BR112016030947B1 - ANTIBODY OR FRAGMENT THEREOF THAT IS CAPABLE OF BINDING AND BLOCKING TREM-1 AND ITS USES - Google Patents

Abstract

ANTIBODY OR FRAGMENT THEREOF THAT IS CAPABLE OF BINDING TREM-1, PHARMACEUTICAL COMPOSITION CONTAINING IT AND ITS USES. The present invention relates to antibodies that are capable of specifically binding and preventing the activation of TREM-1, a protein expressed in monocytes, macrophages and neutrophils with good affinity and low viscosity at clinically relevant concentrations. Such antibodies find use in treating individuals with an inflammatory disease, such as rheumatoid arthritis and inflammatory bowel disease.

Description

TECHNICAL FIELD

[001] The present invention relates to TREM-1 mAbs and the mutation of specifically charged and uncharged negative amino acids involved in both TREM-1 mAb self-interactions and TREM-1 mAb interactions with TREM -1 in order to decrease the viscosity of the mAb solution and maintain target affinity and the present invention relates to uses for such antibodies for therapeutic and pharmaceutical uses.

SEQUENCE LISTINGS OF THE PRESENT INVENTION

[002] SEQ ID NO: 1 represents the amino acid sequence of wild-type human TREM-1 (by weight).

[003] SEQ ID NO: 2 represents the amino acid sequence of the heavy chain of a humanized TREM-1 antibody (mAb 0170 of WO2013/120553).

[004] SEQ ID NO: 3 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0170 of WO2013/120553).

[005] SEQ ID NO: 4 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0317, E27Q, E97S).

[006] SEQ ID NO: 5 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0318, E27Q, E97Q).

[007] SEQ ID NO: 6 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0319, E97S).

[008] SEQ ID NO: 7 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0320, E97Q).

[009] SEQ ID NO: 8 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0321, E27Q).

[0010] SEQ ID NO: 9 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0322, F32A).

[0011] SEQ ID NO: 10 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0323, F32S).

[0012] SEQ ID NO: 11 represents the amino acid sequence of the heavy chain of a humanized TREM-1 antibody (mAb 0324, A59Y).

[0013] SEQ ID NO: 12 represents the amino acid sequence of the heavy chain of a humanized TREM-1 antibody (mAb 0325, N57S).

[0014] SEQ ID NO: 13 represents the amino acid sequence of the heavy chain of a humanized TREM-1 antibody (mAb 0326, A59Y, N57S).

[0015] SEQ ID NO: 14 represents the amino acid sequence of the light chain of a humanized TREM-1 antibody (mAb 0330, F32A, E27Q, E97Q).

[0016] SEQ ID NO: 15 represents the amino acid sequence of the heavy chain of a humanized TREM-1 antibody (mAb 0332, A59Y; 0332 is a combined mAb of SEQ ID NO: 15 as HC and SEQ ID NO 5 as LC , Table 1).

[0017] SEQ ID NO: 16 represents the amino acid sequence of the heavy chain of a humanized TREM-1 antibody (mAb 0333, A59Y; 0333 is a combined mAb of SEQ ID NO: 16 as HC and SEQ ID NO 14 as LC , Table 1).

[0018] SEQ ID NO: 17 represents the full-length cTREM-1 amino acid sequence.

[0019] SEQ ID NO: 18 represents the heavy chain of the Fab region of mAb 0170.

[0020] SEQ ID NO 19 represents the light chain of the Fab region of mAb 0170.

BACKGROUND

[0021] TREM-1 is an activating receptor expressed on monocytes, macrophages and neutrophils. These cells play a central role in chronic inflammatory diseases, releasing cytokines and other mediators that drive inflammation. TREM-1 mRNA and protein expression is upregulated in patients with RA and IBD, and TREM-1-positive cells accumulate at sites of inflammation, correlating with disease severity. The peptidoglycan recognition protein 1 (PGLYRP1) expressed primarily on activated neutrophils is a ligand for TREM-1 and mediates TREM-1 signaling upon binding.

[0022] In vitro, TREM-1 engagement triggers the secretion of pro-inflammatory cytokines including TNF, IL-8 and monocyte chemotactic protein-1. Furthermore, TREM-1 signaling synergizes with multiple Toll-Like Receptors (TLR) to further enhance pro-inflammatory signals. In turn, this upregulates the expression of TREM-1, leading to a vicious cycle amplifying inflammation. Increasing evidence indicates that TLRs contribute to the development and progression of chronic inflammatory diseases such as RA and IBD.

[0023] WO 2013/120553 describes humanized anti-TREM-1 mAbs that inhibit both human and cynomolgus TREM-1 function. However, the viscosity profile of anti-TREM-1 mAbs may complicate the manufacturing process to produce a drug with a value >50 mg/mL and may limit the definition of an optimal dose in the clinic. A high dosage (several mg/kg) of protein therapeutics is often required to achieve an adequate clinical effect and since the vast majority of these therapeutics are administered via subcutaneous administration, the consequence is that self-administration of the therapeutic by the patient is limited to volumes of < 1.5 mL (Shire et al., J. Pharm. Sci. 2004, 93, 1390 to 1402). The development of high concentration protein formulations suitable for patient self-administration is a general obstacle to production and distribution when the protein formulation results in high viscosity of the resulting solution.

[0024] The mAb charge distribution has been studied with regard to the effect on the viscosity behavior of mAb solutions (Ydav et al, Mol. Pharmaceutics 2012, 9, 791 to 802). Weak non-specific charge interactions that persist in dilute solutions have also been shown to influence the viscosity of concentrated mAb solutions (Connolly et al., Biophys, J., 2012, 103, 69 to 78). Remedies for reducing the viscosity of mAb solutions have been to introduce site-directed charge exchange mutations that disrupt direct intermolecular charge-charge interactions (Ydav et al., Mol. Pharmaceutics 2012, 9, 791 to 802) or the addition of salts or counterions (Liu et al., J. Pharm. Sci. 2005, 94, 1928 to 1940, Yadav et al., J. Pharm. Sci. 2010, 99, 1152 to 1168, Yadav et al ., J. Pharm. 2012, 101, 998 to 1011, Kanai et al., J. Pharm. 2008, 97, 4219 to 4227). The addition of salts and counterions may, however, result in adverse effects for the patient in terms of hyperosmolality of the administered solution.

[0025] The antibodies described in the present invention are TREM-1 antibodies generated through site-specific mutation of the CDRs of humanized anti-TREM-1 mAb 0170 described in WO 2013/120553. The described antibodies do not disrupt direct mAb charge-charge intermolecular self-interactions but have a favorable viscosity profile and maintain the target binding profile. The favorable viscosity profile allows the drug product to be produced in high concentrations that may be essential for therapeutic and pharmaceutical use. Such antibodies can have a substantial impact on the quality of life of individuals with sepsis or a chronic inflammatory disease such as rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease.

SUMMARY

[0026] A primary aspect of the present invention relates to an anti-TREM-1 mAb with a viscosity profile in the range expected for monomeric mAbs and which blocks TREM-1 function as potently as mAb0170 of WO 2013/120553. In addition to the advantageous viscosity profiles of the variant mAbs of the present invention, said variants do not exhibit agonism for the TREM-1 receptor.

[0027] One aspect of the present invention is directed to an antibody or fragment thereof that is capable of binding and blocking TREM-1, characterized by the antibody, or an antibody fragment of said antibody, having a viscosity of less than 5 cP in a concentration of 80 mg/mL, preferably less than 4 cP.

[0028] The antibody or fragment thereof may comprise a variant of SEQ ID NO: 3, in which one or more of the negatively charged residues in the CDR1 and CDR3 region of SEQ ID NO: 3 are replaced with amino acid residues uncharged, as described in the present invention.

[0029] The antibody or fragment thereof may comprise a variant of SEQ ID NO: 3 in which the phenylalanine at position 32 of SEQ ID NO 3 is mutated to an amino acid selected from the amino acid residues glycine, serine, threonine, cysteine, alanine, valine, leucine, isoleucine and methionine, preferably selected from alanine, glycine, serine valine and leucine, more preferably in which F32 of SEQ ID NO: 3 is mutated to alanine or serine.

[0030] The antibody or fragment thereof may comprise a variant of SEQ ID NO: 2 in which any of residues Y32, R52, S55, S56, N57, A59, M102, I104 and R106 of SEQ ID NO: 2 or F32, D33, Y34, Y53, R54, D98 of SEQ. ID NO 3 ("Fab-Fab interaction mutations") is replaced by another amino acid, such as a natural amino acid, preferably an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine.

[0031] Another aspect of the present invention is directed to an antibody or fragment thereof that is capable of specifically binding and blocking TREM-1 of SEQ ID NO: 1 and comprises the variants of SEQ ID NO: 2 or SEQ ID NO: 3 or both, wherein the variants are selected from the group consisting of Fab-Fab interacting mutations, Fab-TREM-1 interacting mutations, and "cargo parcel" mutations of SEQ ID NO: 2 or SEQ ID NO: 3 (table 1).

[0032] In one embodiment of an antibody or fragment thereof of the present invention, one or more of the negatively charged residues in the CDR1 and CDR3 regions of SEQ ID NO: 3 are replaced with uncharged amino acid residues. In one embodiment, the negatively charged residues in the CDR1 and CDR3 regions of SEQ ID NO: 3 are replaced with uncharged amino acid residues.

[0033] In one embodiment, an antibody or fragment thereof of the present invention comprises a variant of SEQ ID NO 3 in which any of the "charge portion" residues D1, D30, D33, D74, D98, E27, E97 of SEQ ID NO: 3 is mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine. In one embodiment, more than one of the "charge share" residues D1, D30, D33, D74, D98, E27, E97 of SEQ ID NO 3 is mutated to an amino acid residue selected from the group consisting of glycine , alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine.

[0034] In one embodiment, one of E27 and E97 of the CDR1 and CDR3 regions of SEQ ID NO 3 is replaced by an uncharged amino acid residue, such as an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine. By way of example, E27 of SEQ ID NO 3 may remain unmutated and E97 may be mutated with an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine, plus preferably with an amino acid selected from the group consisting of serine and glutamine. Alternatively, E97 of SEQ ID NO 3 may remain unmutated and E27 may be replaced by an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine, more preferably with an amino acid selected from the group consisting of serine and glutamine.

[0035] In one embodiment, both E27 and E97 of the CDR1 and CDR3 regions of SEQ ID NO 3 are replaced by uncharged amino acid residues, such as an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine. By way of example, E27 of SEQ ID NO 3 can be mutated to glutamine and E97 of SEQ ID NO 3 can be replaced by an amino acid selected from the group consisting of glycine, serine, asparagine, glutamine, threonine, cysteine and tyrosine , more preferably with an amino acid selected from the group consisting of serine and glutamine.

[0036] In another aspect, the present invention provides an antibody or a fragment thereof that comprises a variant of SEQ. ID. NO2 wherein any of residues Y32, R52, S55, S56, N57, A59, M102, I104 and R106 of SEQ ID NO: 2 or F32, D33, Y34, Y53, R54, D98 of SEQ ID NO: 3 (" Fab-Fab interaction mutations") is replaced by another amino acid, such as a natural amino acid, preferably an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, Histidine and tyrosine.

[0037] In one embodiment of the antibody or fragment thereof of the present invention, at least one of residues A59 and N57 of SEQ ID NO: 2 is mutated to an amino acid residue, such as a natural amino acid, preferably a selected amino acid from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine, more preferably serine or tyrosine. By way of example, A59 of SEQ ID NO: 2 may remain unmutated and N57 may be mutated to another amino acid residue, such as a natural amino acid, preferably selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine, more preferably serine or tyrosine. Alternatively, N57 of SEQ ID NO: 2 may remain unmutated and A59 of SEQ ID NO 2 may be mutated to another amino acid residue, such as a natural amino acid, preferably selected from the group consisting of glycine , alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine, more preferably serine or tyrosine. In another embodiment, both A59 and N57 of SEQ ID NO: 2 are mutated to another amino acid residue, such as a natural amino acid, preferably selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine, more preferably serine or tyrosine.

[0038] Another aspect of the present invention is directed towards an improved preclinical evaluation value obtained by introducing site-directed mutations in SEQ ID NO: 3 in the amino acid residues of the "Fab-TREM-1 interaction" to achieve the same mAb affinity for cynomolmous TREM-1 as for human TREM-1 of SEQ ID NO: 1, such as where a Phe residue of SEQ ID NO: 3 is replaced by Ala or Ser.

[0039] The present invention thus provides an antibody or a fragment thereof in which the "Fab-TREM-1 interaction" amino acid residue, F32 of SEQ ID NO: 3, is replaced by an amino acid residue selected from the group consisting of glycine, alanine, serine, threonine, proline and cysteine.

[0040] Another aspect of the present invention is directed to a combined effect of improved viscosity properties and improved preclinical evaluation value obtained through the introduction of site-directed mutations in SEQ ID NO: 2 at the mAb-TREM-interface 1 to achieve the same mAb affinity for (e.g., as described above) in combination with replacing negatively charged residues in the CDR1 and CDR3 regions of SEQ ID NO: 3 with uncharged amino acid residues (e.g. , as described above) .

[0041] In accordance with any of the above-described aspects of the present invention, there is provided an antibody or a fragment thereof in which: I) at least one negatively charged residue of the CDR1 and CDR3 regions of SEQ ID NO: 3 is mutated for an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine; and II) at least one residue of residues Y32, R52, S55, S56, N57, A59, M102, I104 and R106 of SEQ ID NO: 2 or F32, D33, Y34, Y53, R54, D98 of SEQ ID NO: 3 Fab-Fab interaction mutations are mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine and tryptophan, histidine, and tyrosine.

[0042] In one embodiment, an antibody or a fragment thereof is provided, in which: III) one or both E27 and E97 of SEQ ID NO: 3 are mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine; and IV) one or both of A59 and N57 of SEQ ID NO: 2 are mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan , histidine and tyrosine, more preferably serine or tyrosine.

[0043] The present invention further relates to an antibody or fragment thereof that comprises any one of SEQ ID NOs: 4 to 16.

Brief description of the drawings

[0044] Figure 1 illustrates the viscosity profile of the anti-TREM-1 mAb variants and their exponential curve fits.

[0045] Figure 2 illustrates the ability of mAb 0170 variants to inhibit human TREM-1 signaling in the human TREM-1 reporter cell line (BWZ36/hTREM-1) stimulated with PGN and (A) recombinant PGLYRP1 or (B ) PGLYRP1 expressed by activated neutrophils (average of N donors).

[0046] Figure 3 illustrates the ability of mAb 0170 variants to inhibit cynomolgous TREM-1 signaling in the cynomolgous TREM-1 reporter cell line (TE426.27) stimulated with PGN and recombinant PGLYRP1.

[0047] Figure 4 illustrates the ability of mAb 0170 variants to inhibit the release of TNFa from hypoxic M2 macrophages stimulated with PGN and recombinant PGLYRP1.

[0048] Figure 5 illustrates the agonistic potential of plaque-bound mAb 0170 variants to induce TNF release from hypoxic M2 macrophages. MAb 1278 (R&D) was used as a positive control.

[0049] Figure 6 represents the hydrodynamic radius of the anti-TREM-1 mAb variants compared to mAb 0170.

DESCRIPTION

[0050] TREM-1 is a transmembrane protein consisting of 234 amino acids, including a single extracellular immunoglobulin domain and a short cytoplasmic tail with no apparent signaling motif. When activated, TREM-1 associates with the ITAM-containing signaling adapter protein, DAP12. Downstream signaling may include activation of the transcription factor NFAT, triggering upregulation of pro-inflammatory cytokine production.

[0051] The present invention relates to antibodies that are capable of specifically binding and blocking the function of TREM-1. The antibodies of the present invention can block TREM-1 function by reducing/blocking TREM-1 activation and downstream signaling.

[0052] Antibodies according to the present invention can block TREM-1 through one or a combination of several different mechanisms, blocking TREM-1 directly or indirectly. For example, antibodies of the present invention can prevent the natural TREM-1 ligand, peptidoglycan recognition protein 1 (PGLYRP1), from creating a functional complex with TREM-1 and/or antibodies of the present invention can block TREM-1 by preventing Individual TREM-1 to form dimers or multimers. TREM-1 dimerization or multimerization can be reduced or prevented by TREM-1 antibodies that are capable of binding to a portion of TREM-1 that would otherwise reside at the interface of a TREM-1 dimer, thereby preventing individual molecules from of TREM-1 are associated with each other.

[0053] TREM-1 dimerization or multimerization can be reduced or prevented by TREM-1 antibodies that interfere with the interaction of TREM-1 with its ligand. Antibodies according to the present invention can block PGLYRP1-induced TREM-1 activation. PGLYRP1, a highly conserved protein of 196 amino acids in length consisting of a signal peptide and a peptidoglycan-binding domain, is expressed in neutrophils and released upon activation. Antibodies according to the present invention can downregulate the release of pro-inflammatory cytokines from myeloid cells. The antibodies according to the present invention can block the release of TNF, MIP-1beta, MCP-1, IL-1beta, GM.CSF, IL-6 and/or IL-8 from macrophages, neutrophils, tissue cells synovial and/or Reporter, as described in the present invention.

[0054] The antibodies of the present invention may be capable of binding both human TREM-1 and TREM-1 from a species other than a human. The term "TREM-1", as used herein, thus encompasses any natural form of TREM-1 that can be derived from any suitable organism. For example, TREM-1 for use as described in the present invention may be vertebrate TREM-1, such as mammalian TREM-1, such as TREM-1 from a primate (such as a human, a chimpanzee, a cynomolgus monkey or a rhesus monkey), a rodent (such as a mouse or a rat), a lagomorph (such as a rabbit), or an artiodactyl (such as a cow, sheep, pig or camel). Preferably, TREM-1 is SEQ ID NO: 1 (human TREM-1). TREM-1 may be a mature form of TREM-1 such as a TREM-1 protein that has undergone post-translational processing within a suitable cell. Such a mature TREM-1 protein may, for example, be glycosylated. TREM-1 may be a full-length TREM-1 protein.

[0055] The antibodies of the present invention can be monoclonal antibodies, in the sense that they are derived directly or indirectly from a single clone of a B lymphocyte. TREM-1 antibodies can be produced, screened and purified using, for example, the methods described in the Examples of WO2013/120553. In summary, a suitable mouse such as a TREM-1 or TREM-1/TREM-3 knockout (KO) mouse can be immunized with TREM-1, a cell expressing TREM-1, or a combination of both.

[0056] The antibodies of the present invention may be polyclonal in the sense of being a mixture of monoclonal antibodies according to the present invention.

[0057] Primary screening of hybridoma supernatants can be performed using direct ELISA or FMAT and secondary screening can be performed using flow cytometry. Positive hybridoma supernatants can then be screened in a reporter gene assay.

[0058] Antibodies can be expressed recombinantly in prokaryotic or eukaryotic cells. The prokaryotic cell could be E. coli. The eukaryotic cell may be a yeast, insect, or mammalian cell, such as a cell derived from an organism that is a primate (such as a human, chimpanzee, cynomolgus monkey, or rhesus monkey), a rodent (such as a mouse or rat), a lagomorph (such as a rabbit) or an artiodactyl (such as a cow, sheep, pig or camel). Suitable mammalian cell lines include, but are not limited to, HEK293 cells, CHO cells, and HELA cells. TREM-1 antibodies can also be produced by other known methods by a person skilled in the art, such as a phage display or a yeast display.

[0059] Once produced, antibodies can be screened for binding to, for example, full-length TREM-1 or mutants thereof using the methods described in the Examples of WO2013/120553.

[0060] The functional TREM-1 antibodies of the present invention are antibodies that are capable of specifically binding to TREM-1 and that have an effect on TREM-1 activation and downstream signaling by blocking or stimulating TREM-1 and are referred to in the present invention as "TREM-1". The method of identifying a functional TREM-1 antibody comprises (a) culturing a first cell that expresses TREM-1, a signaling protein and a reporter construct; (B) measuring the activity of the first cell when said cell is incubated with a TREM-1 modifying agent; (C) contacting the co-culture of (b) with a TREM-1 antibody; and (d) measuring that the activity of the first cell is lower or higher than the activity measured in (b).

[0061] The "first cell" of (a) may be a cell of hematopoietic origin, such as a myeloid cell, such as a T cell. The signaling protein of (a) may be any signaling protein that is capable of forming a complex with TREM-1. Suitable signaling proteins include DAP10, DAP12, TCR zeta, Fc gamma RIII and an Fc receptor, or part thereof. The reporter construct of (a) can be any construct that is capable of being activated via the signaling protein and generating a recognizable signal. Suitable reporter constructs comprise a transcription factor and a reporter gene. The signaling protein can signal through a transcription factor selected from the group consisting of NFAT and NFkB. The reporter gene is a gene that is not expressed natively in said first cell and may be but is not limited to being a gene encoding p-galactosidase, luciferase, green fluorescent protein (GFP) or chloramphenicol transferase. Said first cell can be transfected with a transcription factor and a reporter gene using methods that are well known in the art.

[0062] The "BWZ/hTREM-1 reporter cell" and the "TE426.27 reporter cell" described in the Examples is an example of a "first cell".

[0063] The modifying agent of (b) can be a TREM-1 ligand or an activated neutrophil. The "TREM-1 antibody" of (c) may be a TREM-1-specific hybridoma supernatant or a purified antibody. The activity measured in (d) is the signal produced through the reporter construct. An example of such signaling is the luminescence caused through NFAT-induced LacZ (β-lactamase luciferase) production.

[0064] The method can be adapted to identify a blocking TREM-1 antibody. The method of identifying a blocking TREM-1 antibody comprises (a) culturing a first cell that expresses TREM-1, a signaling protein and a reporter construct; (B) measuring the activity of the first cell when said cell is incubated with an activated neutrophil; (C) contacting the coculture of the first cell and the activated neutrophil with a TREM-1 antibody; And (d) measure that the activity of the first cell is lower than the activity measured in (b).

[0065] The method can also be adapted to identify a TREM-1 stimulating antibody. The method of identifying a stimulating TREM-1 antibody comprises (a) culturing a first cell that expresses TREM-1, a signaling protein and a reporter construct; (B) measure the activity of the first cell; (C) contact/incubation of said cell with a TREM-1 antibody; And (d) measure that the activity of the first cell is more than the activity measured in (b).

[0066] The present invention relates to blocking TREM-1 antibodies that can be identified using the method described in the present invention, of identifying a blocking antibody. When tested using the method described above and in the Examples, an antibody according to the present invention can, at a concentration of less than 50 μg/mL, such as less than 40 μg/mL, such as less than 30 μg/mL, such as such as less than 20 μg/mL, such as less than 10 μg/mL, such as less than 5 μg/mL, such as less than 1 μg/mL, are capable of reducing by 50% the activity of said first cell, such like 60%, like 70%, like 80%, like 90%, like 95%, like 100%. An antibody according to the present invention may be capable of completely quenching the activity of the first cell. When tested using the method described above and in the Examples, an antibody according to the present invention can, at a concentration of less than 1 μg/mL - such as less than 0.9 μg/mL, such as less than 0.8 μg/mL, Such as less than 0.6 μg/mL, such as less than 0.6 μg/mL, such as less than 0.3 μg/mL, such as less than 0.3 μg/mL mL, such as less than 0.2 μg/mg/mL - be able to extinguish the activity of the first cell.

[0067] The present invention also relates to blocking TREM-1 antibodies that can be identified by means other than the method described in the present invention.

[0068] The term "antibody" refers in the present invention to a protein, derived from a germline immunoglobulin sequence, which is capable of specifically binding to an antigen (TREM-1) or to a portion of the same. The term includes full-length antibodies of any class or isotype (i.e., IgA, IgE, IgG, IgM and/or IgY) and any single chain or fragment thereof. An antibody that specifically binds to an antigen, or portion thereof, may bind exclusively to that antigen, or portion thereof, or may bind to a limited number of homologous antigens or portions thereof. Full-length antibodies typically comprise at least four polypeptide chains: two heavy (H) chains and two light (L) chains that are interconnected through disulfide bonds. One immunoglobulin subclass of particular pharmaceutical interest is the IgG family. In humans, the IgG class can be subdivided into 4 subclasses: IgG1, IgG2, IgG3 and IgG4, based on the sequence of their heavy chain constant regions. Light chains can be divided into two types, kappa and lambda, based on differences in their sequence composition. IgG molecules are composed of two heavy chains, linked through two or more disulfide bonds, and two light chains, each linked to a heavy chain through a disulfide bond. A heavy chain may comprise a heavy chain variable region (VH) and up to three heavy chain constant regions (CH): CH1, CH2 and CH3. A light chain may comprise a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called structural regions (FR). The VH and VL regions are typically composed of three CDRs and four FRs, arranged from amino-terminal to carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy and light chains form a binding domain that is capable of interacting with an antigen, while the constant region of an antibody can mediate binding of the immunoglobulin to host tissues or factors, including but not limited to various cells of the immune system (effector cells), Fc receptors and the first component (Clq) of the classical complement system.

[0069] The antibodies of the present invention can be isolated. The term "isolated antibody" refers to an antibody that has been separated and/or recovered from (one) other component(s) in the environment in which it was produced and/or that has been purified from a mixture of components present in the environment in which it was produced.

[0070] Certain antigen-binding antibody fragments may be suitable in the context of the present invention, since it has been demonstrated that the antigen-binding function of an antibody can be carried out through the fragments of a full-length antibody. The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, such as TREM-1, as described in the present invention. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)S, Fv (typically the VL and VH domains of a single arm of an antibody) (Eng. example, Bird et al., Science 1988, 242: 42S-426 and Huston et al., PNAS 1988; 85: 5879 to 5883), dsFv, Fd (typically the VH and CHI domain) and dAb Fragments (typically a VH domain) ; VH, VL, VhH and V-NAR domains; Monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies and kappa bodies (see, for example, Ill et al., Protein Eng 1997; 10: 949 to 57); camel IgG; IgNAR; As well as one or more isolated CDRs or a functional paratope, wherein the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together to form a functional antibody fragment. Various types of antibody fragments have been described or analyzed in, for example, Holliger and Hudson, Nat Biotechnol 2005; 2S: 1126-1136; WO 2005040219 and published U.S. patent applications 20050238646 and 20020161201. These antibody fragments can be obtained using conventional techniques known to those skilled in the art and the fragments can be screened for utility in the same manner as intact antibodies.

[0071] An antibody of the present invention can be a human antibody or a humanized antibody. The term "human antibody" as used herein is intended to include antibodies having variable regions in which at least a portion of a framework region and/or at least a portion of a CDR region are derived from sequences of human germline immunoglobulin. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences (e.g., a human antibody may have variable regions in which both the framework and CDR regions are derived from germline immunoglobulin sequences). human germline immunoglobulin). The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutation in vivo). .

[0072] Such a human antibody can be a human monoclonal antibody. Such a human monoclonal antibody may be produced by a hybridoma, which includes a B cell obtained from a transgenic non-human animal, for example, a transgenic mouse, having a genome comprising a human heavy chain transgene and a human heavy chain transgene. lightweight fused to an immortalized cell.

[0073] Human antibodies can be isolated from sequence libraries built on selections of human germline sequences, further diversified with diversity of natural and synthetic sequences.

[0074] Human antibodies can be prepared through in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.

[0075] The term "human antibody derivative" refers to any modified form of the human antibody, such as a conjugate of the antibody and another agent or antibody.

[0076] The term "humanized antibody", as used in the present invention, refers to a human/non-human chimeric antibody that contains one or more sequences (CDR regions or parts thereof) that are derived from a non-human immunoglobulin. A humanized antibody is thus a human immunoglobulin (recipient antibody) in which at least the residues of a hypervariable region of the receptor are replaced by residues of a hypervariable region of an antibody of a non-human species (donor antibody) such as a rat, mouse, rabbit or non-human primate, which has the desired specificity, affinity, sequence composition and functionality. In some cases, the FR residues of human immunoglobulin are replaced with corresponding non-human residues. An example of such a modification is the introduction of one or more so-called secondary mutations, which are typically amino acid residues derived from the donor antibody. Humanization of an antibody can be carried out using known recombinant techniques by a person skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. C. Lo). A suitable human receptor structure for the light chain and heavy chain variable domain can be identified by, for example, sequence or structural homology. In an alternative way, fixed receptor frames can be used, for example, based on knowledge of the structure, biophysical and biochemical properties. Receptor structures can be germline derived or derived from a mature antibody sequence. The CDR regions of the donor antibody can be transferred by CDR grafting. The CDR-grafted humanized antibody can be further optimized for affinity, functionality, and biophysical properties by identifying critical framework positions where reintroduction (backmutation) of the amino acid residue of the donor antibody has a beneficial impact on the properties of the humanized antibody. In addition to backmutation derived from donor antibodies, the humanized antibody can be modified through the introduction of germline residues into the CDR or framework regions, elimination of immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0077] Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications are made to further refine the performance of the antibody. In general, a humanized antibody will comprise at least one typically two-variable domain, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and wherein all or substantially all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0078] The term "humanized antibody derivative" refers to any modified form of the humanized antibody, such as a conjugate of the antibody and another agent or antibody.

[0079] The term "chimeric antibody", as used in the present invention, refers to an antibody whose light and heavy chain genes have been constructed, typically through genetic engineering, from variable and constant region genes of immunoglobulin originating from different species. For example, the variable gene segments of a mouse monoclonal antibody can be joined to human constant segments.

[0080] The crystallizable region of the fragment ("Fc region"/"Fc domain") of an antibody is the N-terminal region of an antibody, which comprises the constant CH2 and CH3 domains. The Fc domain can interact with cell surface receptors called Fc receptors, as well as some complement system proteins. The Fc region allows antibodies to interact with the immune system. In one aspect of the present invention, antibodies can be modified to include modifications within the Fc region, typically to alter one or more of their functional properties, such as serum half-life, complement fixation, Fc receptor binding, stability of the protein and/or antigen-dependent cellular cytotoxicity, or its absence, among others. Furthermore, an antibody of the present invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. An IgG1 antibody may carry a modified Fc domain comprising one or more, and perhaps all of the following mutations that will result in decreased affinity for certain Fc receptors (L234A, L235E, and G237A) and reduced C1q-mediated complement fixation (A330S and P331S), respectively (waste numbering according to the EU index).

[0081] The isotype of an antibody of the present invention may be IgG, such as IgG1, such as IgG2, such as IgG4. If desired, the class of an antibody can be "switched" using known techniques. For example, an antibody that was originally produced as an IgM molecule can be class-switched to an IgG antibody. Class switching techniques can also be used to convert one IgG subclass to another, for example: from IgG1 to IgG2 or IgG4; From IgG2 to IgG1 or IgG4; Or from IgG4 to IgG1 or IgG2. Antibody engineering to generate constant region chimeric molecules by combining regions from different IgG subclasses can also be performed.

[0082] In one embodiment, the CH1 hinge region is modified such that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. This approach is further described for example in U.S. Patent No. 5,677,425 to Bodmer et al.

[0083] The constant region can be modified to stabilize the antibody, for example, to reduce the risk of a bivalent antibody separating into two monovalent VH-VL fragments. For example, in a constant region of IgG4, residue S228 (number of residues according to the EU index) can be mutated to a proline residue (P) to stabilize heavy chain disulfide bond formation at the hinge ( see, for example, Angal et al., Mol. Immunol. 1995;

[0084] Antibodies or fragments thereof can also be defined in terms of their complementarity determining regions (CDR). The term "complementarity determining region" or "hypervariable region", when used in the present invention, refers to the regions of an antibody in which the amino acid residues involved in binding to the antigen are located. The hypervariability region or CDRs can be identified as the regions with greatest variability in the amino acid alignments of antibody variable domains. Databases can be used for CDR identification, such as the Kabat database, the CDR, for example, being defined as comprising amino acid residues 24-34 (L1), 50-59 (L2) and 89- 97 (L3) of the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95 -102 (H3) in the heavy chain variable domain; (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) Alternatively, CDRs can be defined as the residues of a " hypervariable circuit" (residues 26 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) Chathia and Lesk, J. Mol. Biol 1987; Typically, the numbering of amino acid residues in this region is performed using the method described in Kabat et al., Supra. Phrases such as "Kabat position", "Kabat residue" and "according to Kabat" in the present invention refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer amino acids or additional amino acids corresponding to a shortening or insertion into a variable domain framework (FR) or CDR. For example, a heavy chain variable domain may include the amino acid insertions (residues 52a, 52b and 52c according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b and 82c, etc. according to Kabat) after Heavy Chain FR Residue 82. Kabat residue numbering can be determined for a given antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence.

[0085] The term "framework region" or "FR" residues refers to VH or VL amino acid residues that are not within the CDRs, as defined in the present invention.

[0086] The antibody mAb 0170 has a variable heavy chain sequence as shown in SEQ ID NO: 2 and a variable light chain sequence as shown in SEQ ID NO: 3. An antibody of the present invention can comprise this variable chain sequence heavy and/or this variable sequence of Light Chain. The mAb 0170 antibody has the CDR sequences shown in amino acids 31 to 35, 50 to 68 and 101 to 110 of SEQ ID NO: 2 and in amino acids 24 to 38, 54 to 60 and 93 to 101 of SEQ ID NO: 3.

[0087] The heavy chain of an antibody according to the present invention may comprise a CDR1 sequence of amino acids 31 to 35 (TYAMH) of SEQ ID NO: 2, wherein one of these amino acids may be replaced by a different amino acid.

[0088] The heavy chain of an antibody according to the present invention may comprise a CDR2 sequence of amino acids 50 to 68 (RIRTKSSNYATYYADSVKD) of SEQ ID NO: 2, wherein one, two or three of these amino acids may be replaced by an amino acid different.

[0089] The heavy chain of an antibody according to the present invention may comprise a CDR3 sequence of amino acids 101 to 110 (DMGQRRQFAY) of SEQ ID NO: 2, wherein one, two or three of these amino acids may be replaced by an amino acid different.

[0090] The light chain of an antibody according to the present invention may comprise a CDR1 sequence of amino acids 24 to 38 (RASESVDTFDYSFLH) of SEQ ID NO: 3, wherein one, two or three of these amino acids may be replaced with an amino acid different.

[0091] The light chain of an antibody according to the present invention may comprise a CDR2 sequence of amino acids 54 to 60 (RASNLES) of SEQ ID NO: 3, wherein one or two of these amino acids may be replaced with a different amino acid.

[0092] The light chain of an antibody according to the present invention may comprise a CDR3 sequence of amino acids 93 to 101 (QQSNEDPYT) of SEQ ID NO: 3, wherein one or two of these amino acids may be replaced with a different amino acid.

[0093] The antibody mAb 0170 has a heavy chain sequence as shown in SEQ ID NO: 2 and a light chain sequence as shown in SEQ ID NO: 3. An antibody of the present invention can comprise this heavy chain sequence or this light chain sequence. The heavy or light chain of the antibody of the present invention, or both, may be a variant of mAb 0170. The mAb 0170 antibody has the CDR sequences shown in amino acids 31 to 35, 50 to 68 and 101 to 110 of SEQ ID NO: 2 and amino acids 24 to 38, 54 to 60 and 93 to 101 of SEQ ID NO: 3. An antibody of the present invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

[0094] The heavy chain of an antibody according to the present invention may comprise a CDRH3 sequence of amino acids 101 to 110 (DMGIRRQFAY) of SEQ ID NO: 2, wherein one, two or three of these amino acids may be replaced by an amino acid different.

[0095] The term "antigen" (Ag) refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag. In the present invention, Ag is designated in a broader sense and is It is generally intended to include target molecules that are specifically recognized by the Ab, thus including fragments or mimics of the molecule used in the immunization process, or other process, for example, phage, used to generate the Ab.

[0096] The term "epitope", as used in the present invention, is defined in the context of a molecular interaction between an "antigen-binding polypeptide", such as an antibody (Ab), and its corresponding antigen (Ag). . Generally, "epitope" refers to the area or region of an Ag to which an Ab specifically binds, that is, the area or region in physical contact with the Ab. Physical contact can be defined using various criteria (e.g., a distance cutoff of 2-6 Â, such as 3 Â, such as 4 Â, such as 5 Â, or accessibility to solvents) for the atoms in the Ab and Ag. A protein epitope may comprise amino acid residues on the Ag that are directly involved in binding to an Ab (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues of The Ag that are effectively blocked by the Ab, i.e. amino acid residues within the "solvent excluded surface" and/or the "footprint" of the Ab.

[0097] The term epitope in the present invention comprises both types of binding region in any particular region of TREM-1 that specifically binds to a TREM-1 antibody. TREM-1 may comprise a number of different epitopes, which may include, without limitation, conformational epitopes consisting of one or more non-contiguous amino acids located close to each other in the mature TREM-1 conformation and post-translational epitopes consisting , either fully or partially, of molecular structures covalently linked to TREM-1, such as carbohydrate groups.

[0098] The epitope for a given antibody (Ab)/antigen (Ag) pair can be described and characterized at different levels of detail using a variety of experimental and computational epitope mapping methods. Experimental methods include mutagenesis, X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, Hydrogen Deuterium eXchange Mass Spectrometry (HX-MS), and various competition binding methods; Methods that are known in the art. As each method is based on a unique principle, the description of an epitope is closely linked to the method by which it was determined. Thus, depending on the epitope mapping method used, the epitope for a given Ab/Ag pair can be described differently.

[0099] At its most detailed level, the epitope for the interaction between Ag and Ab can be described through the spatial coordinates that define the atomic contacts present in the Ag-Ab interaction, as well as information about its relative contributions to thermodynamics binding. At a less detailed level, the epitope can be characterized using the spatial coordinates that define the atomic contacts between the Ag and Ab. At an even less detailed level, the epitope can be characterized by the amino acid residues it comprises, as defined by a specific criterion such as the distance between attachments or access to solvent atoms in the Ab:Ag complex. In less detail, the epitope can be characterized through functions, for example, by binding competition with other Abs. The epitope can also be defined more generally as comprising amino acid residues for which substitution with another amino acid will alter the characteristics of the interaction between Ab and Ag.

[00100] In the context of an X-ray-derived crystal structure defined through the spatial coordinates of a complex between an Ab, for example, a Fab fragment, and its Ag, the term epitope is here unless otherwise specified or contradicted through context, specifically defined as TREM-1 residues characterized by having a heavy atom (i.e., a non-hydrogen atom) at a distance of, for example, 4 Å from a heavy atom in Ab.

[00101] From the fact that epitope descriptions and definitions, dependent on the epitope mapping method used, are obtained with different levels of detail, it follows that the comparison of epitopes for different Abs on the same Ag can be conducted in a similar way to different levels of detail.

[00102] Epitopes described at the amino acid level, for example, determined from an X-ray structure, are considered identical if they contain the same set of amino acid residues. Epitopes are said to overlap if at least one amino acid is shared by the epitopes. Epitopes are said to be separate (unique) if no amino acid residues are shared by the epitopes.

[00103] Epitopes can also be defined indirectly, by comparing the binding kinetics of antibodies to wild-type human TREM-1 with those of human TREM-1 variants that have alanine mutations in anticipated epitopes. Decreased affinity or abrogated binding of an antibody to variants of human TREM-1 in which an amino acid residue has been replaced by an alanine residue indicates that the mutated amino acid contributes to the interaction between said antibody and human TREM-1 type wild. This approach provides negative identification of the epitope. The method is compromised in effectively defining the epitope due to the fact that unraveling or unfolding the protein would give results similar to nullifying the interaction. The analysis can be complemented by comparative gain analysis of functional mutations of an orthologous target protein (e.g., cynomolgus monkey TREM-1), if a cross-reactive antibody exists. The comparison will define the epitope differences between the antibody that does not cross-react with, for example, cynomolgus monkey TREM-1 and the cross-reactive antibody.

[00104] Indirect epitope identification can also be provided by measuring the antibody (or antibody fragment) that binds to variants of the wild-type antigen (TREM-1). If an antibody or fragment thereof binds, for example, to human TREM-1, but not to cynomolgus monkey, and if said antibody or fragment thereof is capable of binding to a partially humanized variant of TREM-1 of cynomolgus monkey, then this recovered binding indicates that the substituted amino acid residue(s) is/are important for the interaction of the antibody with the antigen. Likewise, the increased affinity for humanized variants of cynomolgus monkey TREM-1 of an anti-human TREM-1 antibody (or fragment of the same Fab) that has a weaker binding to cynomolgus monkey TREM-1 compared to human TREM-1, it can provide information about the identity of the residues that make up the binding epitope.

[00105] The effect of the same mutations on any given cross-reacting antibody makes it possible to discriminate between possible protein misfolding (abrogated binding to both antibodies) and loss of interaction in human TREM-1 (binding to one of the antibodies and binding revoked to the other antibody), while unambiguously providing information about the epitope differences between the non-cross-reactive antibody and the cross-reactive antibody at an amino acid level.

[00106] The antibodies of the present invention may be capable of binding human TREM-1 variants as determined using, for example, surface plasmon resonance.

[00107] The antibodies of the present invention may be capable of binding cynomolgus monkey TREM-1 variants as determined using, for example, surface plasmon resonance.

[00108] An antibody of the present invention may be capable of specifically binding to TREM-1, wherein said antibody is capable of specifically binding (i) at least one amino acid residue selected from the group consisting in A21, T22, K23, L24, T25, E26, AND (ii) at least one amino acid residue selected from the group consisting of groups A49, S50, S51, Q52, K53, A54, W55, Q56, I57, I58, R59, D60, G61, E62, M63, (iii) at least one amino acid (R), (I) and (iii) at least one amino acid residue selected from the group consisting of C113, V114, I115, Y116, Q117, P118 and P119 of human TREM-1.

[00109] An antibody of the present invention may be capable of specifically binding to a polypeptide comprising amino acids D38 to F48 of SEQ ID NO: 1 (human TREM-1), as determined using, for example, HX- MS or X-ray diffraction.

[00110] An antibody of the present invention may have 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 using , for example, HX-MS or X-ray diffraction.

[00111] An antibody of the present invention may have an epitope comprising one, two, three or all amino acid residues selected from the group consisting of D42, E46, D92 and H93 of SEQ ID NO: 1 (TREM-1 human), as determined using TREM-1 variants and surface plasmon resonance.

[00112] An antibody of the present invention may have an epitope comprising at least amino acid residues E46 and/or D92 of SEQ ID NO: 1 (human TREM-1), as determined using the TREM-1 variants and resonance of surface plasmon.

[00113] An antibody of the present invention may further comprise one, two or all of the amino acid residues selected from the group consisting of L31, I86 and V101 of SEQ ID NO: 1 (human TREM-1).

[00114] An antibody of the present invention may be capable of specifically binding to a polypeptide comprising amino acid residues E19 to L26 of cynomolgus monkey TREM-1 (SEQ ID NO: 17), as determined using, e.g. , HX-MS or X-ray diffraction.

[00115] An antibody of the present invention may be capable of specifically binding to human TREM-1, wherein the epitope of said antibody comprises one, two, three, four, five, six, seven, eight, nine or all the amino acid residues selected from the Group consisting of groups V39, K40, C41, D42, Y43, L45, E46, K47, F48 and A49 of SEQ ID NO: 1.

[00116] An antibody of the present invention may be capable of specifically binding to human TREM-1, wherein the epitope of said antibody comprises D42 of SEQ ID NO: 1. An antibody of the present invention may be capable of binding in a manner specific to human TREM-1, wherein the Epitope of said antibody comprises E46 of SEQ ID NO: 1. The epitope of said antibody may comprise V39, C41, D42, Y43, L45 of SEQ ID NO: 1. The epitope of said antibody may comprise E46, K47 and A49 of SEQ ID NO: 1. The epitope of said antibody may further comprise F48 of SEQ ID NO: 1.

[00117] The definition of the term "paratope" is derived from the above definition of "epitope" by inverting the perspective. Thus, the term "paratope" refers to the area or region of the antibody to which an antigen binds in a specific way, that is, with which it makes physical contact with the antigen.

[00118] In the context of an X-ray-derived crystal structure defined through the spatial coordinates of a complex between an antibody, such as a Fab fragment, and its antigen, the term paratope is herein unless specified or contradicted by context, specifically defined as Antigen residues characterized by having a heavy atom (i.e., an atom other than hydrogen) within 4 Å of a heavy atom in TREM-1.

[00119] The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair can be identified using routine methods. For example, the general location of an epitope can be determined by evaluating the ability of an antibody to bind to different fragments or variant polypeptides of TREM-1. The specific amino acids within TREM-1 that contact an antibody (epitope) and the specific amino acids in an antibody that contact TREM-1 (paratope) can also be determined using routine methods. For example, the antibody and target molecule can be combined and the Ab:Ag complex can be crystallized. The crystal structure of the complex can be determined and used to identify specific sites of interaction between the antibody and its target.

[00120] Antibodies that bind to the same antigen can be characterized with respect to their ability to bind to their common antigen simultaneously and can be subject to "competitive binding/"compartmentalization." In the present context, the term "compartmentalization" refers It is a method of grouping antibodies that bind to the same antigen. Antibody "compartmentalization" can be based on the competitive binding of two antibodies to their common antigen in assays based on standard techniques such as surface plasmon resonance. (SPR), ELISA or flow cytometry.

[00121] The "compartment" of an antibody is defined using a reference antibody. If a second antibody is unable to bind to an antigen at the same time as the reference antibody, the second antibody is said to belong to the same "compartment" as the reference antibody. In this case, the reference antibody and the second antibody competitively bind to the same part of an antigen and are coined "competing antibodies." If a second antibody is able to bind to an antigen at the same time as the reference antibody, the second antibody is said to belong to a separate "compartment". In this case, the reference antibody and the second antibody do not competitively bind the same part of an antigen and are invented "non-competitive antibodies".

[00122] Antibody "compartmentalization" does not provide direct information about the epitope. Competing antibodies, that is, antibodies belonging to the same "compartment" may have identical epitopes, overlapping epitopes or even separate epitopes. In the latter case, the reference antibody linked to its epitope on the antigen occupies the space necessary for the second antibody to come into contact with its epitope on the antigen ("steric hindrance"). Noncompetitive antibodies generally have separate epitopes.

[00123] An antibody of the present invention can compete with mAb 0170 for binding to human TREM-1. An antibody of the present invention can compete with mAb 0170 for binding to cynomolgus monkey TREM-1. In other words, an antibody of the present invention may belong to the same "compartment" as mAb 0170.

[00124] The term "binding affinity" refers in the present invention to a measurement of the resistance of a non-covalent interaction between two molecules, for example, an antibody, or a fragment thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).

[00125] The binding affinity between two molecules, for example, an antibody, or a fragment thereof, and an antigen, through a monovalent interaction can be quantified by determining the equilibrium dissociation constant (KD). In turn, the KD can be determined by measuring the kinetics of formation and dissociation of the complex, for example, using the SPR method. The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant Ka (or Kon) and the dissociation rate constant Kd (or Koff), respectively. KD is related to Ka and Kd through the equation KD = Kd/Ka.

[00126] Following the above definition, the binding affinities associated with different molecular interactions, such as comparing the binding affinity of different antibodies for a given antigen, can be compared by comparing the KD values for the antibody/complexes. individual antigens.

[00127] An antibody of the present invention can bind human TREM-1 with an affinity (KD) that is 1 x 10-7 M or less, 1 x 10-8 M or less, or 1 x 10-9 M or less, or 1 x 10-10 M or less, 1 x 10-11 M or less, 1 x 10-12 M or less, or 1 x 10-13 M or less, as determined using surface plasmon resonance. An antibody of the present invention can bind to cynomolgus monkey TREM-1 with an affinity (KD) that is 1 x 10-7 M or less, 1 x 10-8 M or less, or 1 x 10-9 M or less. or 1 x 10-10 M or less, 1 x 1011 M or less, 1 x 10-12 M or less, or 1 x 10-13 M or less, as determined using surface plasmon resonance.

[00128] The term "binding specificity" refers in the present invention to the interaction of a molecule such as an antibody, or a fragment thereof, with a single unique antigen, or with a limited number of antigens (or epitopes) highly homologues. In contrast, antibodies that are capable of specifically binding to TREM-1 are not capable of binding to dissimilar molecules. Antibodies according to the present invention may not be able to bind to Nkp44, the p44-related protein of the natural killer cell.

[00129] The specificity of an interaction and the value of an equilibrium binding constant can be determined directly using well-known methods. Standard assays for evaluating the ability of ligands (such as antibodies) to bind to their targets are known in the art and include, for example, ELISAs, Western blots, RIAs and flow cytometry analysis. The binding kinetics and binding affinity of the antibody can also be evaluated using standard assays known in the art, such as SPR.

[00130] A competitive binding assay can be performed in which the binding of the antibody to the target is compared to the binding of the target by another ligand of that target, such as another antibody.

[00131] In another aspect, the present invention provides compositions and formulations comprising the molecules of the present invention, such as the TREM-1 antibodies, polynucleotides, vectors and cells described in the present invention. For example, the present invention provides a pharmaceutical composition comprising one or more TREM-1 antibodies of the present invention, formulated together with a pharmaceutically acceptable carrier.

[00132] Accordingly, an object of the present invention is to provide a pharmaceutical formulation comprising such a TREM-1 antibody that is present in a concentration of 0.25 mg/mL to 250 mg/mL, such as a concentration of 10 to 200 mg/mL, and wherein said formulation has a pH of 2.0 to 10.0, such as a pH of 4.0 to 8.0. The formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well known to a person skilled in the art. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[00133] In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such a formulation is typically a solution or a suspension, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation comprising at least 50% w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50% w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w/w water. .

[00134] In another embodiment, the pharmaceutical formulation is a lyophilized formulation, to which the doctor or patient adds solvents and/or diluents before use.

[00135] In another aspect, the pharmaceutical formulation comprises an aqueous solution of such an antibody and a buffer, wherein the antibody is present at a concentration of 1 mg/mL or greater and wherein said formulation has a pH between about from 2.0 to about 10.0, such as a pH of 4.0 to 8.0.

[00136] The TREM-1 antibodies of the present invention and pharmaceutical compositions comprising such antibodies can be used for the treatment of inflammatory diseases such as the following: inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis ( UC), irritable bowel, Rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Grave's disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, arterial disease coronary heart disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogrens syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, asthma and other autoimmune diseases resulting in acute or chronic inflammation.

[00137] The TREM-1 antibodies of the present invention may be suitable for use in treating individuals with inflammatory bowel disease. Inflammatory bowel disease (IBD) is a disease that can affect any part of the gastrointestinal tract from the mouth to the anus, causing a wide variety of symptoms. IBD mainly causes abdominal pain, diarrhea (which can be bloody), vomiting or weight loss, but it can also cause complications outside the gastrointestinal tract, such as skin rashes, arthritis, eye inflammation, fatigue and poor concentration. Patients with IBD can be divided into two main classes, those with ulcerative colitis (UC) and those with Crohn's disease (CD). CD usually involves the ileum and colon, can affect any region of the intestine, but is often discontinuous (focused areas of disease spread throughout the intestine). UC always involves the rectum (colon) and is more continuous. In CD, inflammation is transmural, resulting in abscesses, fistulas, and strictures, whereas in UC, inflammation is typically confined to the mucosa. There is no known pharmaceutical or surgical cure for Crohn's disease, as some UC patients can be cured through surgical removal of the colon. Treatment options are restricted to controlling symptoms, maintaining remission and preventing relapse. Efficacy in inflammatory bowel disease in the clinic can be measured as a reduction in the Crohn's Disease Activity Index (CDAI) score for CD which is a scoring scale based on laboratory tests and a quality of life questionnaire. In animal models, effectiveness is primarily measured by weight gain and also a disease activity index (DAI), which is a combination of stool consistency, weight, and blood in the stool.

[00138] The TREM-1 antibodies of the present invention may be suitable for use in treating individuals with rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease that affects most if not the entire body and is one of the most common forms of arthritis. It is characterized by inflammation of the joint, which causes pain, stiffness, heat, redness and swelling. This inflammation is a consequence of inflammatory cells that invade the joints, and these inflammatory cells release enzymes that can digest bone and cartilage. As a result, this inflammation can lead to severe bone and cartilage damage and joint deterioration and severe pain, among other physiological effects. The involved joint may lose its shape and alignment, resulting in pain and loss of movement.

[00139] There are several animal models for rheumatoid arthritis known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop an inflammatory arthritis that resembles human rheumatoid arthritis. Since CIA shares similar immunological and pathological features with RA, this makes it a suitable model for screening potential human anti-inflammatory compounds. Efficacy in this model is measured by reducing joint swelling. Efficacy in RA in the clinic is measured through the ability to reduce symptoms in patients, which is measured as a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein levels, and levels of serum factors such as antibodies. anti-citrullinated proteins.

[00140] The TREM-1 antibodies of the present invention may be 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. It is a disease for which there is currently no cure and affects people of all ages. Although individuals with mild psoriasis can often control their disease with topical agents, more than a million patients worldwide require ultraviolet light treatments or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of ultraviolet radiation and the toxicities of many therapies limit their long-term use. Additionally, patients often have psoriasis recurrence and, in some cases, rebound shortly after stopping immunosuppressive therapy. A recently developed model of psoriasis based on the transfer of CD4+ T cells mimics many aspects of human psoriasis and therefore can be used to identify suitable compounds for use in the treatment of psoriasis (Davenport et al., Internat. Immunopharmacol 2: 653 a 672, 2002). Efficacy in this model is measured through reduction in skin pathology using a scoring system. Likewise, efficacy in patients is measured by a reduction in skin pathology.

[00141] The TREM-1 antibodies of the present invention may be suitable for use in treating individuals with psoriatic arthritis. Psoriatic arthritis (PA) is a type of inflammatory arthritis that occurs in a subset of patients with psoriasis. In these patients, skin pathology/symptoms are accompanied by joint swelling similar to that seen in rheumatoid arthritis. It has irregular, raised, red areas of skin inflammation with scale. Psoriasis often affects the tips of the elbows and knees, the scalp, the belly button and around the genital areas or anus. Approximately 10% of patients who have psoriasis also develop an associated inflammation of their joints.

[00142] The term "treatment", as used in the present invention, refers to the medical therapy of any human subject or other animal in need thereof. Said individual is expected to have undergone a physical examination by a physician or veterinarian who has given a provisional or definitive diagnosis that indicates that the use of said treatment is beneficial to the health of said human or other animal. The timing and purpose of said treatment may vary from one individual to another, depending on many factors, such as the status quo of the subject's health. In this way, said treatment can be prophylactic, palliative, symptomatic and/or curative.

[00143] In terms of the present invention, prophylactic, palliative, symptomatic and/or curative treatments may represent separate aspects of the present invention.

[00144] An antibody of the present invention can be administered parenterally, such as intravenously, such as intramuscularly, such as subcutaneously. Alternatively, an antibody of the present invention may be administered via a non-parenteral route, such as perorally or topically. An antibody of the present invention can be administered prophylactically. An antibody of the present invention can be administered therapeutically (on demand).

[00145] In a first aspect of the present invention, the substitution of negatively charged residues of the CDR1 and CDR3 regions of SEQ ID NO: 3 (the variable light chain of mAb 0170) was observed to influence the viscosity of the mAb. In this first aspect of the present invention, the mAb of the present invention is a variant of mAb 0170 having a heavy chain and a light chain, wherein the light chain of mAb 0170, namely SEQ ID NO: 3, comprises mutations in which the charged residues negatively in CDR1 and CDR3 of SEQ ID NO: 3 are replaced with the uncharged residues. Accordingly, one aspect of the present invention is directed to a variant of mAb 0170 comprising replacing any one or any combination of residues D1, D30, D33, D74, D98, E27, E97 of SEQ ID NO: 3 with a residue of amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine. Otherwise, an interesting embodiment of the present invention is directed to an antibody or fragment thereof comprising SEQ ID NO: 2 (or a variant thereof) as a heavy chain and as a light chain comprising a variant of SEQ ID NO: 3 in that any one or any combination of residues D1, D30, D33, D74, D98, E27, E97 of SEQ ID NO: 3 is mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, and tyrosine. These mutations will be referred to as "cargo parcel" mutations. In a preferred embodiment, at least one or both E27 and E97 of the CDR1 and CDR3 regions of SEQ ID NO: 3 are replaced by uncharged amino acid residues, such as an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine. In a preferred embodiment, E27 of the CDR1 and CDR3 regions of SEQ ID NO: 3 is mutated to glutamine and E97 is replaced by an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, And tyrosine, more preferably with an amino acid selected from the group consisting of serine and glutamine. In another embodiment, E27 remains unmutated and E97 is mutated with an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine, more preferably with an amino acid selected from the group consisting of serine and glutamine. In another embodiment, residue E97 remains unmutated and E27 is replaced by an amino acid selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine, more preferably with an amino acid selected from from the group consisting of serine and glutamine.

[00146] Another aspect of the present invention is based on the observation that Fab-Fab dimers were formed due to interactions in the paratope area. Since mAbs comprise two Fabs, it was thought that mAbs would be capable of multimerizing, which could have an impact on viscosity properties. Thus, another aspect of the present invention is directed to mutating residues in the Fab-Fab interaction area of SEQ ID NO: 2 (namely in the variable heavy chain area of mAb 0170) to reduce Fab-Fab dimerization. These mutations are referred to as "Fab-Fab interaction mutations." Accordingly, one aspect of the present invention is directed to replacing any of the residues Y32, R52, S55, S56, N57, A59, M102, I104 and R106 of SEQ ID NO: 2 or F32, D33, Y34, Y53, R54, D98 From SEQ. ID NO 3 with an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine. Otherwise, an interesting embodiment of the present invention is directed to an antibody or fragment thereof comprising a variant of SEQ ID NO: 2 in which any of residues Y32, R52, S55, S56, N57, A59, M102, I104 and R106 of SEQ ID NO: 2 or F32, D33, Y34, Y53, R54, D98 of SEQ. ID NO 3 ("Fab-Fab interaction mutations") is replaced by another amino acid, such as a natural amino acid, preferably an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine , threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine. Preferably, at least one of residues A59 and N57 SEQ ID NO: 2 is mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan , histidine and tyrosine, more preferably serine or tyrosine. In one embodiment, A59 remains unmutated and N57 is mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine, more preferably serine or tyrosine. In another embodiment, N57 remains unmutated and A59 is mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine. In at least one embodiment, both A59 and N57 are mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine, more preferably serine or tyrosine.

[00147] In an interesting embodiment, i) at least one of the negatively charged residues of the CDR1 and CDR3 regions of SEQ ID NO: 3 is mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine , asparagine, glutamine, threonine, cysteine and tyrosine and ii) at least one residue of residues Y32, R52, S55, S56, N57, A59, M102, I104 and R106 of SEQ ID NO: 2 or F32, D33, Y34, Y53 , R54, D98 of SEQ ID NO: 3 (Fab-Fab interaction mutations) is mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine and tryptophan, histidine and tyrosine.

[00148] In a suitable embodiment, one or both E27 and E97 of SEQ ID NO: 3 are mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine and tyrosine and One or both of A59 and N57 of SEQ ID NO: 2 are mutated to an amino acid residue selected from the group consisting of glycine, alanine, serine, asparagine, glutamine, threonine, cysteine, lysine, arginine, tryptophan, histidine and tyrosine, more preferably serine or tyrosine.

[00149] Another aspect of the present invention is based on the observation that an Ala substitution at position Y90 of SEQ. TREM-1 ID NO 1 improved SEQ affinity. ID NO 3 to TRAIN-1. Y90 was found to interact with a phenylalanine residue of SEQ ID NO: 3. Mutation of SEQ ID NO: 3 to improve Fab-TREM-1 interaction is referred to as Fab-TREM-1 interaction mutations. In one embodiment of this aspect of the present invention, the phenylalanine at position 32 of SEQ ID NO: 3 is mutated to an amino acid selected from the amino acid residues glycine, serine, threonine, cysteine, alanine, valine, leucine, isoleucine and methionine, preferably selected from alanine, glycine, serine valine and leucine. In an interesting embodiment, F32 of SEQ ID NO: 3 is mutated to alanine or serine.

[00150] Charge portion mutations in CDR1 and CDR3 of SEQ ID NO: 3 reduced the hydrodynamic radius (Rh) while mutations in the Fab-Fab interaction region increased Rh (Figure 6). Mutations at the “Fab-TREM-1 interaction” site did not influence Rh and had very little effect on viscosity (Figures 6 and 1, Tables 3C and 3E). Table 1: Overview of SEQ ID NO (0170) of generated mAb variants. L = light chain, H = heavy chain.

[00151] Viscosity was determined for the mAb 0170 variants by DLS or microrheology and showed that both "charge parcel" mutations and "Fab-Fab interaction" mutations reduced viscosity. The mAb 0318 variant, which comprises the E27Q and E97Q charge share mutations, was found to have the lowest viscosity.

[00152] The binding kinetics of the mAb 0170 variants in relation to human TREM-1-Fc and cynomolgus TREM-1-Fc were determined. All mAb 0170 variants were found to have similar affinity toward human TREM-1-Fc. Interestingly, the mAb variant, SEQ ID NO: 9 comprising a "Fab-TREM-1 interaction" mutation was found to have an increased affinity towards the cynomolgic TREM-1-Fc (Table 2). Table 2: Afinidac and anti-TREM-1 mAb variants for human T CREM-1-Fc and cynomolgus TREM-1-Fc respectively.

[00153] In an embodiment with improved affinity for TREM-1 and with low viscosity, the mutations of SEQ ID NO: 9 were combined with the mutations in SEQ ID NO: 5 to generate the light chain from SEQ ID NO: 14 and the light chain mutations of SEQ ID NO: 14 were further combined with the heavy chain mutations of SEQ ID NO: 16. The increased affinity for TREM-1 cynomolgus-Fc was retained for the mAb variants comprising the combined mutations of SEQ ID NO: 14 And the mutations did not negatively affect the affinity for human TREM-1-Fc. Accordingly, embodiments comprising "charge parcel" mutations and "Fab-TREM-1 interaction mutations" are provided for by the present invention, as well as embodiments comprising "charge parcel" mutations and "charge parcel" mutations. "Fab-Fab interacting mutations" comprising "Fab-Fab interacting" mutations and "Fab-TREM-1 interacting" mutations and embodiments comprising Fab-Fab interacting mutations, Fab-TREM-1 interaction and cargo portion mutations. Table 3A: Protein Concentrations versus mAb Viscosity Table 3B: Protein Concentrations versus Viscosity of mAb 0319 and 0320 and 321. Table 3C: Protein Concentrations versus Viscosity of mAb 0322, 0323 and 324. Table 3D: Protein Concentrations versus Viscosity of mAb 0325, 0326 and 0330. Table 3E Protein Concentrations versus Viscosity of mAb 0332 and 0333.

[00154] Non-limiting embodiments of the present invention further comprise: 1. Antibody or fragment thereof that is capable of binding and blocking TREM-1 of SEQ ID NO: 1, characterized in that the antibody or an antibody fragment of said antibody having a viscosity of less than 5 cP at a concentration of 50 mg/mL, such as less than 4 cP, preferably less than 3 cP (wherein the method of determining conditions of viscosity versus protein concentration are conventional or as described in the present invention). 2. Antibody or fragment thereof that is capable of binding and blocking TREM-1, characterized in that the antibody, or an antibody fragment of said antibody, has a viscosity of less than 5 cP at a concentration of 80 mg/mL, preferably less than 4 cP (wherein the method of determining viscosity conditions versus protein concentration are conventional or as described in the present invention). 3. Antibody or fragment thereof according to embodiments 1 or 2, wherein the antibody or fragment thereof blocks TREM-1 function with a KD for SEQ ID NO: 1 less than 0.5 nM, such as less than 0.4 nM, preferably 0.3 nM or less. 4. Antibody or fragment thereof wherein the antibody or fragment thereof competitively binds with mAb 0170 for binding to SEQ ID NO: 1 and has a viscosity profile such that: (A) the antibody or an antibody fragment thereof said antibody has a viscosity of less than 5 cP at a concentration of 50 mg/mL, such as less than 4 cP, preferably less than 3 cP; or (B) the antibody, or an antibody fragment of said antibody, has a viscosity of less than 5 cP at a concentration of 80 mg/mL, preferably less than 4 cP. 5. Antibody or fragment thereof that is capable of specifically binding and blocking TREM-1, wherein the antibody or fragment thereof has "Fab-Fab interaction" mutations of SEQ ID NO: 2. 6 Antibody or fragment thereof that is capable of specifically binding and blocking TREM-1 of SEQ ID NO: 1, wherein the antibody or fragment thereof has "cargo portion" mutations, such as wherein. at least one of the negatively charged residues of CDR1 and CDR3 of SEQ ID NO: 3 are replaced with the uncharged residues. 7. Antibody or fragment thereof according to any of the embodiments of the present invention in which the negatively charged amino acids are replaced by amino acid residues that can form hydrogen bonding partners, such as mutation of an Asp residue or Glu to any of Asn, Gln, Ser, and Thr. 8. Antibody or fragment thereof according to any of the embodiments of the present invention having a reduced viscosity compared to mAb 0170, but still having a KD for SEQ ID NO: 1 of less than 0.5 nM. 9. Antibody or fragment thereof according to any of the embodiments of the present invention having a reduced viscosity compared to mAb 0170, a KD with SEQ ID NO: 1 less than 0.5 nM and a KD with SEQ ID NO: 1 : 17 less than 0.6 nM, replacing uncharged amino acids of SEQ ID NO: 2 and/or 3, involved in specific "Fab Fab interactions" with amino acids with altered size or hydrogen bonding potential. 10. Antibody or fragment thereof according to any of the embodiments of the present invention that is capable of specifically binding and blocking TREM-1 of SEQ ID NO: 1 and comprises variants of SEQ ID NO: 2 or SEQ ID NO: 3 or both, where variants are selected from the group consisting of "Fab-Fab interaction" mutations, "Fab-TREM-1 interaction" mutations, and "cargo parcel" mutations of SEQ ID NO: 2 or SEQ ID NO: 3. 11. Antibody or fragment thereof according to any of the embodiments of the present invention comprising "Fab-TREM-1 interaction" mutations, such as wherein a residue Phe of SEQ ID NO: 3 is replaced by Ala or Ser. 12. Antibody or fragment thereof according to any of the embodiments of the present invention that competes with SEQ ID NO: 2 or 3 for binding to SEQ ID NO: 1 and which 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 and one, two or all of the of amino acids E46, K47, F48 of SEQ ID NO: 1. 13. Antibody or fragment thereof according to any of the embodiments of the present invention, which competes for binding with SEQ ID NO: 3 and which comprises an LC having one or both of the glutamate (E) residues of positions 27 and 97 of SEQ ID NO: 3 mutated to serine (S) or glutamine (Q), such as wherein E27Q, E97S, such as wherein both E27 and E97 are mutated to glutamine, or in which E27 remains unmutated and E97 is mutated to S (E27, E97S), or in which E27 remains unmutated and E97 is mutated to Q (E27, E97Q), or in which E97 remains unmutated and E27 is mutated to Q or S, more preferably to Q (E27, E97Q). 14. Antibody or fragment thereof according to any of the embodiments of the present invention comprising SEQ ID NO: 3, wherein the phenylalanine at position 32 (F32) is mutated, such as mutated to A (mAb 0322) or for S (mAb 0323). 15. Antibody or fragment thereof according to any of the embodiments, comprising SEQ ID NO: 2 in which one or both of residue N57 and residue A59 have been mutated, such as in which residue A59 has been mutated to a tyrosine, or where residue N57 has been mutated to a serine, or where residue N57 has been mutated to a serine and residue A59 has been mutated to a tyrosine. 16. Antibody or fragment thereof comprising a variant of SEQ ID NO: 2 in which uncharged amino acids involved in specific Fab Fab interactions are replaced by amino acids with altered size or hydrogen bonding potential, such as in which alanine (A ) or asparagine (N) are substitutions with any of Ser, Thr, Phe, Tyr and Trp. 17. Antibody or fragment thereof that comprises a variant of SEQ ID NO: 3 and that competes for binding with SEQ ID NO: 3 in which one or both glutamate residues (E) of positions 27 and 97 of SEQ ID NO:3 are mutated to Serine (S) or glutamine (Q), such as where both E27 and E97 are mutated to glutamine (318), or where E27 remains unmutated and E97 is mutated to S (E27, E97S) , or wherein E27 remains unmutated and E97 is mutated to Q (E27, E97Q), or wherein E97 remains unmutated and E27 is mutated to Q or S, more preferably to Q (E27, E97Q). 18. Antibody or fragment thereof comprising a variant of SEQ ID NO: 2 and competing for binding with SEQ ID NO: 2, wherein one or both of residue N57 and residue A59 have been mutated, as in that residue A59 has been mutated to a tyrosine, or that residue N57 has been mutated to a serine, or that both residues N57 have been mutated to a serine and residue A59 has been mutated to a tyrosine. 19. Antibody or fragment thereof comprising SEQ ID NO: 4. 20. Antibody or fragment thereof comprising SEQ ID NO: 5. 21. Antibody or fragment thereof comprising SEQ ID NO: 6. 22. Antibody or fragment thereof comprising SEQ ID NO: 7. 23. Antibody or fragment thereof comprising SEQ ID NO: 8. 24. Antibody or fragment thereof comprising SEQ ID NO: 9. 25. Antibody or the fragment thereof comprising SEQ ID NO: 10. 26. Antibody or the fragment thereof comprising SEQ ID NO: 11. 27. Antibody or the fragment thereof comprising SEQ ID NO: 12. 28. Antibody or the fragment thereof of the same comprising SEQ ID NO: 13. 29. Antibody or fragment thereof comprising SEQ ID NO: 14. 30. Antibody or fragment thereof comprising SEQ ID NO: 15. 31. Antibody or fragment thereof comprising SEQ ID NO: 16. 32. Antibody or fragment thereof comprising any of SEQ ID NOs: 4 to 16. 33. Composition comprising an antibody as defined in any of embodiments 1-32. 34. Antibody of the present invention, such as embodiments 1 to 32, for use as a medicament. 35. Antibody of the present invention, such as embodiments 1 to 32, for use in treating a disease selected from an inflammatory disease or an autoimmune disease. 36. Antibody according to embodiment 35, wherein the disease is selected from the group consisting of rheumatoid arthritis and inflammatory bowel disease. 37. Use of an antibody of the present invention, such as any one of embodiments 1 to 32, for the preparation of a medicament for the treatment of a disease selected from the group consisting of an inflammatory disease or an autoimmune disease. 38. Use in accordance with embodiment 35, in which the disease is selected from the group consisting of rheumatoid arthritis and inflammatory bowel disease. 39. Method for treating a disease selected from an inflammatory disease or an autoimmune disease comprising administering an antibody of the present invention (such as an antibody of any of embodiments 1-32), a composition comprising the antibody of the present invention, or a medicament comprising the antibody of the present invention.

EXAMPLES Example 1: Hydrodynamic radius and viscosity of mAb0170 variants

[00155] It was found that the hydrodynamic radius of mAb0170 of WO2013/120553 was higher (9-10 nm) than desired (5-6 nm). The hydrodynamic radius was determined via dynamic light scattering analysis using a 96-well plate with a DynaPro plate reader (Wyatt Inc.). The plates used were Corning 3540 assay plates (Corning). The total sample volume was approximately 20 μL and the temperature was maintained at 25°C for the mAb0170 variants (Figure 6).

[00156] Charge portion mutations in CDR1 and CDR3 of the variable light chain reduced Rh to the level expected for monomeric mAbs while mutations in the Fab-Fab interaction region appear to increase Rh. Mutations in the Fab-TREM-1 interaction site did not influence Rh.

[00157] Viscosity was determined for mAb0170 variants 0317, 0318, 0319, 0320, 0321, 0322, 0323, 0324, 0325, 0326 and 0330 by DLS. The hydrodynamic radius was measured on a Wyatt DynaPro Platereader using Corning 3540 uncoated, clear-bottom black polystyrene microplates and Phosphorex Inc. plain polystyrene nanobeads with an average diameter of 206.5 nm (Reference No. 106). . Protein samples were transferred to a Corning 3540 plate and covered with a top seal. The samples were centrifuged and the hydrodynamic radii of the protein samples were measured on the Wyatt DynaPro DLS plate reader. Each well was added 0.5 μL of polystyrene beads and gently mixed by pipetting. The plate was centrifuged again and the hydrodynamic radii of the beads were measured on the Wyatt DynaPro DLS plate reader.

[00158] The viscosities of the protein samples were calculated as

[00159] Viscosity (protein) = (hydrodynamic radius (spheres, meas) x viscosity (buffer))/hydrodynamic radius (beads, real)

[00160] Where viscosity (protein) is the calculated viscosity of the protein solution, hydrodynamic radius (beads, meas) is the measured hydrodynamic radius of the polystyrene beads in the protein solution, viscosity (buffer) is the viscosity of the buffer and the hydrodynamic radius, Real) is the real average diameter of the beads [He et al., Anal Biochem, 399, 141 to 143, 2010].

[00161] Viscosity was determined using micro-rheology for variants mAb0170 0332 and 0333. For rheological analysis, a 250 μL Hamilton LT syringe (Hamilton-Bonaduz Inc) was used with a Novofine 30G needle. The syringe was placed in a custom-made aluminum holder attached to a platform. The syringe plunger was actuated by a TAXTPlus texture analyzer, which measured the resulting force on the plunger at a predetermined injection speed. Each sample was tested with three different injection speeds. The crosshead speed and measured force were used to calculate the shear rate and shear stress, respectively. Viscosity can be expressed in relation to shear rate as: nprotein = TW/Y = (ΔPD/4L)/((32Q)/(πD3))

[00162] Where nprotein is the viscosity of the protein solution, Y is the apparent shear rate, TW is the shear stress, P is the pressure resulting from the movement of the plunger, Q is the volumetric flow rate of the fluid passing through the needle capillary and D and L are the internal diameter and length, respectively, of the capillary (Allahham et al., 2004; Intl J Pharm 270, 139 to 148). The viscosity, nprotein was calculated from the known values of D, L and Q and the measured value of P.

[00163] Analysis showed that both "charge parcel" mutations and "Fab-Fab interaction" mutations reduced viscosity (Figure 1). The mAb variant SEQ ID NO: 5, which comprises the E27Q and E97Q charge share mutations, was found to have the lowest viscosity.

Example 2: Kinetics of mAb0170 variants

[00164] The binding kinetics of mAb 0170 variants in relation to human TREM-1-Fc and cynomolgus TREM-1-Fc were determined. Binding studies were performed on a ProteOn analyzer (BioRad) that measures molecular interactions in real time via surface plasmon resonance. Experiments were performed at 25°C and samples were stored at 15°C in the sample compartment. The signal (RU, response units) reported by ProteOn is directly correlated to the mass on the individual sensor chip surfaces in the six parallel flow cells. Monoclonal anti-human Fc antibody or polyclonal anti-murine Fc antibody from Biacore human or mouse Fc capture kits were immobilized in horizontal direction onto flow cells of a GLM sensor chip according to the manufacturer's instructions. The final immobilization level of the capture antibody was approximately 2600-6000 RU in each experiment. Capture of purified mouse monoclonal or recombinantly expressed anti-hTREM-1 antibodies was conducted by diluting the antibodies to 510 nM in running buffer (0.15 M Hepes, 10 mM NaCl, 5 mM EDTA, 0.05% surfactant P20, pH 7.4) followed by injection in the vertical direction at 30 μL/min for 60 seconds, creating reference points adjacent to all flow cells with only anti-Fc antibody immobilized. This typically results in final test antibody capture levels of approximately 100-300 RU values and analyte Rmax of 30-90 RU. Binding of hTREM-1 or cTREM-1 proteins was conducted by injecting analyte (antigen) onto all flow cells in a horizontal direction to allow comparative analyzes of binding to different captured anti-TREM-1 antibodies relative to binding to the point of reference. hTREM-1 or cTREM-1 proteins were serially diluted 1:3 to 1.2–100 nM or in running buffer, injected at 100 μL/min for 250 s, and allowed to dissociate for 600 s. The GLM surface was regenerated after each analyte injection cycle through two injections of 18 μg of 10 mM Glycine, pH 1.7 and 50 mM NaOH at 100 μL/min. This regeneration step removed the anti-TREM-1 antibody and any bound TREM-1 protein from the surface of the immobilized capture antibody and allowed subsequent binding of the next pair of interacting samples. The regeneration procedure did not remove the immobilized anti-Fc capture antibody directly from the chip surface.

[00165] The binding affinity between antibodies and the antigen was quantified by determining the equilibrium dissociation constant (KD) determined by measuring the kinetics of formation and dissociation of the complex. The rate constants corresponding to the association and dissociation of a monovalent complex such as Ka (association rate) and Kd (dissociation rate) were recovered by fitting data to the 1:1 Langmuir model using ProteOn evaluation software for data analysis. . KD is related to Ka and Kd through the equation KD = Kd/Ka.

[00166] Binding curves were processed using double reference (subtraction of reference surface signals as well as blank buffer injections over captured anti-TREM-1 antibodies) prior to data analysis. This allowed correction for instrument noise, volume displacement, and drift during sample injections.

[00167] The mAb 0170 variants were all observed to have similar affinity for human TREM-1-Fc as mAb 0170. Interestingly, mAb variants comprising a "Fab-TREM-1 interaction" mutation, such as mAb 0322, had increased affinity for cynomolgus TREM-1-Fc (Table 2).

Example 3: Kinetics and viscosity of mAb 0330 and mAb 0333

[00168] In an attempt to generate a mAb with both improved affinities towards Tyn-1 of cynomolous TREM-1 and having low viscosity, the mutations of SEQ ID NO: 9 were combined with the mutations in SEQ ID NO: 5, which was found to have the lowest viscosity among Variants mAb0170 and with SEQ ID NO: 11, which was found to have a modest effect on viscosity. The increased affinity toward human TREM-1-Fc was maintained for the mAb variant comprising the combined mutations (mAb 0330 and mAb 0333) and the mutations did not affect the affinity for human TREM-1-Fc. The viscosity of mAb0330 and mAb 0333 was markedly reduced compared to mAb 0170 of WO2013/120553 (Figure 1).

Example 4: Cultivation of a stable BWZ'36/hTREM-1 cell line

[00169] BWZ/hTREM-1 were grown in RPMI 1640 without phenol red (Cat # 11835, Gibco, Carlsbad CA, USA), supplemented with 10% FCS (Cat # 16140-071, Gibco, New York, USA) 1% Pen/Strep (Cat # 15070-06, Gibco), 1 mM Sodium Pyruvate (Cat # 11360, Gibco), 5 μM -2ME (Cat # 31350-010, Gibco) and 2 mM L-glutamine , Gibco). No special plates or coatings were required. 10 ml of Versene (Cat # 15040, Gibco) was added to separate the cells which were then transferred to tubes, centrifuged at 1200 rpm for 5 min and washed in fresh RPMI 1640 with phenol red. These cells were then ready for use in an assay or re-culture for further propagation.

Example 5: Functional characterization of mAb 0170 variants

[00170] The ability of anti-TREM-1 mAb 0170 variants to inhibit human TREM-1 signaling was determined using a reporter cell line (BWZ36/hTREM-1) provided by Bioxell. The reporter cell line was stimulated with PGN and TREM-1 ligand PGLYRP1 either as recombinant protein or expressed by activated neutrophils before incubation with mAbs. The potency of the variants to inhibit TREM-1 signaling was found to be clinically relevant and on par with that of mAb 0170 from WO2013/120553 (Figure 2A and B).

[00171] Likewise, the ability to inhibit cynomolgous TREM-1 signaling was determined for the three variants of mAb0170 with the lowest viscosity and two variants observed to have an increased affinity for cynomolgous TREM-1. In this assay using an established reporter cell line TE 426.27, similar potencies of the mAb and mAb0170 variants of WO2013/120553 were observed (Figure 3).

[00172] The potency of mAb variants to block the release of TNF alpha from primary cells from healthy donors was also evaluated. In this assay, monocytes were differentiated into M2 macrophages under hypoxic conditions to increase TREM-1 expression and activated with PGN and recombinant PGLYRP1 before incubation with mAb. The mAb variants were found to be as potent as 0170 in inhibiting TREM-1-mediated TNFα release from hypoxic M2 macrophages (Figure 4).

Example 6 Crystal structure of a Fab 0170-Fab 0170 complex

[00173] The potential of mAb 0170 molecules to interact through specific self-interactions was evaluated from crystallographic analysis of a Fab 0170-Fab 0170 crystal structure.

[00174] Materials: The Fab region of mAb 0170 (SEQ ID NO: 18 and SEQ ID NO: 19) in a buffer consisting of 10 mM phosphate, 2.68 mM KCl, 140 mM NaCl, pH 7, 4 at a protein concentration of 8.5 mg/mL.

[00175] Methods: The Fab region of mAb 0170 was crystallized in a suspension drop vapor diffusion experiment by equilibrating a drop consisting of 2 μL of protein solution mixed with 2 μL of reservoir solution against a reservoir of 0 .5 mL composed of 20% (w/v) PEG 8000, 200 mM K2HPO4. The crystal was transferred to a droplet consisting of 2 μL of 35% (w/v) PEG3350, 200 mM K2HPO4 and mounted on a 0.2 mm diameter litholoop (Molecular Dimensions Limited) and instantly cooled in liquid nitrogen.

[00176] Kabsch, Acta Crystallogr. D66, 133-144 (2010)). The space group of the crystal was P2(1)2(1)2(1), with unit cell parameters, a = 62.4 Â, b = 110.9 Â, c = 158.4 Â. Data were collected to a resolution of 2.40 Å. The structure was solved by molecular replacement using Phenix software (Adams et al, Acta Crystallogr. D66, 213-221 (2010)) as implemented in the CCP4i suite of programs (Potterton et al, Acta Crystallogr, D59, 1131-1137 ( 2003)). The search models were structures of the heavy chain from the pdb entry 1AD0.pdb (85% identity) and the light chain from the pdb entry 2QRG (94% identity). Structure refinement was performed using Refmac5 (Murshudov et al., Acta Crystallogr. D53, 240-255 (1997)) from the CCP4i suite of programs. The Coot 7 version (Emsley et al., Acta Crystallogr. D66, 486-501 (2010)) was used for manual reconstruction and validation of the structure.

Results and discussion

[00177] The crystal structure of the mAb Fab 170 region contained four Fab molecules in the asymmetric unit (the heavy chains were labeled with A and C, the light chains were labeled with B and D). Fab molecules were packaged as dimers with the antigen-binding region of one Fab molecule interacting with the antigen-binding region of another Fab molecule. It was not possible to include and refine the last cysteine from SEQ ID NO: 19 in the structure crystalline, although it was expected to be in a disulfide bond with a Cys of SEQ ID NO: 18 from the heavy chain. There was some indication of excess 2Fo-Fc and Fo-Fc electron density in the area, and it is possible that the disulfide bond is present in a subset of the Fab molecules. There was no significant sigma-weighted 2Fo-Fc electron density for the residues G26, K78-N79, I104-R105 and S138-E141 of SEQ ID NO: 18 in chain A in the asymmetric unit and for E1, G26-F27, M102-R105, S136-E141, S195-K200 of SEQ ID NO: 18 C chain on the other Fab molecule in the asymmetric unit. Significant sigma weighted 2Fo-Fc electron density was also absent for residues D30-Y34 of SEQ ID NO: 19 in chain D. These residues were not included in the crystal structure but were included in the molecular interaction analysis of the Fab-Fab interface by superposition with the crystal structure of the mAb 0170 fragment from document WO2013/120553 revealed the humanized anti-TREM-1 mAb 0170 crystal structure.

[00178] The structure quality parameters of the Fab fragment of mAb 0170 showed a global structure R factor = 24% and the free R factor = 30%. The overall correlation coefficient was 0.93 and the diffraction component precision index, DPI = 0.3 Å (Cruickshank, Acta Crystallogr. D55, 583-601 (1999)). The root mean square deviation of the bond lengths in the structure from the ideal bond lengths = 0.025 Å and the root mean square deviation of the ideal bond angles = 2.326° (Engh and Huber, Acta Crystallogr. A47, 392400 1991) ).

[00179] The analysis of intermolecular distances was carried out using the NCONT program in the CCP4 program (Potterton et al., Acta Crystallogr. D59, 1131 to 1137 (2003)) with a cutoff of 4Å for intermolecular distances (Table 4). The analysis showed that amino acid residues N57 and A59 of SEQ ID NO: 2 belong to the group of amino acids involved in Fab-Fab interactions in the crystal structure. Table 4. Predicted Fab-Fab interactions based on superposition of WO2013/120553 revealed the humanized anti-TREM-1 mAb 0170 crystal structure with the present crystal structure of the observed mAb 0170 region.

Claims (17)

1. Antibody or fragment thereof that is capable of binding and blocking TREM-1, characterized by the fact that it comprises a light chain CDR1 region, a light chain CDR2 region and a light chain CDR3 region, a light chain CDR1 region heavy chain region, a heavy chain CDR2 region and a heavy chain CDR3 region, wherein the light chain CDR1 region, the light chain CDR2 region and the light chain CDR3 region are the light chain CDR1 region, the light chain CDR2 region light chain and the CDR3 region of the light chain present in a light chain selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 14; and wherein the heavy chain CDR1 region, the heavy chain CDR2 region and the heavy chain CDR3 region are the heavy chain CDR1 region, the heavy chain CDR2 region and the heavy chain CDR3 region present in a selected heavy chain of the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 16. 2. Antibody according to claim 1, characterized in that it comprises a constant region. 3. Antibody according to claim 2, characterized in that the constant region is IgG1, IgG2, IgG3 or IgG4. 4. Antibody according to claim 1, characterized by the fact that the CDR regions of the heavy chain are the CDR regions of the heavy chain as set out in SEQ ID NO: 2. 5. Antibody according to claim 1, characterized by the fact that the CDR regions of the light chain are the CDR regions of the light chain as set out in SEQ ID NO: 5. 6. Antibody according to claim 5, characterized by the fact that it comprises an IgG4 constant region. 7. Antibody according to claim 5, characterized by the fact that it comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 5 and the heavy chain comprises the sequence of amino acids as set out in SEQ ID NO: 2. 8. Antibody according to claim 1, characterized by the fact that it comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein: i) the VL is a VL in a light chain selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 14; and ii) the VH is a VH in a heavy chain selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 16. 9. Antibody according to claim 8, characterized by the fact that the VL is a light chain VL as set out in SEQ ID NO: 5 and the VH is a heavy chain VH as set out in SEQ ID NO: 2. 10. Use of an antibody or fragment thereof as defined in any one of claims 1 to 9, characterized in that it is for preparing a medicament for the treatment of autoimmune disease or chronic inflammation. 11. Use according to claim 10, characterized in that the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Sjogrens syndrome , lupus nephritis and systemic lupus erythematosus. 12. Antibody or fragment thereof that is capable of binding and blocking TREM-1, characterized by the fact that it comprises a light chain CDR1 region, a light chain CDR2 region and a light chain CDR3 region, a CDR1 region of heavy chain, a heavy chain CDR2 region and a heavy chain CDR3 region, wherein the light chain CDR1 region, the light chain CDR2 region and the light chain CDR3 region are as set forth in SEQ ID NO: 3; and wherein the heavy chain CDR1 region, the heavy chain CDR2 region and the heavy chain CDR3 region are present in a heavy chain selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO : 13, SEQ ID NO: 15 and SEQ ID NO: 16. 13. Antibody according to claim 12, characterized in that it comprises a constant region. 14. Antibody according to claim 13, characterized in that the constant region is IgG1, IgG2 or IgG4. 15. Antibody according to claim 12, characterized in that it comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein: i) the VL is a VL in a chain light as set forth in SEQ ID NO: 3; and ii) the VH is a VH in a heavy chain selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 16. 16. Use of the antibody according to any one of claims 12 to 15, characterized in that it is for the preparation of a medicine for the treatment of an autoimmune disease or chronic inflammation. 17. Use according to claim 16, characterized in that the autoimmune disease is rheumatoid arthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Sjogren's syndrome, lupus nephritis or systemic lupus erythematosus.