and these can be easily followed by those skilled in the art (see, for example, Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47(2008) 10030-10074). In some embodiments, the reaction of the maleimide moiety with the antibody or a cysteine ​​residue within the moiety is used. For example, in the case of the Fab or Fab' fragment of an antibody, this is a particularly suitable coupling chemistry method and is used. Alternatively, in some embodiments, coupling to the C-terminus of the antibody or moiety is performed. For example, modification of the C-terminus of a protein such as a Fab fragment can be carried out as described in (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7(2009) 3361-3371).

[0189] Generally, site-directed reactions and covalent couplings are based on converting native amino acids to amino acids that have orthogonal reactivity to the reactivity of other functional groups present. For example, specific cysteines in rare sequences can be enzymatically converted in aldehydes (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). Furthermore, it is possible to perform desired amino acid modifications by utilizing specific enzymatic reactions between native amino acids in a given sequence and specific enzymes (see, for example, Taki, M. et al., Prot.Eng.Des.Sel.17(2004) 119-126; Gautier, A. et al. Chem.Biol.15(2008) 128-136; and Bordusa, F., Highlights in Bioorganic Chemistry(2004) 389-403, which utilizes protease catalysis of CN bonds). Site-specific reactions and covalent coupling can also be achieved by selectively reacting terminal amino acids with appropriate modification reagents.

[0190] Site-directed covalent coupling can also be achieved using the reactivity between the N-terminal cysteine ​​and benzonitrile (Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48(2009) 9658-9662).

[0191] Native chemical ligation can also depend on the C-terminal cysteine ​​residue (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0192] US6437095 B1 describes a binding method based on a more rapid reaction between cysteine ​​located in negatively charged amino acids and cysteine ​​located in positively charged amino acids.

[0193] The portion may be a synthetic peptide or a peptide mimetic. When polypeptides are chemically synthesized, amino acids with orthogonal chemical reactivity may be incorporated during such synthesis (see, for example, de Graaf, AJet al., Bioconjug. Chem. 20(2009) 1281-1295). A wide variety of orthogonal functional groups are of interest and can be introduced into synthetic peptides, so linking such peptides to linkers has become a standard chemical method.

[0194] To obtain single-labeled polypeptides, conjugates using a 1:1 stoichiometric ratio can be separated from other conjugate byproducts by chromatography. This procedure can be easily carried out by using dye-labeled bond pair elements and charged linkers. By using this type of labeled, strongly negatively charged bond pair element, differences in charge and molecular weight can be used for selection, thus easily separating single-bonded polypeptides from unlabeled polypeptides and polypeptides supporting multiple linkers. Fluorescent dyes may be useful for purifying complexes from unbonded components such as labeled monovalent substances.

[0195] In some embodiments, the portion attached to the anti-TREM-1 antibody is selected from the group consisting of a binding portion, a labeling portion, and a bioactive portion.

[0196] The anti-TREM-1 antibodies described herein can also be conjugated to therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA subgroove binding agents, DNA intercalators, DNA crosslinking agents, histone deacetylase inhibitors, nuclear transport inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as 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: 129), 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. Patents No. 7,087,600, 6,989,452, and 7,129,261, PCT International Patent Application Publications WO02 / 096910, WO07 / 038658, WO07 / 051081, WO07 / 059404, WO08 / 083312, and WO08 / 103693, and U.S. Patent Application Publications 20060024317, 20060004081, and 20060247295.

[0197] Anti-TREM-1 antibodies, such as those described herein, can also be used to detect human TREM-1, such as human TREM-1 in tissue or tissue samples. The antibodies can be used, for example, in ELISA assays or flow cytometry. In some embodiments, the anti-TREM-1 antibody is in contact with cells, such as cells in tissue, for a time suitable for specific binding to occur, and then a reagent, such as an antibody for detecting the anti-TREM-1 antibody, is added. Exemplary assays are presented in the Examples. The anti-TREM-1 antibody may be a fully human antibody or a chimeric antibody, such as an antibody having a human variable region and a mouse constant region or part thereof. Exemplary methods for detecting TREM-1, such as human TREM-1, in a sample (a sample of cells or tissue) include: (i) contacting the sample with an anti-TREM-1 antibody for a sufficient time to allow the anti-TREM-1 antibody to specifically bind to TREM-1 in the sample; and (2) contacting the sample with a detection reagent, such as an anti-TREM-1 antibody, or an antibody that specifically binds to the Fc region of the anti-TREM-1 antibody, thereby detecting TREM-1 bound to the anti-TREM-1 antibody. A washing step 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 labeled or bound to a detection agent; a separate detection agent can be used.

[0198] Other uses of anti-TREM-1 antibodies, such as monotherapy or combination therapy, are presented separately in this specification, for example, in the section relating to combination therapy.

[0199] IV. Bispecific molecules The anti-TREM-1 antibodies described herein can be used to form bispecific molecules. The anti-TREM-1 antibody or its antigen-binding moiety may be derivatized or conjugated to another functional molecule, such as another peptide or protein (e.g., a ligand for another antibody or receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. For example, the anti-TREM-1 antibody may be conjugated to an antibody or scFv that specifically binds to any protein that can be used as a potential target for combination therapy, such as the proteins described herein (e.g., antibodies against IP-10 or TNF-α). The antibodies described herein may, in practice, be derivatized or conjugated to multiple other functional molecules to produce a polyspecific molecule that binds to three or more different binding sites and / or target molecules. Such polyspecific molecules are also intended to be included in the term “bispecific molecule” as used herein. To generate the bispecific molecules described herein, the antibodies described herein may be functionally conjugated to one or more other conjugating molecules, such as another antibody, antibody fragment, peptide, or conjugation mimetic, so that the bispecific molecules are obtained as a result (e.g., by chemical coupling, gene fusion, non-covalent bond, or by other means).

[0200] Accordingly, this specification provides a two-specificity molecule comprising at least one first binding specificity to TREM-1 and a second binding specificity to a second target epitope. In some embodiments described herein, the two-specificity molecule is polyspecific and may further comprise a third binding specificity.

[0201] In some embodiments, the bispecific molecules described herein include binding specificity in at least one antibody or antibody fragment, such as Fab, Fab', F(ab')2, Fv, or single-chain Fv(scFv). The antibody may be a light-chain or heavy-chain dimer, or any smallest fragment such as Fv, or a single-chain construct as described in U.S. Patent No. 4,946,778 by Ladner et al.

[0202] Human monoclonal antibodies are preferred, but other antibodies that may be used in the bispecific molecules described herein include mouse monoclonal antibodies, chimeric monoclonal antibodies, and humanized monoclonal antibodies.

[0203] The two-specific molecules described herein can be prepared by linking constitutive binding specificities using methods known in the art. For example, each binding specificity of the two-specific molecules may be generated separately and then linked to one another. When the binding specificity is a protein or peptide, various coupling agents or crosslinking agents may be used for covalent bonding. Examples of crosslinking 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, Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include those described by Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Some binders are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, Illinois).

[0204] If the binding specificity is that of an antibody, they may be bound via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region is modified to contain an odd number, preferably one, sulfhydryl residue before binding.

[0205] Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is an mAb x mAb, mAb x Fab, mAb x (scFv)2, Fab x F(ab')2, or ligand x Fab fusion protein. The bispecific antibody may comprise an antibody containing scFv at the C-terminus of each heavy chain. The bispecific molecules described herein may be a single-chain molecule containing one single-chain antibody and a binding determinant, or a single-chain bispecific molecule containing two binding determinants. The bispecific molecule may comprise at least two single-chain molecules. Methods for producing bispecific molecules are described, for example, in U.S. Patents 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498, and 5,482,858.

[0206] The binding of bispecific molecules to a specific target can be confirmed using methods recognized in this field, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blotting assays. Each of these assays generally detects the presence of a specific target protein-antibody complex by employing a labeling reagent (e.g., antibody) specific to the complex in question.

[0207] V. Kit Kits comprising one or more anti-TREM-1 antibodies, their antigen-binding moieties, bispecific molecules, or immunoconjugates thereof, as described herein, are provided herein. In some embodiments, a pharmaceutical pack or kit is provided herein, comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more antibodies or their antigen-binding moieties, and optionally an instruction manual. In some embodiments, the kit contains the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein.

[0208] VI. Compositions and Formulations This specification further provides compositions (e.g., pharmaceutical compositions) and formulations comprising one or more anti-TREM-1 antibodies (including polynucleotides, vectors, and cells encoding and / or expressing anti-TREM-1 antibodies) disclosed herein. For example, in one embodiment, this disclosure provides a pharmaceutical composition comprising one or more anti-TREM-1 antibodies disclosed herein, which is formulated with a pharmaceutically acceptable carrier.

[0209] As used herein, “pharmaceutically acceptable carrier” includes all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or dermal 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 substance that protects the compound from the action of acids that can inactivate it and from other natural conditions.

[0210] Therefore, one object of this disclosure is to provide a pharmaceutical formulation that improves the stability of anti-TREM-1 antibodies and consequently allows for long-term storage. In some embodiments, the pharmaceutical formulations disclosed herein comprise (a) an anti-TREM-1 antibody, (b) a buffer, (c) a stabilizer, (d) a salt, (e) a volume extender, and / or (f) a surfactant. In some embodiments, the pharmaceutical formulation is stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years, or longer. In some embodiments, the formulation is stable when stored at 4°C, 25°C, or 40°C.

[0211] cushioning agent A buffer useful in the present invention may be a weak acid or base used to maintain the acidity (pH) of the solution close to a select value after the addition of another acid or base. A suitable buffer can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may also depend on the pH of the formulation. Common buffers, but not limited to, include histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) together with an isotonic agent and includes potential pH adjustment using acids or bases known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained at about 2 to about 10, or about 4 to about 8.

[0212] Stabilizer Stabilizers are added to pharmaceuticals to stabilize the product. Such agents can stabilize proteins in a variety of different ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine, carbohydrates such as glucose, sucrose, trehalose, raffinose, or maltose, polyols such as glycerol, mannitol, sorbitol, cyclodextrin, or dextran of any type and molecular weight, or PEG. In one aspect of the invention, the stabilizer is selected to maximize the stability of the immobilized polypeptide in a lyophilized preparation. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0213] Bulking agent Bulking agents can be added to pharmaceuticals to add volume and mass to the product, thereby facilitating accurate dosing and handling. Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate. Surfactant

[0214] Surfactants are amphiphilic substances having a hydrophilic group and a hydrophobic group. Surfactants can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxides, polypropylene oxides, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0215] In some embodiments, the pharmaceutical formulation of the disclosure contains: (a) an anti-TREM-1 antibody at about 0.25 mg / mL to 250 mg / mL (e.g., 10 to 200 mg / mL); (b) Approximately 20 mM histidine, (c) Approximately 150 mM sucrose, (d) Approximately 25 mM arginine, and (e) Approximately 50 mM NaCl.

[0216] The formulation may further comprise one or more of a buffer system, preservatives, isotonicity agents, chelating agents, stabilizers, and / or surfactants, and various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is known to those skilled in the art. Remington: The Science and Practice of Pharmacy, 19 th edition, 1995, see.

[0217] In some embodiments, the pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may include colloids, dispersions, emulsions, and multilayered substances. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0218] In some embodiments, the pharmaceutical formulation is a lyophilized formulation, and a solvent and / or diluent is added to the formulation by a physician or patient prior to use.

[0219] The pharmaceutical compositions described herein may also be administered in combination with other agents, i.e., in combination with other agents. For example, a combination therapy may include an anti-TREM-1 antibody described herein in combination with at least one other therapeutic agent. Examples of therapeutic agents that may be used in combination therapy include other compounds, drugs, and / or agents used to treat diseases or disorders (e.g., inflammatory disorders). Such compounds, drugs, and / or agents may include, for example, anti-inflammatory agents or antibodies that interfere with or reduce the production of inflammatory cytokines. In some embodiments, therapeutic agents include anti-IP-10 antibodies, anti-TNF-α antibodies (e.g., adalimumab (HUMIRA®), golimumab (SIMPONI®), infliximab (REMICADE®), certolizumab pegol (CIMZIA®)), interferon beta-1a (e.g., AVONEX®, REBIF®), interferon beta-1b (e.g., BETASERON®, EXTAVIA®), glatiramer acetate (e.g., COPAXONE®, GLATOPA®), mitoxantrone (e.g., NOVANTRONE®), nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids, and combinations thereof.

[0220] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” means a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxic effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as non-toxic organic acids such as aliphatic monocarboxylic acids and aliphatic dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanoates, aromatic acids, aliphatic sulfonic acids, and aromatic sulfonic acids. Examples of base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0221] The pharmaceutical compositions described herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfite, and sodium sulfite; (2) lipid-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0222] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained by the use of coating substances such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0223] These compositions may further contain adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial growth may be ensured by both performing the sterilization procedures described above and by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, sustained absorption of the injectable drug may be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin.

[0224] Examples of pharmaceutically acceptable carriers include sterile aqueous solutions or sterile dispersions, and sterile powders for the immediate preparation of sterile injection solutions or sterile dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is anticipated. The pharmaceutical compositions may or may not contain preservatives. Supplementary active compounds may be incorporated into the compositions.

[0225] Therapeutic compositions are typically sterile and must be stable under manufacturing and storage conditions. Compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or a suitable mixture thereof. Appropriate fluidity may be maintained by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, compositions may contain sugars such as mannitol and sorbitol, polyols, or isotonic agents such as sodium chloride. Sustained absorption of injectable compositions may be achieved by including absorption-delaying agents in the composition, such as monostearate or gelatin.

[0226] Sterile injection solutions may be prepared by mixing the required amount of the active compound in a suitable solvent with one or a combination of the components listed above, as needed, and then by sterile microfiltration. Generally, dispersions are prepared by combining the active compound with a sterile vehicle containing a basic dispersion medium and other components required from the substances listed herein. In the case of sterile powders for the preparation of sterile injection solutions, some preparation methods are vacuum drying and freeze-drying, by which powders of the active component and any additional desired components are obtained from the previously sterile-filtered solution.

[0227] The amount of active ingredient that can be mixed with a carrier material to prepare a single dosage form varies depending on the target being treated and the specific mode of administration. Generally, the amount of active ingredient that can be mixed with a carrier material to prepare a single dosage form is the amount of the composition that produces the therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01 percent to about 99 percent of the active ingredient, or, when combined with a pharmaceutically acceptable carrier, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of the active ingredient.

[0228] The drug regimen is adjusted to produce the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, or the dose may be divided into several doses administered over time, or the dose may be relatively reduced or increased as specified by the requirements of the treatment situation. For ease of administration and dose uniformity, it is particularly beneficial to formulate parenteral compositions in unit dosage forms. As used herein, a unit dosage form refers to a physically distinct unit adapted as a unit form dose for the subject being treated. Each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications of the unit dosage forms described herein are influenced by and directly depend on (a) the inherent characteristics of the active compound and the specific therapeutic effect achieved, and (b) the limitations inherent in the field of formulation of such active compounds with respect to the therapeutic sensitivity of the individual.

[0229] For example, for the administration of anti-TREM-1 antibodies described herein, the dose is in the range of approximately 0.0001 to 100 mg / kg of host body weight, more commonly 0.01 to 5 or 10 mg / kg. For example, the dose may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or in the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. An exemplary administration regimen for the anti-TREM-1 antibody described herein comprises intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight, wherein the antibody is administered using one of the following dosing schedules: (i) six doses every four weeks, followed by doses every three months; (ii) doses every three weeks; (iii) one dose of 3 mg / kg body weight, followed by 1 mg / kg body weight every three weeks.

[0230] In some embodiments, the anti-TREM-1 antibody is administered in a fixed dose (fixed dosing 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.

[0231] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within a specified range. Antibodies are usually administered multiple times. The interval between single doses may be, for example, weekly, monthly, every three months, or annually. The interval may also be irregular and is determined by measuring the blood levels of antibodies against the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of approximately 1–1000 μg / ml, while in others it is approximately 25–300 μg / ml.

[0232] Antibodies may be administered as sustained-release formulations, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies have the longest half-lives, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic applications, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for their lifetime. For therapeutic applications, relatively high doses at relatively short intervals may be required until disease progression slows or stops, and the patient shows partial or complete improvement of disease symptoms. After that, the patient may be administered a prophylactic regimen.

[0233] The actual dose levels of the active ingredients in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient effective in achieving a desirable therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dose level depends on various pharmacokinetic factors, including the activity of the particular composition described herein or its ester, salt, or amide used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health status and medical history of the patient being treated, and similar factors known in the medical field.

[0234] The compositions described herein may be administered via one or more routes of administration using one or more of the various methods known in the art. As will be appreciated by those skilled in the art, the route of administration and / or mode of administration will vary depending on the desired result. Routes of administration of the anti-TREM-1 antibodies described herein include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration by injection or infusion. As used herein, the term "parenteral administration" means a mode of administration other than enteral and topical administration and is usually effected by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intra-articular, intra-orbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subdural, intrathecal, epidural and substernal injections and infusions.

[0235] Alternatively, the antibodies described herein may potentially be administered via, for example, topical, epidermal or mucosal routes of administration, such as nasal, oral, vaginal, rectal, sublingual, or parenteral routes such as topical.

[0236] The active compounds may be prepared with carriers that protect the compound from rapid release, for example, controlled release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0237] The therapeutic compositions may be administered using medical devices known in the art. For example, in certain embodiments, the therapeutic compositions described herein may be administered using needle-free subcutaneous injection devices such as those disclosed in U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of known implants and modules for use with the anti-TREM-1 antibody described herein include: U.S. Patent No. 4,487,603, which discloses a microinfusion pump for implants for drug delivery at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a drug delivery pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implant infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0238] In some embodiments, the anti-TREM-1 antibodies described herein may be formulated to ensure appropriate distribution in vivo. For example, the blood-brain barrier (BBB) ​​is excluded if the compound is highly hydrophilic. The therapeutic compounds described herein may be formulated in liposomes to ensure reliable passage across the BBB (e.g., in brain tumors, as needed). For methods of producing liposomes, see, for example, U.S. Patents 4,522,811, 5,374,548, and 5,399,331. The liposomes may contain one or more portions that are selectively delivered to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) J.Clin.Pharmacol. 29:685). Examples of targeted sites include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 by Low et al.), mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038), antibodies (PGBloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180), surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090), and also K. Keinanen; M. Laukkanen (1994) FEBS. See also Lett.346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0239] VII. Use and Method The anti-TREM-1 antibodies of this disclosure, and compositions containing such antibodies (e.g., pharmaceutical compositions, formulations, polynucleotides, vectors, and cells), may be used to treat inflammatory diseases (e.g., by inhibiting TREM-1 activity).

[0240] Accordingly, in one embodiment, the present disclosure provides a method for treating an inflammatory disease in a target in need, comprising administering a therapeutically effective dose of anti-TREM-1 antibody to the target. Examples of inflammatory diseases that can be treated with the anti-TREM-1 antibody of this disclosure include inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type 1 diabetes mellitus, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, and other autoimmune diseases resulting from either acute or chronic inflammation.

[0241] In one embodiment, an anti-TREM-1 antibody is suitable for use in the treatment of individuals with inflammatory bowel disease (IBD). Inflammatory bowel disease (IBD) is a disease in which all parts of the digestive tract from mouth to anus can be affected, causing a variety of symptoms. IBD primarily causes abdominal pain, diarrhea (sometimes bloody), vomiting, or weight loss, but can also cause extra-gastrointestinal complications such as skin rashes, arthritis, eye inflammation, fatigue, and lack of concentration. IBD patients can be divided into two main classes: those with ulcerative colitis (UC) and those with Crohn's disease (CD). CD generally involves the ileum and colon, and all areas of the intestinal tract can be affected, but is often discontinuous (areas of concentrated affected areas spread throughout the entire intestinal tract). UC always involves the rectum (colon) and is more continuous. In CD, inflammation is transmural, resulting in abscesses, fistulas, and strictures. In UC, on the other hand, inflammation is typically limited to the mucosa. There are no known medical or surgical treatments for Crohn's disease, although some UC patients may be cured by surgical removal of the colon. Treatment options are limited to symptom control, maintenance of remission, and prevention of relapse. Clinical effectiveness in inflammatory bowel disease may be measured as a reduction in the score of Crohn's Disease Activity Index (CDAI) for CD, which is a scale based on clinical examinations and the quality of lifestyle questionnaires. In animal models, effectiveness is primarily measured by weight gain and also by the disease activity index (DAI), which is a combination of stool consistency, body weight, and bloody stools.

[0242] In one embodiment, the anti-TREM-1 antibody of this disclosure is suitable for use in the treatment of individuals with rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease affecting almost the entire body, if not the whole, and is one of the most common forms of arthritis. It is characterized by inflammation of the joints, causing pain, stiffness, heat, redness, and swelling. This inflammation is a result of inflammatory cells infiltrating the joints, which release enzymes that can digest bone and cartilage. As a result, this inflammation can cause significant damage to the bone and cartilage, leading to joint deterioration and severe pain, among other physiological effects. Affected joints may lose their shape and bite, resulting in pain and loss of movement. Several animal models of rheumatoid arthritis are known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop inflammatory arthritis similar to that of human rheumatoid arthritis. Because CIA shares similar immunological and pathological characteristics with RA, it is a suitable model for screening promising human anti-inflammatory compounds. Efficacy in this model is measured by a reduction in joint swelling. In clinical RA, efficacy is measured by the ability to reduce the patient's symptoms, which are 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.

[0243] In one embodiment, the anti-TREM-1 antibody disclosed herein is suitable for use in the treatment of individuals with psoriasis. Psoriasis is a T-cell-mediated inflammatory disorder of the skin that can cause considerable discomfort. Currently, there is no cure, and it affects people of all ages. Individuals with mild psoriasis can often control their condition with topical agents, but more than one million patients worldwide require ultraviolet light therapy or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of UV irradiation, and the toxicity of many treatments, limit their long-term use. Furthermore, patients typically experience relapses of psoriasis, sometimes rebounding immediately after discontinuation of immunosuppressive therapy. A recently developed psoriasis model based on CD4+ T-cell transplantation mimics many aspects of human psoriasis and can be used to identify compounds suitable for use in the treatment of psoriasis (Davenport et al., Internat. Immunopharmacol 2:653-672, 2002). Efficacy in this model is measured by a reduction in skin pathology using a scoring system. Similarly, effectiveness in patients is measured by a reduction in skin conditions.

[0244] In one embodiment, an anti-TREM-1 antibody is suitable for use in the treatment of individuals with psoriatic arthritis. Psoriatic arthritis (PA) is a type of inflammatory arthritis that occurs in a subgroup of psoriasis patients. In these patients, the skin pathology / symptoms involve swelling of the joints, similar to the symptoms seen in rheumatoid arthritis. It is characterized by desquamating, mottled, raised, and reddish areas of skin inflammation. Psoriasis often affects the tips of the elbows and knees, the scalp, the navel, and the genital area and around the anus. Approximately 10% of psoriasis patients also develop joint-related inflammation.

[0245] To the extent of this disclosure, prophylactic, palliative, symptomatic, and / or curative treatments may represent other aspects of this disclosure. The antibodies of the present invention can be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously. Alternatively, the antibodies of the present invention can be administered via an oral route, for example, orally or topically. The antibodies of the present invention can be administered prophylactically. The antibodies of the present invention can be administered therapeutically (as required).

[0246] The following examples are provided for illustrative purposes only and are not intended to limit them. The contents of all references cited throughout this specification are expressly incorporated herein by reference. [Examples]

[0247] Example 1: Preparation of anti-TREM-1 antibody Six cohorts of transgenic mice expressing human antibodies (each cohort containing 2-4 mice) were immunized with either recombinant TREM-1 extracellular domain, TREM-1 Jurkat cell line, or a cell membrane preparation of TREM-1 Jurkat cell line. Spleens, lymph nodes, and bone marrow were collected from the immunized mice and used to construct four immune antibody scFv (single-stranded variable fragment) libraries. Briefly, mRNA was extracted from the collected cells and reverse-transcribed to generate cDNA. Antibody variable region genes were PCR-amplified from cDNA using a primer cocktail, assembled using duplication-extension PCR, and then constructed the scFv libraries. The scFv library was expressed and selected using mRNA display (Xu L et al. (2002) Chemistry & Biology 9:933; Roberts RW and JW Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297; Kurz et al. (2000) Nucleic Acids Res. 28(18):E83). The first round was performed to enrich TREM-1 specific antibodies by selecting the mRNA-displayed scFv library against recombinant TREM-1 extracellular domain Fc fusion protein, followed by capture on protein G magnetic beads. The results of the first round were incorporated through subsequent rounds of mRNA display, and the library was split into two groups: (1) all TREM-1-bound scFv were enriched by successive selection rounds against recombinant TREM-1 extracellular domain Fc fusion protein followed by capture on protein G magnetic beads. (2) A series of selection rounds were performed on recombinant TREM-1 extracellular domain Fc fusion proteins pre-incubated with mAb170, followed by capture on protein G magnetic beads, to enrich antibodies against novel epitopes (epitope manipulation group). The sequences of the final results from both selection groups were analyzed, promising unique clones were cloned, and they were expressed as full-length immunoglobulin G (IgG). These IgG antibodies contained an IgG1.1f constant region with mutations that reduced effector function.These IgG antibodies were used in subsequent characterization and assays.

[0248] Example 2: Analysis of binding competition between epitope-modified anti-TREM-1 antibodies to human TREM-1. To analyze the functional characteristics of epitope-modified anti-TREM-1 antibodies, we evaluated their ability to inhibit the binding of mAb170 and PGRP to human TREM-1. Briefly, mAb 170 was directly labeled with AlexaFluor 647 dye using the reagent manufacturer's protocol. The antibodies tested against mAb170 were conjugated on huTREM1-expressing Jurkat cells at 4°C for 1 hour. After washing the cells, directly labeled mAb170 was added to the cells at 300 pM. After further incubation at 4°C for 30 minutes, the cells were washed and analyzed by FACS using standard methods. Unlabeled mAb 170 was used as a control for 100% inhibition.

[0249] As shown in Figures 3 and 5A, the unepitoped anti-TREM-1 antibody (black diamonds in both Figures 3 and 5A) inhibited the binding of mAb170 to TREM-1, as expected. In contrast, the epitope-modified anti-TREM-1 antibody (gray circles in both Figures 3 and 5A) failed to inhibit the binding of mAb170 to TREM-1. This result confirms that the epitope-modified anti-TREM-1 antibody binds to human TREM-1 at a different epitope than the mAb170 antibody.

[0250] The ability of epitope-modified anti-TREM-1 antibodies to inhibit the binding of PGRP to human TREM-1 was far more diverse compared to mAb170. As shown in Figure 5A, mAb170, along with the majority of non-epitope-modified antibodies, was able to efficiently inhibit the interaction between TREM-1 and its natural ligand, PGRP. However, for epitope-modified antibodies, only a small number were able to inhibit the binding of PGRP to TREM-1 with similar efficiency to mAb170 (circles in Figure 5A). For most epitope-modified antibodies, the inhibition rates ranged from approximately 90% to as low as less than 10%.

[0251] Example 3: Analysis of inhibition of THP-1 cell activation by epitope-modified anti-TREM-1 antibody To evaluate the antagonistic capabilities of epitope-modified anti-TREM-1 antibodies, their ability to inhibit the release of inflammatory cytokines from activated human cells was assessed. Briefly, human monocyte THP-1 cells were stimulated in a culture medium containing plate-bound PGRP1 and soluble peptidoglycan lacking TLR2 activity, either in the presence or absence of anti-TREM-1 antibodies (epitope-modified or non-epitope-modified).

[0252] As shown in Figure 3, most of the unepitoped anti-TREM-1 antibodies (black circles) inhibited THP-1 cell activation, with IC50 values ​​below approximately 100 nM. However, only one of the tested epitope-modified anti-TREM-1 antibodies (gray circle) had an IC50 value below 100 nM. This result appears to be consistent with the data from Example 2, which indicated that only a small number of epitope-modified antibodies were able to efficiently inhibit the binding of PGRP to human TREM-1. Figure 4 shows the amino acid sequences of the heavy chain variable region CDR3 for the anti-TREM-1 antibodies shown in Figure 3.

[0253] Example 4: Sequence analysis of epitope-modified anti-TREM-1 antibody To further characterize the epitope-modified anti-TREM-1 antibody, the human germline genes corresponding to the VH and VK regions of the antibody were determined. The sequences were then grouped according to the heavy chain V gene family and the HCDR3 sequence.

[0254] As shown in Figure 5B, the VH regions of the epitope-modified antibodies (gray circles in Figure 5A) corresponded to human germline genes 1-18, 1-69, 3-09, 3-13, 3-33, 4-59, and 5-51. The VL regions mainly corresponded to human germline genes L15, L4, L6, L10, L1, and A27. The epitope-modified antibody that most effectively inhibited PGRP binding to TREM-1 (circle in Figure 5A - see the lower right quadrant) had VH regions corresponding to human germline genes 1-69, 3-33, and 4-59, as well as VL regions corresponding to human germline genes L4 and A27.

[0255] In contrast, the epitope-unmanipulated anti-TREM-1 antibodies shown in Figure 5A (black diamonds) had VH antibodies corresponding to human germline genes 1-08 and 1-69, and VL antibodies corresponding to human germline genes L15 and L4. Of these, the antibody that best inhibited the binding of both PGRP and mAb170 to TREM-1 (see the square in Figure 5A - upper right quadrant) had VH and VL antibodies corresponding to human germline genes 1-69 and L15, respectively. For comparison, the VH and VL of mAb170 correspond to 3-73 and B3, respectively. [Table 1] TIFF0007870246000002.tif52152 [Table 2] TIFF0007870246000004.tif203155 TIFF0007870246000005.tif203155 TIFF0007870246000006.tif203155 TIFF0007870246000007.tif78148 Table 3 TIFF0007870246000009.tif153170 Table 4 TIFF0007870246000011.tif230170 TIFF0007870246000012.tif222170 TIFF0007870246000013.tif223170 TIFF0007870246000014.tif218170 TIFF0007870246000015.tif225170 TIFF0007870246000016.tif78170 Table 5 Table 6 TIFF0007870246000019.tif186170

Claims

1. An isolated antibody that specifically binds to triggering receptor expressed on myeloid cells-1 (TREM-1) and comprises a heavy chain variable region (VH) and a light chain variable region (VL), with heavy chains CDR1, CDR2, and CDR3 in the VH, and light chains CDR1, CDR2, and CDR3 in the VL, (a) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 26, 27, and 28, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 29, 30, and 31, respectively. (b) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 32, 33, and 34, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 37, respectively. (c) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 32, 33, and 34, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 29, 30, and 38, respectively. (d) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 45, 46, and 47, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 48, and 49, respectively. (e) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 50, 51, and 52, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 37, respectively. (f) The heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 136, 137, and 138, respectively, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs. 35, 36, and 139, respectively, or (g) The antibody wherein the heavy chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs: 136, 137, and 138, and the light chains CDR1, CDR2, and CDR3 each contain the amino acid sequences described in SEQ ID NOs: 35, 36, and 103.

2. (a) The VH comprises an amino acid sequence that is 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 described as SEQ ID NO: 13, 15, 23, 25, or 130, and / or (b) The antibody according to claim 1, wherein the VL comprises an amino acid sequence that is 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 described as SEQ ID NO: 14, 16, 17, 24, 131, or 132.

3. (a) The VH includes sequence number 13, and the VL includes sequence number 14. (b) The VH includes sequence number 15, and the VL includes sequence number 16. (c) The VH includes sequence number 15, and the VL includes sequence number 17. (d) The VH includes sequence number 23, and the VL includes sequence number 24. (e) The VH includes sequence number 25, and the VL includes sequence number 16. (f) The VH includes sequence number 130, and the VL includes sequence number 131, or (g) The VH includes sequence number 130, and the VL includes sequence number 132. The antibody according to claim 1 or 2.

4. Further comprising a heavy chain (HC) steady region and a light chain (LC) steady region, (a) The HC constant region includes an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 123, SEQ ID NO: 122, SEQ ID NO: 124, or SEQ ID NO: 125, and / or (b) The antibody according to any one of claims 1 to 3, wherein the LC constant region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

126.

5. A bispecific molecule comprising the antibody according to any one of claims 1 to 4, bound to a molecule having a second binding specificity.

6. A nucleic acid encoding the antibody according to any one of claims 1 to 4.

7. A vector comprising the nucleic acid described in claim 6.

8. A cell containing the vector described in claim 7.

9. An immunoconjugate comprising an antibody according to any one of claims 1 to 4 or a bispecific molecule according to claim 5, conjugated to a reagent.

10. A composition comprising an antibody according to any one of claims 1 to 4, a bispecific molecule according to claim 5, a nucleic acid according to claim 6, a vector according to claim 7, a cell according to claim 8, or an immunoconjugate according to claim 9, and a carrier.

11. A kit comprising an antibody according to any one of claims 1 to 4, a bispecific molecule according to claim 5, a nucleic acid according to claim 6, a vector according to claim 7, a cell according to claim 8, or an immunoconjugate according to claim 9, and instructions for use.

12. A pharmaceutical composition for inhibiting TREM-1 activity in a target, comprising an antibody according to any one of claims 1 to 4, a bispecific molecule according to claim 5, a nucleic acid according to claim 6, a vector according to claim 7, a cell according to claim 8, or an immunoconjugate according to claim 9.

13. In the subject, a pharmaceutical composition for treating inflammatory diseases or autoimmune diseases comprises an antibody according to any one of claims 1 to 4, a bispecific molecule according to claim 5, a nucleic acid according to claim 6, a vector according to claim 7, a cell according to claim 8, or an immunoconjugate according to claim 9, wherein the inflammatory disease or autoimmune disease is inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, vasculitis, sepsis, systemic A pharmaceutical composition selected from the group consisting of inflammatory response syndrome (SIRS), type 1 diabetes mellitus, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma, other autoimmune diseases resulting from either acute or chronic inflammation, and any combination thereof.

14. The pharmaceutical composition according to claim 12 or 13, further comprising one or more additional therapeutic agents.

15. The pharmaceutical composition according to claim 14, wherein the one or more additional therapeutic agents are anti-IP-10 antibodies or anti-TNF-α antibodies.

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