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Patent Analysis of

Antibodies that bind peptidoglycan recognition protein 1

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10150809

Application Number

US15/483390

Application Date

10 April 2017

Publication Date

11 December 2018

Current Assignee

NOVO NORDISK A/S

Original Assignee (Applicant)

NOVO NORDISK A/S

International Classification

A61K39/395,C07K16/28,C07K16/00,C07K16/18

Cooperative Classification

C07K16/18,C07K2317/76,C07K2317/565

Inventor

STENNICKE, VIBEKE WESTPHAL,READ, CHRISTINE BRENDER,KUIJPER, JOSEPH LEON,TANG, XIAOTING,HEIPEL, MARK,HJORTH, SIV ANNEGRETHE

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US10150809 Antibodies bind peptidoglycan recognition 1 US10150809 Antibodies bind peptidoglycan recognition 2 US10150809 Antibodies bind peptidoglycan recognition 3
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Abstract

Disclosed herein is a method for identifying TREM-1's ligand and antibodies, or fragments thereof, which are capable of modifying the function of TREM-1's ligand. Antibodies that reduce or block TREM-1 activation may be identified and selected using this method. Antibodies that bind to TREM-1's ligand and reduce TREM-1 activity may be suitable for use as medicaments.

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Claims

1. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein the antibody comprises a CDRH1 sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 15, a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 15, a CDRH3 sequence as set forth in amino acid residues 99 to 108 of SEQ ID NO: 15, a CDRL1 sequence as set forth in amino acid residues 24 to 34 of SEQ ID NO: 16, a CDRL2 sequence as set forth in amino acid residues 51 to 56 of SEQ ID NO: 16, and a CDRL3 sequence as set forth in amino acid residues 89 to 97 of SEQ ID NO: 16.

2. The antibody of claim 1, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 15 and the variable light chain of SEQ ID NO: 16.

3. The antibody of claim 1, which comprises a constant region.

4. The antibody of claim 3, wherein the antibody is IgG1, IgG2, IgG3, or IgG4.

5. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein the antibody comprises a CDRH1 sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 19, a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 19, a CDRH3 sequence as set forth in amino acid residues 99 to 109 of SEQ ID NO: 19, a CDRL1 sequence as set forth in amino acid residues 24 to 33 of SEQ ID NO: 20, a CDRL2 sequence as set forth in amino acid residues 49 to 55 of SEQ ID NO: 20, and a CDRL3 sequence as set forth in amino acid residues 88 to 96 of SEQ ID NO: 20.

6. The antibody of claim 5, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 19 and the variable light chain of SEQ ID NO: 20.

7. The antibody of claim 5, which comprises a constant region.

8. The antibody of claim 7, wherein the antibody is IgG1, IgG2, IgG3, or IgG4.

9. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein the antibody comprises a CDRH1 sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 23, a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 23, a CDRH3 sequence as set forth in amino acid residues 99 to 108 of SEQ ID NO: 23, a CDRL1 sequence as set forth in amino acid residues 24 to 33 of SEQ ID NO: 24, a CDRL2 sequence as set forth in amino acid residues 49 to 55 of SEQ ID NO: 24, and a CDRL3 sequence as set forth in amino acid residues 88 to 96 of SEQ ID NO: 24.

10. The antibody of claim 9, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 23 and the variable light chain of SEQ ID NO: 24.

11. The antibody of claim 9, which comprises a constant region.

12. An isolated 1F95 antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein the antibody comprises a CDRH1 sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 27, a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 27, a CDRH3 sequence as set forth in amino acid residues 99 to 106 of SEQ ID NO: 27, a CDRL1 sequence as set forth in amino acid residues 24 to 33 of SEQ ID NO: 28, a CDRL2 sequence as set forth in amino acid residues 49 to 54 of SEQ ID NO: 28, and a CDRL3 sequence as set forth in amino acid residues 87 to 95 of SEQ ID NO: 28.

13. The antibody of claim 12, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 27 and the variable light chain of SEQ ID NO: 28.

14. The antibody of claim 12, which comprises a constant region.

15. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein the antibody comprises a CDRH1 sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 31, a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 31, a CDRH3 sequence as set forth in amino acid residues 99 to 109 of SEQ ID NO: 31, a CDRL1 sequence as set forth in amino acid residues 24 to 35 of SEQ ID NO: 32, a CDRL2 sequence as set forth in amino acid residues 51 to 57 of SEQ ID NO: 32, and a CDRL3 sequence as set forth in amino acid residues 90 to 98 of SEQ ID NO: 32.

16. The antibody of claim 15, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 31 and the variable light chain of SEQ ID NO: 32.

17. The antibody of claim 15, which comprises a constant region.

18. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1)PGLYRP I and reducing PGLYRP1-mediated TREM-1 activity, wherein the antibody comprises a CDRH1 sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 35, a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 35, and a CDRH3 sequence as set forth in amino acid residues 99 to 109 SEQ ID NO: 35, a CDRL1 sequence as set forth in amino acid residues 24 to 34 of SEQ ID NO: 36, a CDRL2 sequence as set forth in amino acid residues 50 to 56 of SEQ ID NO: 36, and a CDRL3 sequence as set forth in amino acid residues 89 to 96 of SEQ ID NO: 36.

19. The antibody of claim 18, wherein the antibody comprises the variable heavy chain of SEQ ID NO: 35 and the variable light chain of SEQ ID NO: 36.

20. The antibody of claim 18, which comprises a constant region.

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Claim Tree

  • 1
    1. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein
    • the antibody comprises
    • 2. The antibody of claim 1, wherein
      • the antibody comprises
    • 3. The antibody of claim 1, which comprises
      • a constant region.
  • 5
    5. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein
    • the antibody comprises
    • 6. The antibody of claim 5, wherein
      • the antibody comprises
    • 7. The antibody of claim 5, which comprises
      • a constant region.
  • 9
    9. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein
    • the antibody comprises
    • 10. The antibody of claim 9, wherein
      • the antibody comprises
    • 11. The antibody of claim 9, which comprises
      • a constant region.
  • 12
    12. An isolated 1F95 antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein
    • the antibody comprises
    • 13. The antibody of claim 12, wherein
      • the antibody comprises
    • 14. The antibody of claim 12, which comprises
      • a constant region.
  • 15
    15. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1) and reducing PGLYRP1-mediated TREM-1 activity, wherein
    • the antibody comprises
    • 16. The antibody of claim 15, wherein
      • the antibody comprises
    • 17. The antibody of claim 15, which comprises
      • a constant region.
  • 18
    18. An isolated antibody capable of binding to Peptidoglycan recognition protein 1 (PGLYRP1)PGLYRP I and reducing PGLYRP1-mediated TREM-1 activity, wherein
    • the antibody comprises
    • 19. The antibody of claim 18, wherein
      • the antibody comprises
    • 20. The antibody of claim 18, which comprises
      • a constant region.
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Description

FIELD OF THE INVENTION

The present invention relates to the field of immunology. More particularly, the present invention relates to a method for the identification of the ligand that is able to stimulate Triggering Receptor Expressed on Myeloid cells (TREM-1), as well as antibodies that bind TREM-1's ligand. Such antibodies are capable of modulating myeloid cell activation and, therefore, inflammatory responses.

BACKGROUND

Peptidoglycan recognition protein 1 (otherwise known as PGLYRP1, PGRP-S, TNFSF3L, PGRP, TAG7 and PGRPS) is expressed in neutrophils and released upon their activation. PGLYRP1 is highly abundant in diseased tissue and has been shown to play an important role in the clearance of bacterial infections by the innate immune system. The family of PGLYRP proteins (PGLYRP1, PGLYRP2, PGLYRP3, PGLYRP4) all interact with bacterial peptidoglycans (PGNs) but there are important differences in the proteins' PGN binding sites. PGLYRP1 has an additional groove hypothesized to constitute a binding site for an unknown effector or signalling protein (J. Mol. Biol. 347:683-691 (2005)). PGLYRP1 is a highly conserved, 196 amino acid long protein consisting of a signal peptide and a peptidoglycan binding domain.

The identification of a signalling mechanism mediated by PGLYRP1 is important in order to understand and thereby manipulate the function of this protein in various infectious and inflammatory diseases.

TREM-1, likewise, has well-described effects in immune modulation but, thus far, the mechanism leading to TREM-1 mediated immune function has not been understood. TREM-1 is a receptor expressed on myeloid cells, such as monocytes, macrophages and neutrophils. It is a transmembrane protein consisting of 234 amino acids, including a single extracellular immunoglobulin domain and a short cytoplasmic tail. TREM-1 has no apparent signalling motif but, when activated, forms dimers/multimers and mediates signalling by associating with the ITAM-containing signalling adaptor protein, DAP12. Downstream signalling may include phosphorylation of Syk and Zap70. Downstream signalling may include activation of the NFAT, ELK, NK-kappaB transcription factor. When TREM-1 is activated, it triggers the release of pro-inflammatory cytokines, such as TNF-α (TNF-alpha), IL-8 and monocyte chemotactic protein-1, from myeloid cells.

TREM-1 is upregulated in patients with sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) and increasing evidence supports the theory that TREM-1 contributes to the development and progression of inflammatory diseases. The blocking of TREM-1 signalling has furthermore been shown to have therapeutic activity in in vivo mouse models of RA and IBD.

The mode of action of TREM-1 activation has remained elusive because the ligand that activates TREM-1 is not known in the art. Therefore, there is a need in the art for a means of identifying TREM-1's ligand. There is a need in the art for a method of identifying a molecule, such as an antibody, that is capable of reducing, blocking, or interfering with the interaction of TREM-1 with its ligand. There is a need in the art for a molecule, such as an antibody, that is capable of binding TREM-1's ligand and thus reducing, blocking, or interfering with the stimulation of TREM-1 by its ligand. There is a need in the art for a molecule, such as an antibody, that is capable of binding TREM-1's ligand. There is a need in the art for a molecule, such as an antibody, that is capable of binding TREM-1's ligand and thus blocking TREM-1 activation and signalling. There is a need in the art for a molecule, such as an antibody, that is capable of binding TREM-1's ligand and thus reducing or blocking cytokine release from a myeloid cell expressing TREM-1.

Disclosed herein is a method and assay for identifying TREM-1's ligand and molecules, such as antibodies, that are capable of binding the ligand of TREM-1. Described herein are antibodies that are capable of influencing TREM-1 activation. Thus, the antibodies disclosed herein are suitable for use as pharmaceuticals. Antibodies that bind the ligand of TREM-1 and that reduce or block the interaction of TREM-1 with its ligand may have a substantial impact upon the quality of life of individuals with chronic inflammatory diseases such as rheumatoid arthritis, psoriatic arthritis and inflammatory bowel diseases.

SUMMARY

The invention relates to a method for identifying TREM-1's ligand and molecules, such as antibodies, that bind to TREM-1's ligand, herein identified as being PGLYRP1. The invention also relates to PGLYRP1 antibodies that may be identified by means of the invented method. Thus, the invention relates to PGLYRP1 antibodies that are capable of modifying the activation of TREM-1 by PGLYRP1, such as PGLYRP1 antibodies that are capable of reducing TREM-1 activity (signalling and/or activation) by PGLYRP1. Antibodies that reduce TREM-1 activity may be used for the treatment of inflammation.

The method for identifying a TREM-1 ligand comprises (a) culturing a cell expressing TREM-1, a signalling protein for TREM-1 and a reporter construct that is activated by said signalling protein; (b) detecting, preferably quantifying, the activity of said cell expressing TREM-1 when it is contacted with a cell, a compound or a fluid, such as a biological fluid or a tissue, that triggers TREM-1 activation; (c) contacting the culture of (b) with a TREM-1-activating component; (d) isolating the component that binds TREM-1 and (e) characterising the isolated component. The ligand for TREM-1, identified by means of the current invention as being PGLYRP1, may be used to modify the activity of TREM-1.

The method for identifying a molecule that specifically binds PGLYRP1 and that modifies TREM-1 mediated cellular activity, comprises: (a) culturing the cell according to any one of embodiments 1-18; (b) detecting, preferably quantifying, the activity of said cell expressing TREM-1 when it is contacted with PGLYRP1 and, optionally, a multimerisation agent such as PGN; (c) contacting the culture of (b) with a molecule that specifically binds PGLYRP1; and (d) detecting, preferably quantifying, that the activity of said cell expressing TREM-1 is less than or more than its activity as measured in (b).

The method for identifying a PGLYRP1 antibody that modifies TREM-1 mediated cellular activity comprises: (a) culturing a cell expressing TREM-1, a signalling protein for TREM-1 and a reporter construct that is activated by said signalling protein; (b) detecting, preferably quantifying, the activity of said cell expressing TREM-1 when it is contacted with PGLYRP1 and, optionally, in combination with a multimerising agent such as PGN; (c) contacting the culture of (b) with an antibody that binds PGLYRP1; and (d) detecting, preferably quantifying, that the activity of said cell expressing TREM-1 is less than or more than its activity as measured in (b).

One method of identifying a PGLYRP1 antibody that decreases TREM-1 mediated cellular activity comprises: (a) culturing a cell such as a T-cell expressing TREM-1, a signalling protein such as DAP12 and a reporter gene such as luciferase or beta-galactosidase; (b) incubating said cell with an activated neutrophil and, optionally, in combination with a multimerising agent such as PGN (c) detecting, preferably quantifying, the luminescence of said cell; (d) contacting the culture of the cell and the activated neutrophil with a PGLYRP1 antibody; and (e) detecting, preferably quantifying, that the luminescence of said cell is less than the activity measured in (c).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that neutrophils activate the BWZ-TREM-1 reporter cell line. The BWZ reporter cell line expresses human TREM-1 and mediates activation of a NFAT-linked beta (β)-galactosidase reporter gene that may be quantitated by luminescence using the Beta-Glo Assay System kit from Promega, Denmark. The figure illustrates activation of the reporter cell line when cultured with neutrophils in the presence of either cytokine cocktails containing TNF-alpha (α), IL-6, IFN-gamma (γ) and GM-CSF or Toll-like Receptor (TLR) activating ‘cocktails’ (TLRL and TLR2 mix (tlrl-kit2hm, Invivogen, Sigma-Aldrich, Denmark), as well as in the presence of separate components of these activation mixtures.

FIGS. 2A-2D show the flow cytometric staining of PGN-stimulated neutrophils with a TREM-1-tetramer recombinant protein. Control protein does not bind (FIG. 2A), whereas a TREM-1-tetramer (SEQ ID NO: 2) binds to a subset of PGN-activated neutrophils (FIG. 2B), and this can be competed by another TREM-1 protein (FIG. 2D) but not by a control protein (FIG. 2C), confirming the specificity of the interaction.

FIG. 3A and FIG. 3B: PGLYRP1 is identified by Immunoprecipitation (IP)/Mass spectroscopy (MS) with TREM-1. Soluble TREM-Fc was incubated with PGN-activated neutrophils, crosslinked, immunoprecipitated followed by cleanup, trypsin digestion and mass spectroscopy. The immunoprecipitation of the TREM-1 binding proteins resulted in 3 specific proteins, 73 proteins overlap with the control protein, and 72 were precipitated by the control protein alone (background (FIG. 3A)). The table (FIG. 3B) shows the results from the TREM-1 specific immunoprecipitation and subsequent mass spectroscopy.

FIG. 4A and FIG. 4B show that soluble TREM-1 binds to PGLYRP1. Shown both by flow cytometric staining with TREM-1 on HEK293 transfectants expressing recombinant PGLYRP1 (FIG. 4A) and by Biacore and ForteBio analyses of the interaction between PGLYRP1 and a TREM-1 tetramer (SEQ ID NO: 2). Soluble human PGLYRP1 bound immobilized human TREM-1 in the presence and absence of 10 μg/ml soluble E. coli peptidoglycan (a PGN) (FIG. 4B). PGN by itself also bound immobilized human PGLYRP1 (FIG. 4C), and soluble human TREM-1 bound to immobilized PGLYRP1 before and after the PGLYRP1 surface was exposed to and bound by PGN (FIG. 4D).

FIG. 5A and FIG. 5B show activation of a TREM-1 reporter cell line by recombinant PGLYRP1. Stimulation of a TREM-1 reporter cell line with recombinant PGLYRP1 (cat. no. 2590-PG-050 R&D Systems Minneapolois Minn., USA), as well as PGLYRP1 generated in-house (SEQ ID NO: 1) leads to a dose dependent response in the presence of PGN (FIG. 5A). This PGLYRP1-induced response can be specifically blocked by TREM-1-Fc fusion protein (FIG. 5B) validating a TREM-1 specific signal.

FIGS. 6A-6G show that monoclonal anti-PGLYRP1 antibodies can block the TREM-1 response in the reporter assay stimulated with PGN-activated neutrophils. Examples of antibody hybridoma clone supernatants screened in various plates for their ability to block the activation signal. A few antibodies (those below the dotted line, such as F10, F95) are able to block this signal. Black dots represent isotype control in each plate (FIG. 6A). FIG. 6B shows testing from another fusion given rise to 2 additional blocking PGLYRP1 antibodies M-hPGRPS-2F5 and -2F7. Testing of commercially available anti-PGLYRP1 mAbs show that they are not able to block this signal even at high doses, whereas the polyclonal, commercially available PGLYRP1 pAb (AF2590, R&D Systems Minneapolois Minn., USA) can. FIG. 6C shows a comparison of the commercially available anti-PGLYRP1 mAb 188C424 from Thermo Scientific (Thermo Scientific, Waltham Mass., USA) with an isotype control (MAB002) and the polyclonal anti-PGLYRP1 pAb (AF2590). FIG. 6D shows a comparison of the commercially available anti-PGLYRP1 mAb 4H230 (US Biological, Salem Mass., USA) with an isotype control (MAB002) and the polyclonal anti-PGLYRP1 pAb (AF2590). FIG. 6E shows a comparison of the commercially available anti-PGLYRP1 mAb 9A319 (US Biological, Salem Mass., USA) with an isotype control (MAB002) and the polyclonal anti-PGLYRP1 pAb (AF2590), FIG. 6F shows a comparison of the commercially available anti-PGLYRP1 mAb 6D653 (Santa Cruz Biotechnology, Santa Cruz Calif., USA) with an isotype control (MAB002) and the polyclonal anti-PGLYRP1 pAb (AF2590). The slight decrease in activity seen for the SC 6D653 mAb appears to be due to the azide-containing formulation since a PGLYRP1 independent signal triggering TREM-1 with 1 ug/ml platebound anti-TREM-1 mAb shows the same phenomenon (FIG. 6G).

FIG. 7 shows that the TREM-1 ligand present in human RA synovial fluid is able to stimulate TREM-1.

    • FIG. 7 shows that plate-bound agonistic anti-TREM-1 mAb (R&D MAB1278, Minneapolis, Minn., USA) stimulates TREM-1 (star) and that an RA synovial fluid (SF) sample to which PGN has been added displayed hTREM-1 ligand activity which can be neutralized by a PGLYRP1 polyclonal antibody (AF2590), indicating that TREM-1 activity is PGLYRP1 dependent.

FIG. 8 shows the overall structure of the Type II PGLYPRI construct. IC represents the intracellular domain, TM the transmembrane domain—both of which originate from MDL-1 protein sequence, in combination with EC (extracellular) domain of hPGLYRP1.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 represents the amino acid sequence of mature, full length hPGLYRP1 peptide sequence.

SEQ ID NO: 2 represents a recombinant protein sequence containing the following elements: human TREM-1 ECD, linker peptide, human TREM-1 ECD, human allotype IgG1 Fc with 7 mutations differing from wild type. (L15A, L16E, G18A, A111S, P112S, D137E, L139M)

SEQ ID NO: 3 represents human allotype IgG1 Fc with 5 mutations differing from wild type: L15A, L16E, G18A, A111S, P112S.

SEQ ID NO: 4 represents a recombinant protein sequence containing the following elements from N to C terminus: 6×HIS tag, two copies of the streptavidin binding protein domain (SBP), GS linker, C terminus of cartilage oligomeric protein (COMP), GS linker, hDCIR-ECD.

SEQ ID NO: 5 represents a recombinant protein sequence containing the following elements: hTREM-1-ECD, GS linker, C terminus of cartilage oligomeric protein (COMP), GS linker, two copies of the streptavidin binding protein domain (SBP), 6×HIS tag.

SEQ ID NO: 6 represents a recombinant protein sequence containing the following elements in order from N to C terminus, human CD83 ECD, G4Sx3 linker peptide, human CD83 ECD, human allotype IgG1 Fc mutant.

SEQ ID NO: 7 represents full length human PGLYRP1 coding cDNA sequence with C terminal GPI signal sequence. This sequence was cloned into pcDNA3.1zeo(+) (Invitrogen: V860-20, Carlsbad, Calif., USA) via EcoR1 and Xho1 restriction sites.

SEQ ID NO: 8 represents mature full length hTREM-1 ECD (aa. 21-200).

SEQ ID NO: 9 represents cDNA for a CD33 leader tandem hTREM extracellular domains separated by a G4Sx3 linker. A synthetic cDNA with a 5′ EcoRI restriction site, a GCCACC Kozak sequence the CD33 leader sequence followed by the extracellular domain of human TREM-1 (aa17-200) with a interspaced KpnI restriction site and glycine-glycine-glycine-serine spacer repeated three times (G4Sx3) followed by an additional copy of the extracellular domain of human TREM-1 (aa17-200) and an Apa1 site to allow cloning.

SEQ ID NO: 10 represents pentameric hTREM-COMP-SBP38x2-6HIS. EcoR1 site and Kozak are 5′ and BamH1 site is 3′ of ORF.

SEQ ID NO: 11 represents hCD83 tetramer cloned into pJSV002-hFc6mut vector with EcoR1 and Apa1. EcoR1 site and Kozak are 5′ and Apa1 site is 3′ of ORF.

SEQ ID NO: 12 represents pentameric 6HIS-SBP38x2-COMP-hDCIR. EcoR1 site and Kozak 5′ and BamH1 3′ of ORF.

SEQ ID NO: 13 represents the nucleic acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F36, mAb 0182).

SEQ ID NO: 14 represents the nucleic acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F36, mAb 0182).

SEQ ID NO: 15 represents the amino acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F36, mAb 0182).

SEQ ID NO: 16 represents the amino acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F36, mAb 0182).

SEQ ID NO: 17 represents the nucleic acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F10).

SEQ ID NO: 18 represents the nucleic acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F10).

SEQ ID NO: 19 represents the amino acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F10).

SEQ ID NO: 20 represents the amino acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F10).

SEQ ID NO: 21 represents the nucleic acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 22 represents the nucleic acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 23 represents the amino acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 24 represents the amino acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 25 represents the nucleic acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 26 represents the nucleic acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 27 represents the amino acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 28 represents the amino acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 29 represents the nucleic acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 30 represents the nucleic acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 31 represents the amino acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 32 represents the amino acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 33 represents the nucleic acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 34 represents the nucleic acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 35 represents the amino acid sequence of the variable heavy chain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 36 represents the amino acid sequence of the variable light chain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 37 represents the amino acid sequence of Type II 1.0 PGLYRP1.

SEQ ID NO: 38 represents the amino acid sequence of Type II 2.0 PGLYRP1.

SEQ ID NO: 39 represents the amino acid sequence of an epitope tag.

SEQ ID NO: 40 represents the amino acid sequence of full-length human PGLYRP2.

SEQ ID NO: 41 represents the amino acid sequence of full-length human PGLYRP3.

SEQ ID NO: 42 represents the amino acid sequence of full-length human PGLYRP4.

SEQ ID NO: 43 represents the nucleic acid sequence of hCD33-hTreml ECD(aa17-200)-Fc6mut.

SEQ ID NO: 44 represents the amino acid sequence of hCD33-hTreml ECD(aa17-200)-Fc6mut.

SEQ ID NO: 45 represents the nucleic acid sequence for hCD33-hTremL1 ECD(aa16-162)-Fc6mut.

SEQ ID NO: 46 represents the amino acid sequence of hCD33-hTremL1 ECD(aa16-162)-Fc6mut.

SEQ ID NO: 47 represents the nucleic acid sequence for hCD33-hTremL2 ECD(aa19-268)-Fc6mut.

SEQ ID NO: 48 represents the amino acid sequence of hCD33-hTremL2 ECD(aa19-268)-Fc6mut

SEQ ID NO: 49 represents the nucleic acid sequence of hCD33-hTREM2-Fc6mut dimer

SEQ ID NO: 50 represents the amino acid sequence of the hCD33-hTREM2-Fc6mut dimer.

SEQ ID NO: 51 represents the nucleic acid sequence of a primer.

SEQ ID NO: 52 represents the nucleic acid sequence of a primer.

SEQ ID NO: 53 represents the amino acid sequence of hCD33.

DESCRIPTION

The invention relates to a method for the identification of molecules such as antibodies that are capable of specifically binding TREM-1's signalling partner, herein identified as being PGLYRP1, and influencing PGLYRP1 binding with its signalling partner, TREM-1. PGLYRP1 may be used to modify the activity of TREM-1. Hence, the invention relates to molecules, such as antibodies, that influence inflammatory responses that are mediated by PGLYRP1. Antibodies that are capable of binding to PGLYRP1 and that influence TREM-1 activation and signalling have been created and identified.

A method or assay for the identification of TREM-1's ligand and for identification of molecules, such as antibodies, that are capable of specifically binding PGLYRP1 and reducing or blocking PGLYRP1 activation of TREM-1 may be created as follows:

A first cell or population of first cells is transfected with genes encoding TREM-1 or fragments thereof, a signalling protein and a reporter construct. The cell may be of haematopoietic origin, such as a myeloid cell, it may be a T-cell or it may be any other cell type that is capable of being transfected and expressing such molecules. The signalling protein may be any protein that is capable of transmitting or conveying a signal, either directly or indirectly, from TREM-1 to the reporter construct and may include DAP10, DAP12, TCR zeta, Fc gammaRIII, an Fc receptor, or any other protein that is capable of transmitting or conveying a signal from TREM-1 to the reporter construct. Alternatively, the signalling protein may be a TREM-1/signalling chimera molecule. The reporter construct comprises a transcription factor and a reporter gene, which in turn encodes a reporter protein that produces a detectable signal, such as a quantifiable signal. The transcription factor may be NFAT or NFkB or any other suitable transcription factor known in the art. The reporter gene may encode beta (β)-galactosidase, luciferase, green fluorescent protein (GFP), chloramphenicol transferase or any other reporter protein capable of producing a detectable signal. One suitable cell line that may be used for this bioassay is the BWZ.36/hTREM-1:DAP12:NFAT-LacZ T-cell (herein also identified as the “BWZ/hTREM-1 reporter cell”), the creation of which is described in detail in the examples. When activated, the BWZ/hTREM-1 reporter cell produces beta (β)-galactosidase, the production of which may be measured using equipment or kits known in the art, such as Beta Glow™ (Promega E4720, Madison, Wis., USA).

The first cell or population of first cells may be activated by incubation with PGLYRP1 and, optionally, a multimerisation agent. The optional multimerisation agent acts as a scaffold for PGLYRP1 and may be peptidoglycan (PGN), neutrophil extracellular traps (NETs), hyaloronic acid, a proteoglycan structure such as versican, aggrecan, decorin or fibrin, or any other naturally occurring matrix structure or molecule that is able to multimerise or present PGLYRP1. The first cell may be activated by incubation with one or more second cell(s) that express PGLYRP1 on its/their surface, or intracellularly. An example of intracellular expression may be the storage of PGLYRP1 in secretory granules. The second cell may thus be any cell (or population of cells) that expresses or is transfected with a gene encoding PGLYRP1 and that expresses PGLYRP1 on its surface. Such a second cell may be a prokaryotic or a eukaryotic cell, such as a mammalian cell, such as a CHO cell, a BHK cell or a HEK cell. The second cell may also be an activated neutrophil. Neutrophils may be obtained from the whole blood or tissue of an individual and used either in bulk or as purified neutrophils. Any agent that mimics the bacterial activation of neutrophils, such as peptidoglycan (PGN) from the cell wall of a bacterium, such as PGN-SA, PGN-EB, PGN-EC, PGN-BS (InVivogen, tlrl-pgnsa, SanDiego, Calif.), may be used to activate a neutrophil.

The activity of the first cell or population of first cells is then detected and, preferably, measured.

The culture of the first cell and the second cell(s) expressing PGLYRP1 and/or the culture of the first cell incubated with PGLYRP1 and, optionally, a multimerisation agent such as PGN, is contacted with an antibody that has been raised against PGLYRP1. The activity of the first cell or population of first cells is detected, and preferably measured.

In this way, antibodies that are capable of binding TREM-1's ligand, PGLYRP1, and that influence the interaction of PGLYRP1 with TREM-1 may be identified. PGLYRP1 antibodies that cause an increase in the activity of the first cell enhance the interaction of PGLYRP1 with TREM-1 and are herein identified as “stimulating PGLYRP1 antibodies”. PGLYRP1 antibodies that cause a decrease in the activity of the first cell reduce, interfere, or block the interaction of PGLYRP1 with TREM-1 and are herein identified as “inhibitory PGLYRP1 antibodies”. Inhibitory PGLYRP1 antibodies reduce or block TREM-1 activation and signalling.

Hence, the present invention relates to a method of characterising the function of PGLYRP1 antibodies. Antibodies capable of specifically binding PGLYRP1 and that have any effect upon TREM-1 activation and downstream signalling are herein referred to as “functional PGLYRP1 antibodies”. Consequently, the term “functional PGLYRP1 antibodies” is intended to encompass both stimulating PGLYRP1 antibodies and inhibitory PGLYRP1 antibodies.

Furthermore, the present invention relates to antibodies that are capable of specifically binding PGLYRP1 and reducing, interfering with, or blocking its interaction with TREM-1, hence reducing TREM-1 activation and downstream signalling. Antibodies of the invention may have an immunoregulatory function, reducing the cytokine production of myeloid cells expressing TREM-1. For example, antibodies of the invention may reduce or prevent release of TNF-alpha (α), IL-1beta (b), IL-6, IFN-gamma (γ), MIP-1beta (b), MCP-1, IL-8 and/or GM-CSF from myeloid cells such as macrophages and/or neutrophils and/or myeloid cells in diseased tissue such as synovial tissue. Antibodies of the invention may be capable of down-regulating neutrophil responses.

PGLYRP1 antibodies according to the invention may reduce or block TREM-1 activation by means of one or a combination of several different mechanisms, affecting TREM-1 directly or indirectly. Antibodies of the invention may prevent PGLYRP1 from creating a functional complex with TREM-1.

Antibodies of the invention may block PGLYRP1 function by reducing or blocking TREM-1 activation and downstream signalling.

The present invention also relates to inhibitory PGLYRP1 antibodies that may be identified by other means than the method disclosed herein.

Antibodies of the invention may be capable of binding both human PGLYRP1 and PGLYRP1 from a species other than a human being. The term “PGLYRP1”, as used herein, thus encompasses any naturally occurring form of PGLYRP1 which may be derived from any suitable organism, such as an invertebrate species or a vertebrate species. PGLYRP1 for use as described herein may be vertebrate PGLYRP1, such as mammalian PGLYRP1, such as PGLYRP1 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 a cow, sheep, pig or camel), among others. Preferably, the PGLYRP1 is human PGLYRP1 (SEQ ID NO: 1). The PGLYRP1 may be a mature form of PGLYRP1 such as a PGLYRP1 protein that has undergone post-translational processing within a suitable cell. Such a mature PGLYRP1 protein may, for example, be glycosylated. The PGLYRP1 may be a full length PGLYRP1 protein. The PGLYRP1 may be a splice variant.

Antibodies of the invention may also be capable of specifically binding variants of PGLYRP1 such as SEQ ID NO: 37 (Type II 1.0 PGLYRP1) and/or SEQ ID NO: 38 (Type II 1.0 PGLYRP1).

Antibodies of the invention may be capable of influencing, such as inhibiting/reducing/blocking, the activity (signalling and/or activation) of both human TREM-1 and TREM-1 from another species than a human being. The term “TREM-1”, as used herein, thus encompasses any naturally occurring form of TREM-1 which may be derived from any suitable organism. For example, TREM-1 for use as described herein may be vertebrate TREM-1, such as mammalian TREM-1, such as TREM-1 from a primate (such as a human, a chimpanzee, a cynomolgous monkey or a rhesus monkey); a rodent (such as a mouse or a rat), a lagomorph (such as a rabbit), or an artiodactyl (such a cow, sheep, pig or camel), among others. Preferably, the TREM-1 is human TREM-1. The 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. The TREM-1 may be a full length TREM-1 protein. The TREM-1 may be a splice variant.

The term “antibody” herein refers to a protein, derived from a germline immunoglobulin sequence, which is capable of specifically binding to PGLYRP1 or a portion thereof. The term includes full length antibodies of any isotype (that is, IgA, IgE, IgG, IgM and/or IgY) and any single chain or fragment thereof. An antibody that specifically binds to PGLYRP1, or portion thereof, may bind exclusively to PGLYRP1, or portion thereof, or it may bind to a limited number of homologous antigens, or portions thereof.

Antibodies of the invention may be monoclonal antibodies, in the sense that they may be directly or indirectly derived from a single clone of a B lymphocyte. An antibody of the invention may be a monoclonal antibody, with the proviso that it is not 188C424 (Thermo Scientific), 4H230 or 9A319 (US Biological) or Clone 6D653 (Santa Cruz Biotechnology).

Antibodies of the current invention may be isolated. The term “isolated antibody” refers to an antibody that has been separated and/or recovered from another/other component(s) of its natural environment and/or purified from a mixture of components in its natural environment.

Antibodies may be recombinantly expressed in prokaryotic cells, eukaryotic cells or an acellular system derived from cellular extracts. The prokaryotic cell may 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, 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 a cow, sheep, pig or camel). Suitable mammalian cell lines include, but are not limited to, HEK293 cells, CHO cells and HELA cells. PGLYRP1 antibodies may also be produced by means of other methods known to the person skilled in the art, such as a phage display or a yeast display. Antibodies of the invention may be raised in vivo by immunising a suitable mammal with PGLYRP1, a cell expressing PGLYRP1 or a combination of both.

PGLYRP1 antibodies may be produced, screened and purified using, for example, the methods described in the Examples. In brief, any suitable mouse, including a PGLYRP1 knock-out (KO) mouse or a TREM-1 KO mouse, may be immunised with PGLYRP1, a cell expressing PGLYRP1 or a combination of both. Primary screening of hybridoma supernatants may be performed using direct ELISA or FMAT and secondary screening may be performed using flow cytometry. Positive hybridoma supernatants, as well as purified antibodies, may then be screened for binding to, for example, full length PGLYRP1. Positive hybridoma supernatants or purified antibodies may then be tested for their ability to reduce or block PGLYRP1-stimulation of TREM-1-bearing cells. The method of the current invention may be used for this purpose.

Full-length antibodies of the invention may comprise at least four polypeptide chains: that is, two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. One immunoglobulin sub-class of particular pharmaceutical interest is the IgG family, which may be sub-divided into isotypes IgG1, IgG2, IgG3 and IgG4. IgG molecules are composed of two heavy chains, interlinked by two or more disulfide bonds, and two light chains, each attached to a heavy chain by a disulfide bond. A heavy chain may comprise a heavy chain variable region (VH) and up to three heavy chain constant (CH) regions: CH1, CH2 and CH3. A light chain may comprise a light chain variable region (VL) and a light chain constant region (CL). VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). VH and VL regions are typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy and light chains form a [binding] domain that is capable of interacting with an antigen (PGLYRP1), whilst the constant region of an antibody may 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.

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), single-chain Fv (scFv; see e.g. Bird et al., Science 1988; 242:42S-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (typically the VH and CHI domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., III et al. Protein Eng 1997; 10: 949-57); camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, where the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 2S: 1126-1136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201.

Certain antigen-binding fragments of antibodies may be suitable in the context of the current invention, as it has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-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 human PGLYRP1 or PGLYRP1 from another species, as described herein. Examples of antigen-binding fragments include Fab, Fab′, F(ab)2, F(ab′)2, F(ab)S, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv; see e.g. Bird et al., Science 1988; 242:42S-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (typically the VH and CHI domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., III et al. Protein Eng 1997; 10:949-57); camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, where the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 2S:1126-1136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

An antibody of the invention may be a human antibody or a humanised 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 human germline immunoglobulin sequences. (For example, a human antibody may have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.) Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

Such a human antibody may 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 nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

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

Human antibodies may be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.

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.

The term “humanised antibody”, as used herein, refers to a human/non-human chimeric antibody that contains one or more sequences (CDR regions) derived from a non-human immunoglobulin. A humanised antibody is, thus, a human immunoglobulin (recipient antibody) in which at least residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as from a mouse, rat, rabbit, or non-human primate, which have the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. An example of such a modification is the introduction of one or more so-called back-mutations.

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

The term “humanised antibody derivative” refers to any modified form of the humanised antibody, such as a conjugate of the antibody and another agent or antibody.

The term “chimeric antibody”, as used herein, refers to an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes that originate from different species. For example, the variable segments of genes from a mouse monoclonal antibody may be joined to human constant segments.

The fragment crystallisable region (“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 may interact with cell surface receptors called Fc receptors, as well as some proteins of the complement system. The Fc region enables antibodies to interact with the immune system. In one aspect of the invention, antibodies may be engineered to include modifications within the Fc region, typically to alter one or more of its functional properties, such as serum half-life, complement fixation, Fc-receptor binding, protein stability and/or antigen-dependent cellular cytotoxicity, or lack thereof, among others. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Preferably, a modified Fc domain comprises one or more, and perhaps all of the following mutations that will result in decreased affinity to certain Fc receptors (L234A, L235E, and G237A) and in reduced C1q-mediated complement fixation (A330S and P331S), respectively (residue numbering according to the EU index).

The isotype of an antibody of the invention may be IgG, such as IgG1, such as IgG2, such as IgG4. If desired, the class of an antibody may be “switched” by known techniques. For example, an antibody that was originally produced as an IgM molecule may be class switched to an IgG antibody. Class switching techniques also may 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. Engineering of antibodies to generate constant region chimeric molecules, by combination of regions from different IgG subclasses, can also be performed.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further for instance in U.S. Pat. No. 5,677,425 by Bodmer et al.

The constant region may further be modified to stabilise the antibody, e.g., to reduce the risk of a bivalent antibody separating into two monovalent VH-VL fragments. For example, in an IgG4 constant region, residue S241 may be mutated to a proline (P) residue to allow complete disulphide bridge formation at the hinge (see, e.g., Angal et al., Mollmmunol. 199S; 30:105-8).

Antibodies or fragments thereof may also be defined in terms of their complementarity-determining regions (CDRs). The term “complementarity-determining region” or “hypervariable region”, when used herein, refers to the regions of an antibody in which amino acid residues involved in antigen binding are situated. The CDRs are generally comprised of amino acid residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and/or those residues from a “hypervariable loop” (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as “Kabat position”, “Kabat residue”, and “according to Kabat” herein 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 or additional amino acids corresponding to a shortening of, or insertion into, a framework (FR) or CDR of the variable domain. For example, a heavy chain variable domain may include amino acid insertions (residue 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. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

The term “framework region” or “FR” residues refer to those VH or VL amino acid residues that are not within the CDRs, as defined herein.

An antibody of the invention may comprise a CDR region from one or more of the specific antibodies disclosed herein, such as a CDR region from within SEQ ID NOs: 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35 or 36.

The 1F36 antibody has a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 16. An antibody of the invention may comprise this variable heavy chain sequence and/or this variable light chain sequence. The 1F36 antibody has the CDR sequences shown at amino acids 31 to 35, 50 to 66 and 99 to 108 of SEQ ID NO: 15 and amino acids 24 to 34, 51 to 56 and 89 to 97 of SEQ ID NO: 16. An antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (SYWMN) of SEQ ID NO: 15, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (MIHPSDSETRLNQKFKD) of SEQ ID NO: 15, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 108 (DYSDYDGFAY]) of SEQ ID NO: 15, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

An antibody according to the invention may comprise: a CDRL1 sequence that corresponds to amino acid residues 24 to 34 (RASQSISDYLH) of SEQ ID NO: 16, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 51 to 56 (ASQSIS) of SEQ ID NO: 16, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 89 to 97 (QNGHSFPLT) of SEQ ID NO: 16, wherein one or two of these amino acid residues may be substituted with a different amino acid.

The 1F10 antibody has a heavy chain as shown in SEQ ID NO: 19 and a light chain as shown in SEQ ID NO: 20. An antibody of the invention may comprise this variable heavy chain sequence and/or this variable light chain sequence. The 1F10 antibody has the CDR sequences shown at amino acids 31 to 35, 50 to 66 and 99 to 109 of SEQ ID NO: 19 and amino acids 24 to 33, 49 to 55 and 88 to 96 of SEQ ID NO: 20. An antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (DYNMY) of SEQ ID NO: 19, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (YIDPYNGDTSYNQKFKG) of SEQ ID NO: 19, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 109 (GDYGNPFYLDY) of SEQ ID NO: 19, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

An antibody according to the invention may comprise a CDRL1 sequence that corresponds to amino acid residues 24 to 33 (SVSSSVNYMY) of SEQ ID NO: 20, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 49 to 55 (DTSKLPS) of SEQ ID NO: 20, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 88 to 96 (QQWTSNPPT) of SEQ ID NO: 20, wherein one or two of these amino acid residues may be substituted with a different amino acid.

The 1F105 antibody has a heavy chain as shown in SEQ ID NO: 23 and a light chain as shown in SEQ ID NO: 24. An antibody of the invention may comprise this variable heavy chain sequence and/or this variable light chain sequence. The 1F105 antibody has the CDR sequences shown at amino acids 31 to 35, 50 to 66 and 99 to 108 of SEQ ID NO: 23 and amino acids 24 to 33, 49 to 55 and 88 to 96 of SEQ ID NO: 24. An antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (DTYIH) of SEQ ID NO: 23, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (RIDPANDDTKYDPNFQG) of SEQ ID NO: 23, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 108 (SDNSDSWFAY) of SEQ ID NO: 23, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

An antibody according to the invention may comprise: a CDRL1 sequence that corresponds to amino acid residues 24 to 33 (SVSSSVNFMN) of SEQ ID NO: 24, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 49 to 55 (DTSKLAP) of SEQ ID NO: 24, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 88 to 96 (HQWSSYSLT) of SEQ ID NO: 24, wherein one or two of these amino acid residues may be substituted with a different amino acid.

The 1F95 antibody has a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 28. An antibody of the invention may comprise this variable heavy chain sequence and/or this variable light chain sequence. The 1F95 antibody has the CDR sequences shown at amino acids 31 to 35, 50 to 66 and 99 to 106 of SEQ ID NO: 27 and amino acids 24 to 33, 49 to 54 and 87 to 95 of SEQ ID NO: 28. An antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (DYNMH) of SEQ ID NO: 27, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (YVDPYDGGTSSNQKFKG) of SEQ ID NO: 27, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 106 (EVPYYFDY) of SEQ ID NO: 27, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

An antibody according to the invention may comprise: a CDRL1 sequence that corresponds to amino acid residues 24 to 33 (VASSSVTYMY) of SEQ ID NO: 28, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 49 to 54 (THPLAS) of SEQ ID NO: 28, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 87 to 95 (PHWNTNPPT) of SEQ ID NO: 28, wherein one or two of these amino acid residues may be substituted with a different amino acid.

The 2F5 antibody has a heavy chain as shown in SEQ ID NO: 31 and a light chain as shown in SEQ ID NO: 32. An antibody of the invention may comprise this variable heavy chain sequence and/or this variable light chain sequence. The 2F5 antibody has the CDR sequences shown at amino acids 31 to 35, 50 to 66 and 99 to 109 of SEQ ID NO: 31 and amino acids 24 to 33, 49 to 55 and 88 to 96 of SEQ ID NO: 32. An antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (DYYMY) of SEQ ID NO: 31, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (AISDDSTYTYYPDSVKG) of SEQ ID NO: 31, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 109 (GGYGNLYAMDY) of SEQ ID NO: 31, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

An antibody according to the invention may comprise: a CDRL1 sequence that corresponds to amino acid residues 24 to 35 (TASSSVSSSYLH) of SEQ ID NO: 32, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 51-57 (STSNLAS) of SEQ ID NO: 32, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 90-98 (HQYHRSPFT) of SEQ ID NO: 32, wherein one or two of these amino acid residues may be substituted with a different amino acid.

The 2F7 antibody has a heavy chain as shown in SEQ ID NO: 35 and a light chain as shown in SEQ ID NO: 36. An antibody of the invention may comprise this variable heavy chain sequence and/or this variable light chain sequence. The 2F5 antibody has the CDR sequences shown at amino acids 31 to 35, 50 to 66 and 99 to 109 of SEQ ID NO: 35 and amino acids 24 to 34, 50 to 56 and 89 to 96 of SEQ ID NO: 36. An antibody of the invention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (NYVMH) of SEQ ID NO: 35, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (WINPFNDGTNYNENFKN) of SEQ ID NO: 35, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 109 (SGFITTLIEDY) of SEQ ID NO: 35, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

An antibody according to the invention may comprise: a CDRL1 sequence that corresponds to amino acid residues 24 to 34 (KASESVGSFVS) of SEQ ID NO: 36, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 50 to 56 (GASNRYT) of SEQ ID NO: 36, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 89 to 96 (GQYYTHPT) of SEQ ID NO: 36, wherein one or two of these amino acid residues may be substituted with a different amino acid.

The term “antigen” (Ag) refers to the molecular entity used to immunise an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag. Herein, Ag is termed more broadly and 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, e.g. phage display, used for generating the Ab.

The term “epitope”, as used herein, 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 on an Ag to which an Ab specifically binds, i.e. the area or region in physical contact with the Ab. Physical contact may be defined using various criteria (e.g., a distance cut-off of 2-6 Å, such as 3 Å, such as 4 Å, such as 5 Å; or solvent accessibility) for atoms in the Ab and Ag molecules. A protein epitope may comprise amino acid residues in the Ag that are directly involved in binding to a Ab (also called the immunodominant component of the epitope) and other amino acid residues, which are not directly involved in binding, such as amino acid residues of the Ag which are effectively blocked by the Ab, i.e. amino acid residues within the “solvent-excluded surface” and/or the “footprint” of the Ab.

The term epitope herein comprises both types of binding region in any particular region of PGLYRP1 that specifically binds to a PGLYRP1 antibody. PGLYRP1 may comprise a number of different epitopes, which may include, without limitation, conformational epitopes which consist of one or more non-contiguous amino acids located near each other in the mature PGLYRP1 conformation and post-translational epitopes which consist, either in whole or part, of molecular structures covalently attached to PGLYRP1, such as carbohydrate groups. PGLYRP1 may also comprise linear epitopes.

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. The 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 relies on a unique principle, the description of an epitope is intimately linked to the method by which it has been determined. Thus, depending on the epitope mapping method employed, the epitope for a given Ab/Ag pair may be described differently.

At its most detailed level, the epitope for the interaction between the Ag and the Ab can be described by the spatial coordinates defining the atomic contacts present in the Ag-Ab interaction, as well as information about their relative contributions to the binding thermodynamics. At a less detailed level, the epitope can be characterized by the spatial coordinates defining the atomic contacts between the Ag and Ab. At an even less detailed level the epitope can be characterized by the amino acid residues that it comprises as defined by a specific criteria such as the distance between or solvent accessibility of atoms in the Ab:Ag complex. At a further less detailed level the epitope can be characterized through function, e.g. by competition binding with other Abs. The epitope can also be defined more generically as comprising amino acid residues for which substitution by another amino acid will alter the characteristics of the interaction between the Ab and Ag.

In the context of an X-ray derived crystal structure defined by spatial coordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag, the term epitope is herein, unless otherwise specified or contradicted by context, specifically defined as PGLYRP1 residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of, eg., 2-6 Å, such as 3 Å, such as 4 Å, such as 5 Å from a heavy atom in the Ab.

From the fact that descriptions and definitions of epitopes, dependent on the epitope mapping method used, are obtained at different levels of detail, it follows that comparison of epitopes for different Abs on the same Ag can similarly be conducted at different levels of detail.

Epitopes described at the amino acid level, e.g. determined from an X-ray structure, are said to be 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 residue are shared by the epitopes.

The definition of the term “paratope” is derived from the above definition of “epitope” by reversing the perspective. Thus, the term “paratope” refers to the area or region on the Ab to which an Ag specifically binds, i.e. with which it makes physical contact to the Ag.

In the context of an X-ray derived crystal structure, defined by spatial coordinates of a complex between an Ab, such as a Fab fragment, and its Ag, the term paratope is herein, unless otherwise specified or contradicted by context, specifically defined as Ag residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4 Å from a heavy atom in PGLYRP1.

The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair may be identified by routine methods. For example, the general location of an epitope may be determined by assessing the ability of an antibody to bind to different fragments or variant PGLYRP1 polypeptides. The specific amino acids within PGLYRP1 that make contact with an antibody (epitope) and the specific amino acids in an antibody that make contact with PGLYRP1 (paratope) may also be determined using routine methods. For example, the antibody and target molecule may be combined and the Ab:Ag complex may be crystallised. The crystal structure of the complex may be determined and used to identify specific sites of interaction between the antibody and its target.

Antibodies that bind to the same antigen can be characterised with respect to their ability to bind to their common antigen simultaneously and may be subjected to “competition binding”/“binning”. In the present context, the term “binning” refers to a method of grouping antibodies that bind to the same antigen. “Binning” of antibodies may be based on competition binding of two antibodies to their common antigen in assays based on standard techniques such as surface plasmon resonance (SPR), ELISA or flow cytometry.

An antibody's “bin” 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 “bin” as the reference antibody. In this case, the reference and the second antibody competitively bind the same part of an antigen and are coined “competing antibodies”. If a second antibody is capable of binding to an antigen at the same time as the reference antibody, the second antibody is said to belong to a separate “bin”. In this case, the reference and the second antibody do not competitively bind the same part of an antigen and are coined “non-competing antibodies”.

Antibody “binning” does not provide direct information about the epitope. Competing antibodies, i.e. antibodies belonging to the same “bin” may have identical epitopes, overlapping epitopes or even separate epitopes. The latter is the case if the reference antibody bound to its epitope on the antigen takes up the space required for the second antibody to contact its epitiope on the antigen (“steric hindrance”). Non-competing antibodies generally have separate epitopes.

An antibody according to the current invention may be capable of competing with 1F10 for binding to PGLYRP1. An antibody according to the current invention may be capable of competing with 1F36/mAb 0182 for binding to PGLYRP1. An antibody according to the current invention may be capable of competing with 1F95 for binding to PGLYRP1. An antibody according to the current invention may be capable of competing with 1F105/mAb 0184 for binding to PGLYRP1. An antibody according to the current invention may be capable of competing with 2F5 for binding to PGLYRP1. An antibody according to the current invention may be capable of competing with 2F7 for binding to PGLYRP1. Hence, an antibody according to the current invention may belong to the same bin as any one or more of these antibodies.

The term “binding affinity” herein refers to a measurement of the strength of a non-covalent interaction between two molecules, e.g. an antibody, or fragment thereof, and an antigen. The term “binding affinity” is used to describe monovalent interactions (intrinsic activity).

Binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determining the equilibrium dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method. The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and dissociation rate constant kd (or koff), respectively. KD is related to ka and kd through the equation KD=kd/ka.

Following the above definition, binding affinities associated with different molecular interactions, such as comparison of the binding affinity of different antibodies for a given antigen, may be compared by comparison of the KD values for the individual antibody/antigen complexes.

A PGLYRP1 antibody of the invention may have a KD for its target (PGLYRP1) of 1×10−6M or less, 1×10−7M or less, 1×10−8M or less, or 1×10−9M or less, or 1×10−10M or less, 1×10−11M or less, 1×10−12M or less or 1×10−13M or less.

An antibody according to the current invention may be able to compete with another molecule, such as a naturally occurring ligand or receptor or another antibody, for binding to PGLYRP1. Therefore, an antibody according to the current invention may be able to bind PGLYRP1 with a greater affinity that that of another molecule also capable of binding PGLYRP1. The ability of an antibody to compete with a natural ligand/receptor for binding to an antigen may be assessed by determining and comparing the KD value for the interactions of interest, such as a specific interaction between an antibody and an antigen, with that of the KD value of an interaction not of interest.

The term “binding specificity” herein refers to the interaction of a molecule such as an antibody, or fragment thereof, with a single exclusive antigen, or with a limited number of highly homologous antigens (or epitopes). Antibodies that are capable of specifically binding to PGLYRP1 are not capable of binding dissimilar molecules. Antibodies according to the invention may not be able to bind PGLYRP family members such as PGLYRP2, PGLYRP3 and PGLYRP4. Antibodies according to the invention may not be able to bind human PGLYRP family members such as human PGLYRP2, human PGLYRP3 and human PGLYRP4.

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

A competitive binding assay can be conducted 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.

In another aspect, the present invention provides compositions and formulations comprising molecules of the invention, such as the PGLYRP1 antibodies, polynucleotides, vectors and cells described herein. For example, the invention provides a pharmaceutical composition that comprises one or more PGLYRP1 antibodies of the invention, formulated together with a pharmaceutically acceptable carrier.

Accordingly, one object of the invention is to provide a pharmaceutical formulation comprising such a PGLYRP1 antibody which is present in a concentration from 0.25 mg/ml to 250 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0. The formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabiliser, or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilisers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

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 multi-phase 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.

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

In a further aspect, the pharmaceutical formulation comprises an aqueous solution of such an antibody, and a buffer, wherein the antibody is present in a concentration from 1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0.

The PGLYRP1 antibodies of the present invention and pharmaceutical compositions comprising such antibodies may be used for the treatment of inflammatory diseases such as the following: inflammatory bowel disease (IBD), Crohns disease (CD), ulcerative colitis (UC), irritable bowel syndrome, 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, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atoptic dermatitis, vitiligo, graft versus host disease, Sjogrens's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, asthma and other autoimmune diseases that are a result of either acute or chronic inflammation. The PGLYRP1 antibodies of the present invention and pharmaceutical compositions comprising such antibodies may be used for the treatment of cardiovascular disease, stroke, ischemia reperfusion injury, pneumonia, sepsis and cancer.

PGLYRP1 antibodies of the invention are suitable for use in the treatment of individuals with inflammatory bowel disease. Inflammatory Bowel Disease (IBD) is a disease that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms. IBD primarily causes abdominal pain, diarrhea (which may be bloody), vomiting, or weight loss, but may also cause complications outside of the gastrointestinal tract such as skin rashes, arthritis, inflammation of the eye, fatigue, and lack of concentration. Patients with IBD can be divided into two major classes, those with ulcerative colitis (UC) and those with Crohn's disease (CD). While CD generally involves the ileum and colon, it can affect any region of the intestine but is often discontinuous (focused areas of disease spread throughout the intestine), UC always involves the rectum (colonic) and is more continuous. In CD, the inflammation is transmural, resulting in abscesses, fistulas and strictures, whereas in UC, the inflammation is typically confined to the mucosa. There is no known pharmaceutical or surgical cure for Crohn's disease, whereas some patients with UC can be cured by 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 may be measured as a reduction in the Crohn's Disease Activity Index (CDAI) score for CD which is scoring scale based on laboratory tests and a quality of life questionnaire. In animal models, efficacy is mostly measured by increase in weight and also a disease activity index (DAI), which is a combination of stool consistency, weight and blood in stool.

PGLYRP1 antibodies of the invention are suitable for use in the treatment of individuals with rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease that affects nearly if not all of the body and is one of the most common forms of arthritis. It is characterized by inflammation of the joint, which causes pain, stiffness, warmth, redness and swelling. This inflammation is a consequence of inflammatory cells invading the joints, and these inflammatory cells release enzymes that may digest bone and cartilage. As a result, this inflammation can lead to severe bone and cartilage damage and to joint deterioration and severe pain amongst other physiologic effects. The involved joint can lose its shape and alignment, resulting in pain and loss of movement.

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

PGLYRP1 antibodies of the invention are 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 one 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. Moreover, patients usually have recurrence of psoriasis, 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 compounds suitable for use in treatment of psoriasis (Davenport et al., Internat. Immunopharmacol 2: 653-672, 2002). Efficacy in this model is a measured by reduction in skin pathology using a scoring system. Similarly, efficacy in patients is measured by a decrease in skin pathology.

PGLYRP1 antibodies of the invention are suitable for use in the treatment of individuals with psoriatic arthritis. Psoriatic arthritis (PA) is a type of inflammatory arthritis that occurs in a subset of patients with psoriasis. In these patients, the skin pathology/symptoms are accompanied by joint swelling, similar to that seen in rheumatoid arthritis. It features patchy, raised, red areas of skin inflammation with scaling. Psoriasis often affects the tips of the elbows and knees, the scalp, the navel and around the genital areas or anus. Approximately 10% of patients who have psoriasis also develop an associated inflammation of their joints.

The term “treatment”, as used herein, refers to the medical therapy of any human or other animal subject in need thereof. Said subject is expected to have undergone physical examination by a medical or veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said treatment is beneficial to the health of said human or other animal subject. The timing and purpose of said treatment may vary from one individual to another, according to many factors, such as the status quo of the subject's health. Thus, said treatment may be prophylactic, palliative, symptomatic and/or curative.

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

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

EXEMPLARY EMBODIMENTS

1. A cell expressing TREM-1, a signalling protein for TREM-1 and a reporter construct that is activated by said signalling protein.

2. The cell according to embodiment 1, wherein the cell is of haematopoetic origin.

3. The cell according to embodiment 2, wherein the cell is a myeloid cell.

4. The cell according to embodiment 2, wherein the cell is a T-cell.

5. The cell according to any one of embodiments 1-4, wherein the signalling protein is DAP10.

6. The cell according to any one of embodiments 1-4, wherein the signalling protein is DAP12.

7. The cell according to any one of embodiments 1-4, wherein the signalling protein is TCR zeta.

8. The cell according to any one of embodiments 1-4, wherein the signalling protein is Fc gamma RIII.

9. The cell according to any one of embodiments 1-4, wherein the signalling protein is a Fc receptor.

10. The cell according to any one of embodiments 1-9, wherein the reporter construct comprises a transcription factor and a reporter gene.

11. The cell according to embodiment 10, wherein said transcription factor is NFAT.

12. The cell according to embodiment 11, wherein said transcription factor is NFkB.

13. The cell according to any one of embodiments 10-12, wherein said reporter gene encodes β-galactosidase.

14. The cell according to any one of embodiments 10-12, wherein said reporter gene encodes luciferase.

15. The cell according to any one of embodiments 10-12, wherein said reporter gene encodes green fluorescent protein (GFP).

16. The cell according to any one of embodiments 10-12, wherein said reporter gene is a gene that encodes chloramphenicol transferase.

17. The cell according to any one of embodiments 1-4, 6, 10-11 and 13, which is a BWZ.36/hTREM-1:DAP12:NFAT-LacZ T-cell.

18. A method of stimulating the cell according to any one of embodiments 1-17, comprising contacting said cell with PGLYRP1.

19. A method of stimulating the cell according to any one of embodiments 1-17, comprising contacting said cell with PGLYRP1 and PGN.

20. The method according to any one of embodiments 18-19, wherein said PGLYRP1 is expressed by a cell.

21. The method according to embodiment 20, wherein the cell expressing PGLYRP1 is a prokaryotic cell.

22. The method according to embodiment 20, wherein the cell expressing PGLYRP1 is a eukaryotic cell.

23. The method according to embodiment 22, wherein the cell expressing PGLYRP1 is a mammalian cell.

24. The method according to embodiment 23, wherein the cell expressing PGLYRP1 is an activated neutrophil.

25. The method according to embodiment 23, wherein the cell expressing PGLYRP1 is a HEK cell.

26. A method of identifying a TREM-1 ligand, comprising: (a) culturing the cell according to any one of embodiments 1-18; (b) detecting, preferably quantifying, the activity of said cell expressing TREM-1 when it is contacted with a cell, a fluid such as a biological fluid or a tissue that triggers TREM-1 activation; (c) contacting the culture of (b) with a TREM-1 protein; (d) isolating the component that binds TREM-1 and (e) characterising the isolated component.

27. A method of identifying a molecule that specifically binds PGLYRP1 and that modifies TREM-1 mediated cellular activity, comprising: (a) culturing the cell according to any one of embodiments 1-18; (b) detecting, preferably quantifying, the activity of said cell expressing TREM-1 when it is contacted with PGLYRP1 and, optionally, a multimerisation agent such as PGN; (c) contacting the culture of (b) with a molecule that specifically binds PGLYRP1; and (d) detecting, preferably quantifying, that the activity of said cell expressing TREM-1 is less than or more than its activity as measured in (b).

28. A method of identifying a PGLYRP1 antibody, or fragment thereof, that modifies TREM-1 mediated cellular activity, comprising: (a) culturing the cell according to any one of embodiments 1-18; (b) detecting, preferably quantifying, the activity of said cell expressing TREM-1 when it is contacted with PGLYRP1 and, optionally, a multimerisation agent such as PGN; (c) contacting the culture of (b) with an antibody that binds PGLYRP1; and (d) detecting, preferably quantifying, that the activity of said cell expressing TREM-1 is less than or more than its activity as measured in (b).

29. The method according to embodiment 27, wherein said PGLYRP1 antibody, or fragment thereof, decreases TREM-1 mediated cellular activity and wherein the activity of the first cell when measured in (d) is less than its activity when measured in (b).

30. The method according to embodiment 27, wherein said PGLYRP1 antibody, or fragment thereof, increases TREM-1 mediated cellular activity and wherein the activity of the first cell when measured in (d) is more than its activity when measured in (b).

31. The PGLYRP1 antibody or fragment thereof that is identified by means of the method according to any one of embodiments 28-30.

32. A PGLYRP1 antibody or fragment thereof which is capable of specifically binding PGLYRP1 and reducing PGLYRP1-mediated TREM-1 activity.

33. The antibody, or fragment thereof, according to any one of embodiments 31-32, which is capable of reducing the release of one or more cytokines from cells that express TREM-1.

34. The antibody or fragment thereof according to any one of embodiments 31-33, which is a monoclonal antibody.

35. The antibody or fragment thereof according to any one of embodiments 31-34, which is a humanised antibody.

36. The antibody or fragment thereof according to any one of embodiments 31-34, which is a human antibody.

37. The antibody or fragment thereof according to any one of embodiments 31-34, which is a chimeric antibody.

38. The antibody according to any one of embodiments 31-37, wherein the isotype of said antibody is IgG.

39. The antibody according to embodiment 38, wherein said isotype is IgG1, IgG2 or IgG4.

40. The antibody according to embodiment 37, wherein the isotype of said antibody is IgG1.

41. The antibody according to embodiment 37, wherein the isotype of said antibody is IgG4.

42. The antibody or fragment thereof according to any one of embodiments 31-41, which is capable of competing with antibody 1F10 for binding to PGLYRP1.

43. The antibody or fragment thereof according to any one of embodiments 31-41, which is capable of competing with antibody 1F36/mAb 0182 for binding to PGLYRP1.

44. The antibody or fragment thereof according to any one of embodiments 31-41, which is capable of competing with antibody 1F95 for binding to PGLYRP1.

45. The antibody according to any one of embodiments 31-41, which is capable of competing with antibody 1F105/mAb 0184 for binding to PGLYRP1.

46. The antibody according to any one of embodiments 31-41, which is capable of competing with antibody 2F5 for binding to PGLYRP1.

47. The antibody according to any one of embodiments 31-41, which is capable of competing with antibody 2F7 for binding to PGLYRP1.

48. The antibody or fragment thereof according to any one of embodiments 31-47, which is capable of specifically binding SEQ ID NO: 37 (Type II 1.0 PGLYRP1) and/or SEQ ID NO: 38 (Type II 2.0 PGLYRP1).

49. The antibody or fragment thereof according to any one of embodiments 31-48, which has a KD for its target that is 1×10−6M or less, 1×10−7M or less, 1×10−8M or less, or 1×10−9M or less, or 1×10−10M or less, 1×10−11M or less, 1×10−12M or less or 1×10−13M or less, when determined using surface plasmon resonance.

50. The antibody according to any one of embodiments 31-49, the heavy chain of which comprises: a CDRH1 sequence corresponding to amino acid residues 31 to 35 (SYWMN) of SEQ ID NO: 15, wherein one of said amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence corresponding to amino acids 50 to 66 (MIHPSDSETRLNQKFKD) of SEQ ID NO: 15, wherein one, two or three of said amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence corresponding to amino acid residues 99 to 108 (DYSDYDGFAY]) of SEQ ID NO: 15, wherein one, two or three of said amino acid residues may be substituted by a different amino acid.

51. The antibody according to any one of embodiments 31-49, the heavy chain of which comprises: a CDRH1 sequence corresponding to amino acid residues 31 to 35 (DYNMY) of SEQ ID NO: 19, wherein one of said amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence corresponding to amino acids 50 to 66 (YIDPYNGDTSYNQKFKG) of SEQ ID NO: 19, wherein one, two or three of said amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence corresponding to amino acid residues 99 to 109 (GDYGNPFYLDY) of SEQ ID NO: 19, wherein one, two or three of said amino acid residues may be substituted by a different amino acid.

52. The antibody according to any one of embodiments 31-49, the heavy chain of which comprises: a CDRH1 sequence corresponding to amino acid residues 31 to 35 (DTYIH) of SEQ ID NO: 23, wherein one of said amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence of amino acids 50 to 66 (RIDPANDDTKYDPNFQG) of SEQ ID NO: 23, wherein one, two or three of said amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence of amino acid residues 99 to 108 (SDNSDSWFAY) of SEQ ID NO: 23, wherein one, two or three of said amino acid residues may be substituted by a different amino acid.

53. The antibody according to any one of embodiments 31-49, the heavy chain of which comprises: a CDRH1 sequence corresponding to amino acid residues 31 to 35 (DYNMH) of SEQ ID NO: 27, wherein one of said amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence corresponding to amino acids 50 to 66 (YVDPYDGGTSSNQKFKG) of SEQ ID NO: 27, wherein one, two or three of said amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence corresponding to amino acid residues 99 to 106 (EVPYYFDY) of SEQ ID NO: 27, wherein one, two or three of said amino acid residues may be substituted by a different amino acid.

54. The antibody according to any one of embodiments 31-49, the heavy chain of which comprises: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (DYYMY) of SEQ ID NO: 31, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (AISDDSTYTYYPDSVKG) of SEQ ID NO: 31, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 109 (GGYGNLYAMDY) of SEQ ID NO: 31, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

55. The antibody according to any one of embodiments 31-49, the heavy chain of which comprises: a CDRH1 sequence that corresponds to amino acid residues 31 to 35 (NYVMH) of SEQ ID NO: 35, wherein one of these amino acid residues may be substituted by a different amino acid residue; and/or a CDRH2 sequence that corresponds to amino acids 50 to 66 (WINPFNDGTNYNENFKN) of SEQ ID NO: 35, wherein one, two or three of these amino acids may be substituted by a different amino acid residue; and/or a CDRH3 sequence that corresponds to amino acid residues 99 to 109 (SGFITTLIEDY) of SEQ ID NO: 35, wherein one, two or three of these amino acid residues may be substituted by a different amino acid.

56. The antibody according to any one of embodiments 50-55, the light chain of which comprises: a CDRL1 sequence corresponding to amino acid residues 24 to 34 (RASQSISDYLH) of SEQ ID NO: 16, wherein one, two or three of said amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence corresponding to amino acid residues 51 to 56 (ASQSIS) of SEQ ID NO: 16, wherein one or two of said amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence corresponding to amino acid residues 89 to 97 (QNGHSFPLT) of SEQ ID NO: 16, wherein one or two of said amino acid residues may be substituted with a different amino acid.

57. The antibody according to any one of embodiments 50-55, the light chain of which comprises: a CDRL1 sequence corresponding to amino acid residues 24 to 33 (SVSSSVNYMY) of SEQ ID NO: 20, wherein one, two or three of said amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence corresponding to amino acid residues 49 to 55 (DTSKLPS) of SEQ ID NO: 20, wherein one or two of said amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence corresponding to amino acid residues 88 to 96 (QQVVTSNPPT) of SEQ ID NO: 20, wherein one or two of said amino acid residues may be substituted with a different amino acid.

58. The antibody according to any one of embodiments 50-55, the light chain of which comprises: a CDRL1 sequence corresponding to amino acid residues 24 to 33 (SVSSSVNFMN) of SEQ ID NO: 24, wherein one, two or three of said amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence corresponding to amino acid residues 49 to 55 (DTSKLAP) of SEQ ID NO: 24, wherein one or two of said amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence corresponding to amino acid residues 88 to 96 (HQWSSYSLT) of SEQ ID NO: 24, wherein one or two of said amino acid residues may be substituted with a different amino acid.

59. The antibody according to any one of embodiments 50-55, the light chain of which comprises: a CDRL1 sequence corresponding to amino acid residues 24 to 33 (VASSSVTYMY) of SEQ ID NO: 28, wherein one, two or three of said amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence corresponding to amino acid residues 49 to 54 (THPLAS) of SEQ ID NO: 28, wherein one or two of said amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence corresponding to amino acid residues 87 to 95 (PHWNTNPPT) of SEQ ID NO: 28, wherein one or two of said amino acid residues may be substituted with a different amino acid.

60. The antibody according to any one of embodiments 50-55, the light chain of which comprises: a CDRL1 sequence that corresponds to amino acid residues 24 to 35 (TASSSVSSSYLH) of SEQ ID NO: 32, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 51-57 (STSNLAS) of SEQ ID NO: 32, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 90-98 (HQYHRSPFT) of SEQ ID NO: 32, wherein one or two of these amino acid residues may be substituted with a different amino acid.

61. The antibody according to any one of embodiments 50-55, the light chain of which comprises: a CDRL1 sequence that corresponds to amino acid residues 24 to 34 (KASESVGSFVS) of SEQ ID NO: 36, wherein one, two or three of these amino acid residues may be substituted with a different amino acid; and/or a CDRL2 sequence that corresponds to amino acid residues 50 to 56(GASNRYT) of SEQ ID NO: 36, wherein one or two of these amino acid residues may be substituted with a different amino acid; and/or a CDRL3 sequence that corresponds to amino acid residues 89 to 96 (GQYYTHPT) of SEQ ID NO: 36, wherein one or two of these amino acid residues may be substituted with a different amino acid.

62. The antibody according to any one of embodiments 31-61 for use as a medicament.

63. The antibody according to any one of embodiments 31-62 for use in the treatment of an inflammatory disease.

64. An antibody that is capable of specifically binding PGLYRP1 and reducing PGLYRP1-mediated TREM-1 activity for use as a medicament.

65. An antibody that is capable of specifically binding PGLYRP1 and reducing PGLYRP1-mediated TREM-1 activity for use in the treatment of an inflammatory disease.

66. Use according to any one of embodiments 62-65, wherein the inflammatory disease is an autoimmune disease.

67. Use according to embodiment 66, wherein the autoimmune disease is rheumatoid arthritis (RA).

68. Use according to embodiment 66, wherein the autoimmune disease is inflammatory bowel disease (IBD) and/or ulcerative colitis.

69. Use according to embodiment 66, wherein the autoimmune disease is psoriatic arthritis (PA).

70. Use according to embodiment 62 or 64 in the treatment of cardiovascular disease, stroke, ischemia reperfusion injury, sepsis and/or cancer.

The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES

Example 1: Generation of a BWZ.36 HumanTREM-1:DAP12 Stable Cell Line

The BWZ.36/hTREM-1:DAP12:NFAT-LacZ cell line (herein also referred to as the “BWZ/hTREM-1 reporter cell”) was derived from BW5147 T cells (Mus musculus thymus lymphoma cell line, ATCC TIB-47, LGC Standards, Middelsex, UK) and contains a LacZ reporter construct regulated by four copies of the NFAT promoter element (see Karttunen, J. & Shastri, N. (1991) Proc. Natl. Acad. Sci. USA 88, 3972-3976 and Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834). TREM/DAP12/pMX-IRES vector (encoding 786 bp of TREM-1 from a SmaI site to BamHI site using TREM-1 cDNA (Gene Bank Ref. ID: NM_018643.2, Sino Biological Inc., Beijing, China) as template and oligo 5′ TAGTAGGGATCCGCTGGTGCACAGGAAGG (SEQ ID NO: 51) and 5′ TAGTAGGCGGCCGCTTCGTGGGCCTAGGGTAC (SEQ ID NO: 52) as primers cloned into pIREShyg vector GenBank Accession #U89672 (Cat. no. 6061-1, Clontech Laboratories, CA, USA) was transfected in PLAT-E packaging cell line (provided by W. Yokoyama, Washington University; alternatively, Cat. no. RV-101, Cell Biolabs Inc, Bio-Mediator KY, Vantaa, Finland) using Superfect transfection reagent (Cat. no. 301305, Qiagen Nordic, Denmark). PLAT-E supernatants containing TREM/DAP12/pMX-IRES viral particles were used to infect BWZ.36 cells as follows: 2×105 BWZ.36 cells were cultured in 6 well plates and the medium was replaced with 1.5 ml of supernatant containing the viral particles +8 mg/ml of polybrene. After 6-8 hours, 1.5 ml of normal medium was added to the plate and the cells were incubated for an additional 24 hours. BWZ.36 cell lines stably expressing TREM-1 were stained with anti TREM-1 monoclonal antibody (clone 21C7; Bouchon et al, 2000, J. Immunol vol. 164 page 4991-4995) and isolated by cell sorting.

Example 2: Creation of a Bioassay for the Identification of Cells Expressing a Ligand for TREM-1

A TREM-1 reporter cell line was generated by transfecting the NFAT-lacZ bearing cell line BWZ.36 (Sanderson S, Int. Immun. 1994) with hTREM-1 and DAP12 as described in Example 1. This BWZ.36/hTREM-1:DAP12:NFAT-LacZ cell line (herein also referred to as a BWZ/hTREM-1 reporter cell) is highly responsive to antibody-mediated cross linking of TREM-1, giving ˜40-fold induction of the NFAT-driven LacZ production when stimulated with 1-10 μg/ml plate bound commercially available anti-TREM-1 antibody, as compared to the isotype control. NFAT-driven LacZ production in the reporter cells may be assayed using a luminescence based kit, Beta Glow™ (Promega E4720, Madison, Wis.). Plates were coated with isotype control or aTREM-1 MAB1278 (Conc 3 ug/ml in PBS, 100 ul/well) (R&D Systems, Minneapolis, USA) at 4C for 16 hours or for 2 hr at 37° C., 5% CO2 and the BWZ/hTREM-1 reporter cells were detached by adding 10 ml Versene (catalog number #15040, Gibco, Carlsbad Calif., USA), spun at 400 g for 5 min and washed in PBS and media (RPMI-1640 w/o phenol red; cat. number 11835, Gibco, Carlsbad Calif., USA) before adding to the coated plates (1×106 cells/ml, 4×104 cells/well) to total volume 100 ul and incubated overnight (16-20 hours) at 37° C., 5% CO2.

These TREM-1 responsive cells were used to identify cells expressing TREM-1 ligand. One such cell turned out to be neutrophils from the whole blood. The neutrophils of healthy donors were purified by means of Ficoll and dextran sedimentation and stimulated with PGN (InVivogen, tlrl-pgnsa, SanDiego, Calif., USA) overnight. Briefly, the BWZ/hTREM-1 reporter cells were added to the activated neutrophil cultures in a 1:3 ratio of reporter cell:neutrophils. The assay was run in Poly-D-Lysine coated Black cell culture plates (#356640 from BD Biosciences, San Jose, Calif., USA). TREM-1 activation was read out after 24 hours of culture using the BetaGlo reagent (E4720 from Promega, Madison, Wis., USA) and luminescence measured using a TopCount Luminescence counter from Perkin Elmer.

The vitro stimulated neutrophils possessed a ligand that was able to induce TREM-1 signalling, and neutrophils from the whole blood of healthy donors were purified by Dextran sedimentation and stimulated overnight with multiple reagents. The only reagent that was able to stimulate a TREM-1 responsive signal from neutrophils was PGN-SA (Invivogen, tlrl-pgnsa, SanDiego, Calif., USA) which mimic bacterial activation of the cells. These activated neutrophils were then used to stimulate the BWZ/hTREM-1 reporter cell line, by co-culturing the cells. Briefly, the BWZ/hTREM-1 reporter cells were added to the activated neutrophil cultures in a 1:3 ratio of reporter cell:neutrophils. The assay was run in Poly-D-Lysine coated black cell culture plates (cat. no. 356640 BD Biosciences, San Jose, Calif., USA). TREM-1 activation was read out after 24 hours of culture using the BetaGlo reagent (cat. no. E4720, Promega, Madison, Wis., USA) and luminescence measured using a TopCount Luminescence counter from (Perkin Elmer, Waltham Mass., USA). As shown in FIG. 1, significant induction of reporter activity was observed in BWZ/hTREM-1 cells co-cultured with the PGN-stimulated neutrophils. This induction was highly dependent on neutrophil activation. No response was seen with resting neutrophils (bar 2 neutrophils). Likewise, no response was seen when stimulating BWZ/hTREM-1 reporter cells with PGN-SA in the TLRL cocktail in the absence of neutrophils (bar 1-TLRL), demonstrating that the response is not merely a direct effect of PGN on the BWZ/hTREM-1 cells. Activating neutrophils with cytokine cocktails (bar 3+4 neutrophils+cytokines) did not provide the correct TREM-1 activation signal either.

Example 3: Binding of Soluble TREM-1 to PGN-Activated Neutrophils

PGN-stimulated neutrophils were able to induce TREM-1 activation, indicating the presence of a TREM-1 stimulating factor in the PGN-stimulated neutrophil cultures. In order to confirm the presence of a TREM-1-interacting protein on the neutrophils, PGN-stimulated neutrophils were stained with a recombinant TREM-1-tetrameric protein and analysed by flow cytometry. Briefly, granulocytes were isolated from human whole blood obtained from Astarte Biologics (Redmond, Wash., USA) via a Ficoll-dextran sedimentation method. Blood was stratified on FicollPaque (17-0840-03, GE Healthcare, Piscataway, N.J., USA) gradient with a ratio of 3 parts of Ficoll and 4 parts of blood in a 50 ml tube, then centrifuged at 400×g for 30 minutes at 22° C., without brake. The intermediate PBMC band was gently removed by aspiration. The granulocytes stratified on the packed RBC were aspirated and transferred to a 50 ml polypropylene tube. The granulocytes and contaminating RBCs were diluted to 40 ml with 1× PBS and followed by addition of 10 ml 4% DEXTRAN 500 (Sigma, 31392, St Louis, Mo., USA) in PBS solution. After mixing by gentle inversion, the tubes were left at 22° C. for 20-30 min. A granulocyte rich supernatant was then transferred into a fresh tube and centrifuged at 250×g, 5 min, 22° C.; the supernatant was aspirated and discarded. Contaminating RBCs were removed with an osmotic lysis, briefly, the cell pellet was resuspended in 7.5 ml of 0.2% NaCl; gently mixed for 55-60 seconds and 17.5 ml of a 1.2% NaCl. solution was added. The volume was then brought to 50 ml with PBS and spun at 250×g for 5 min, the pellet was resuspended in 7.5 ml of 0.2% NaCl to repeat the lysis a second time. The final granulocyte pellet was resuspended in RPMI/10% FBS.

Isolated granulocytes were cultured at a density of 3.8 E6/ml in RPMI/10% FBS+10 μg/ml of PGN-SA (Invivogentlrl-pgnsa, San Diego, Calif., USA) for 7 days. Cells were pelleted by centrifugation and resuspended in PBS/2% FBS for staining. Resuspended granulocytes were then plated in a 96 well plate (round bottom) at a density of 100,000/well in the presence or absence of 2 μg/ml of probe, with or without 100 μg/ml (50×) of a specific or irrelevant competitor protein. Cells were incubated with probe/competitor for 1 hour at 4 C in a volume of 50 μl/well. At the end of the incubation, 150 μl/well of PBS/2% FBS was added and the cells were pelleted. The pelleted cells were resuspended in 50 μl/well of goat anti hFc F(ab′)2/PE conjugate (Jackson ImmunoResearch 109-116-098, West Grove, Pa., USA) and incubated at 4° C. for 30 minutes. 150 μl/well of PBS/2% FBS was added and the cells pelleted. Pelleted cells were further washed in 200 ul/well PBS/2% FBS and pelleted. Washed cells were then resuspended in 100 μl/well of fixative (1:1 PBS: Cytofix. 554655, BD Biosciences, San Jose, Calif., USA) and incubated 5 minutes at room temperature. 100 μl/well of PBS/2% FBS was added to fixed cells, then the cells were pelleted. The stained/fixed cells were then resuspended in 100 μl/well of PBS/2% FBS for flow cytometric analysis on a LSR 11 flow cytometer. (BD Biosciences, San Jose, Calif., USA).


Probe Set:
Fc mut
5.36 mg/ml
SEQ ID NO: 3
hTREM-1 tet/Fc mut
1.07 mg/ml
SEQ ID NO: 2
Competitors for hTREM-1 tet/Fc mut:
50X DCIR COMP
 0.3 mg/ml
SEQ ID NO: 4
50X TREM COMP
1.14 mg/ml
SEQ ID NO: 5

Histograms were created from the flow cytometric data. The background fluorescence from the goat anti hFc/PE conjugate was shown in each histogram as the background and was designated “PE”. An identical marker was drawn on each histogram to designate the percentage of cells that bound to the secondary goat anti hFc/PE conjugated antibody. The negative control Fc mut protein showed 2% positive binding cells, which was identical to the background fluorescence seen with the goat anti hFc/PE conjugate alone (FIG. 2A). When cells were stained with 2 μg/ml hTREM-1/tetramer they were 39% positive (FIG. 2B). When the TREM-1 tetramer binding was done in the context of 100 μg/ml DCIR COMP protein, the cells were 41% positive confirming that the DCIR COMP did not compete for binding with TREM-1 tetramer (FIG. 2C). When the TREM-1 tetramer binding was done in the presence of 100 μg/ml of TREM-1 COMP, the cells were 10% positive showing that the TREM COMP protein did compete with TREM-1 tetramer for binding to the cells (FIG. 2D).

Example 4: Identification of PGLYRP1 as a Neutrophil-Expressed TREM-1 Ligand

PGLYRP1 was identified as a TREM-1 ligand through the use of immunoprecipitation coupled with mass spectroscopy (IP-MS). Soluble TREM-1 tetramer was used as an affinity “bait” molecule to identify a ligand. Briefly, TREM-1-tetramer-Fc (SEQ ID NO: 2) and separately CD83-Fc (SEQ ID NO: 5) were each incubated at final concentrations of 100 μg/ml with 270 million human neutrophils, purified by dextran sedimentation as described above, in 1 mL PBS at 4° C., 90 minutes with mild shaking. After pelleting, the cells were resuspended in 1 mL PBS buffer with the inclusion of the crosslinker 3,3′-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) (Thermo Scientific: 21578, Rockford, Ill., USA), at a concentration of 2 mM and incubated 30 minutes at room temperature. Cells were washed 3× with 1 mL PBS followed by lysis in 1 mL RIPA buffer (Thermo Scientific, 89901, Rockford, Ill., USA). The lysate was centrifuged at 15,000×g for 10 minutes at 4° C. to remove insoluble materials. Neutrophil proteins cross-linked to Fc coupled probes were immunoprecipitated from the supernatant using Protein A Mag Sepharose™ beads (GE Healthcare Life Sciences, 28-9670-56, Piscataway, N.J., USA). Briefly, 50 μL of beads were first washed with 200 μL PBS, then resuspended in 1 mL of cell lysate, incubated 60 minutes at 4° C., magnetically captured, and sequentially washed 2× with 200 μl RIPA buffer then 3× with 200 μL PBS. Upon removing PBS from the final magnetic capture, proteins were eluted from the magnetic beads using 200 μL buffer containing 8 M Urea, 100 mM Tris (pH 8.0), and 15 mM TCEP (Thermo Scientific, 77720, Rockford, Ill., USA) and incubated at room temperature for 30 minutes, beads were captured and supernatant was transferred to a Microcon Ultracel YM-30 filter (Millipore, 42410, Billerica, Mass., USA). Samples were spun at 14,000×g, 20° C., 30-60 minutes until no liquid remained on the top of the filter membrane. The retained proteins were then alkylated with 100 μL 50 mM IAA (iodoacetamide) in 8 M Urea for 30 minutes in dark at room temperature. The filter was washed 2× with 100 μL 50 mM NH4HCO3 and then transferred to a new collection tube. 1 μg trypsin (cat. no. V5111, Promega, Madison Wis., USA) in 60 μL 50 mM NH4HCO3 was added followed by incubation at 37° C. overnight. The tryptic digest was collected by centrifugation at 14,000×g for 30 minutes followed by washing the filter with 50 μL 50 mM NH4HCO3. 10 μL of the digest was analyzed by LC/MS/MS using an LTQ-Orbitrap-XL mass spectrometer (Thermo Scientific, Waltham, Mass., USA). The data was searched against IPI human database (v3.81) using SEQUEST-Sorcerer engine (4.0.4 build) (SageN, Milpitas, Calif., USA) and then post processed with Scaffold 3 (Proteome Software, Portland, Oreg., USA) to filter protein IDs with a false discovery rate of 1%. After negative control subtraction, PGLYRP1 was found to be a high-confidence protein specifically associated with hTREM-1 tetramer. The immunoprecipitation in the neutrophils showed that out of the 148 identified proteins, 72 proteins were immunoprecipitated by the control construct (CD83) alone, 73 of the proteins were identical for TREM-1 and CD83, whereas only three were TREM-1 specific (FIG. 3A and FIG. 3B). The experiment was subsequently repeated using neutrophils from a different donor and PGLYRP1 was again identified as specifically interacting with hTREM-1.

Example 5: Purification of Human PGLYRP1 Expressed from HEK293 6E

A recombinant protein sequence was constructed by fusing the human CD33 signal peptide sequence (SEQ ID NO: 53) with the human mature PGLYRP1 coding sequence (SEQ ID NO: 1). The resulting open reading frame was cloned into pcDNA3.1/Zeo(+) vector (Life Technologies, Carlsbad Calif., USA) after a CMV promoter. The pcDNA3.1-hPGLYRP1 construct was then transfected into HEK293 6E cells with 293Fectin™ (Life Technologies, Carlsbad Calif., USA) following the vendor's protocol. 5 days after transfection, the culture supernatant containing secreted human PGLYRP1 was harvested by centrifugation (15,000 rpm×20 min, 4 □C) and then cleared by the filtration with 0.22 μm cellulose nitrate membrane. The cleared supernatant was first diluted 10 fold into 20 mM sodium citrate pH 5.0 and then applied to a Hitrap SP HP 5 ml column (17-1151-01 GE Healthcare, Uppsala, Sweden), followed by a 5 column volume wash with 20 mM sodium citrate pH5.0. The bound human PGLYRP1 was then eluted with a 0-100% linear gradient of 20 mM sodium citrate pH 5.0, 1M NaCl in 30 column volumes. The fractions containing dimer and monomer forms of human PGLYRP1 were pooled separately and concentrated to less than 4 ml by Amicon ultra 15 centrifugal units (UFC800324 3,000 kDa MWCO, Millipore, Hellerup, Denmark). Dimer and monomer pools were further polished and buffer-exchanged to Phosphate Buffered Saline (PBS) by a Hiload 26/60 Superdex 75 318 ml column (17-1070-01 GE Healthcare, Uppsala, Sweden). After concentrating, the final protein concentrations were determined by measuring 280 nm absorbance with a NANODROP UV spectrometer. Protein purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Example 6: Refolding and Purification of Human PGLYRP1 Expressed from E. Coli

Human PGLYRP1 was expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Bacteria were harvested by centrifugation, resuspended in 50 mM Tris-HCl pH8.0, 500 mM NaCl, 5 mM EDTA, 0.5% Triton X-100 and disrupted by sonication. The insoluble pellet was washed three times with 50 mM Tris, pH 8.0, 1% TritonX-100, 2 M urea and once with 50 mM Tris pH 8.0, then solubilized in 50 mM Tris-HCl, 6M guanidine hydrochloride, pH7.4, 1 mM DTT (final protein concentration 20 mg/ml). For in vitro folding, solubilized human PGLYRP1 inclusion bodies were diluted into 50 mM Tris, pH 8.0, 2 mM EDTA, 5 mM cysteamine, 0.5 mM cystamine, 0.4 M arginine (final protein concentration 1 mg/ml). After overnight at 4° C., the folding mixture was cleared by centrifugation/filtration and then diluted 12 fold into 10 mM MES pH 3.5 to lower the conductivity and pH (final pH ˜5.8, conductivity ˜6 mS/cm). The diluted folding mixture was then applied to a Hitrap SP HP 5 ml column (17-1151-01 GE Healthcare, Uppsala, Sweden), followed by a 5 column volume wash with 50 mM MES pH 5.8. The bound human PGLYRP1 was then eluted with a 0˜60% linear gradient of 50 mM MES pH 5.8, 1 M NaCl in 20 column volume. The fractions containing refolded human PGLYRP1 were pooled and concentrated to less than 4 ml by Amicon ultra 15 centrifugal units (UFC800324 3,000 kDa MWCO, Millipore, Hellerup, Denmark). A Hiload 26/60 Superdex 75 318 ml column ((17-1070-01 GE Healthcare, Uppsala, Sweden) was then used to polish and buffer-exchange the proteins to Phosphate Buffered Saline (PBS). Majority of refolded human PGLYRP1 proteins was in monomer form. After concentrating, the final protein concentration was determined by measuring 280 nm absorbance with a NANODROP UV spectrometer. Protein purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Example 7: Immunisation of Mice and Identification of mAbs

Purified human PGLYRP1 was used to immunize mice in order to raise antibodies. Briefly, mice were immunised 3 times with 20 μg recombinant PGLYRP1 per immunisation. The first immunisation was subcutaneous using Complete Freunds Adjuvant (cat. no. 3018, Statens Serum Institut, Copenhagen, Denmark). The following two immunisations were intraperitoneal using Incomplete Freunds Adjuvant (cat. no. 3016, Statens Serum Institut, Copenhagen, Denmark). Ten days after the last immunisation, cheek blood was drawn and the sera were tested against PGLYRP1 in a direct ELISA.

Example 8: Binding of Soluble TREM-1 to PGLYRP1

One common method of validating a novel protein-protein interaction is through reconstituting the interaction using recombinant reagents. To this end recombinant human PGLYRP1 was expressed with a C-terminal epitope signalling the posttranslational addition of a glycosylphosphatidylinositol (GPI) structure. Proteins containing terminal GPI structures are targeted for display on the plasma membrane. This commonly applied technique allows otherwise soluble proteins to be displayed and tested for binding by flow cytometric techniques. In FIG. 4A, HEK-2936E cells were transfected with pCDNA 3.1/zeo(+) hPGLYRP1-GPI (SEQ ID NO: 7) 3 μg of DNA was diluted into 100 μl of Optimem (Cat. no. 31985062, Life Technologies, Carlsbad, Calif., USA). 4 μl of 293fectin (Cat. no. 51-0031, Life Technologies, Carlsbad, Calif., USA) was added to 100 μl of Optimem (Cat. no. 31985062, Life Technologies, Carlsbad, Calif., USA) and incubated at 22° C. for 5 minutes. The DNA/Optimem mix and the 293fectin/Optimem mix were combined and incubated for an additional 20 minutes at room temperature. HEK-2936E cells were diluted to 3 mls at 1 e6/ml in Freestyle media (Cat. no. 12338, Life Technologies, Carlsbad, Calif., USA) and plated in a 6-well dish (Cat. no. 35-3046, Biosciences San Jose, Calif., USA) and then the DNA/293fectin mix was added dropwise to the cells. Cells were incubated for 48 hours at 37° C. with shaking. 1 μg/ml of human TREM-1-G4Sx3-TREM-1/Fc6mut (SEQ ID NO: 2) was diluted in PBS/2% FBS and 50 μl of probe was added to 80,000 transfected HEK293-6E cells in a round bottom 96-well plate (Cat. no. 3799, Costar, Lowell, Mass., USA). Cells were incubated at 4° C. for 1 hr followed by 2× washes with 200 μl PBS/2% FBS. 50 μl of 1 μg/ml PE goat anti-human Fc (Cat. no. 109-116-098, Jackson ImmunoResearch, West Grove, Pa., USA) was added and incubated for an additional hour followed by 2×PBS/2% FBS washes. Cells were fixed for 5 min in 1:1 PBS diluted BDCytofix (Cat. no. 554655, BD Biosciences, San Jose, Calif., USA) and incubated for 5 mins followed by a 200 μl PBS/2% FBS wash. Cells were resuspended in 100 μl PBS/2% FBS before being analyzed on a FACS LSRII (BD Biosciences, San Jose, Calif.). The results of this experiment are shown in FIG. 4A where unstained cells are shown by solid black line (G03). TREM-1-G4Sx3-TREM-1/Fc6mut staining of mock transfected cells is shown dashed line, and shows no binding relative to unstained cells. Finally, cells transfected with human PGLYRP1-GPI robustly bind TREM-1-G4Sx3-TREM-1/Fc6mut shown with the dotted line. Quantitation of binding is expressed as mean florescent intensity, MFI.

In addition to flow cytometry, protein-protein interactions are also commonly assessed by measuring surface plasmon resonance (SPR). A BiacoreT200 (GE Healthcare, Piscataway, N.J., USA) instrument was used to analyze the interaction between human TREM-1 & human PGLYRP1 and also between human PGLYRP1 & sonicated, soluble E. coli peptidoglycan (Cat. no. tlrl-ksspgn, Invivogen, San Diego, Calif., USA). All assays were performed at 25° C. at flow rates of 20-30 μL/minute in 1×HBS-P running buffer (Cat. no. BR-1006-71, GE Healthcare, Piscataway, N.J., USA).

In FIG. 4B, 4641.1 RU of human TREM-1 tetramer (SEQ ID NO: 2) was amine coupled to a Biacore CM5 chip (Cat. no. BR-1005-30, BR-1000-50, GE Healthcare, Piscataway, N.J., USA) following the manufacturer's recommended methods. PGLYRP1 (Cat. no. 2590-PG, R&D Systems, Minneapolis, USA) was diluted to 150 nM in 1×HBS-P running buffer (Cat. no. BR-1006-71, GE Healthcare, Piscataway, N.J., USA) in the presence or absence of 10 μg/mL soluble E. coli peptidoglycan (Cat. no. tlrl-ksspgn, Invivogen, San Diego, Calif., USA) and injected over the TREM-1 surface. Data is reference subtracted vs. an activated and blocked (with ethanolamine) reference surface. Though PGLYRP1 binds TREM-1 both in presence and absence of PGN, there appears to be a significant avidity effect when PGLYRP1 is crosslinked by PGN.

In FIG. 4C, 274.8 RU of PGLYRP1 dimer (SEQ ID NO: 1) were amine coupled to a Biacore CM5 chip (Cat. no. BR-1005-30, BR-1000-50, GE Healthcare, Piscataway, N.J.). Soluble E. coli peptidoglycan (Cat. no. tlrl-ksspgn, Invivogen, San Diego, Calif., USA) was diluted to 10 μg/mL in 1×HBS-P running buffer (Cat. no. BR-1006-71, GE Healthcare, Piscataway, N.J., USA) and injected over the PGLYRP1 surface. Data is reference subtracted vs. an activated and blocked (with ethanolamine) reference surface.

In FIG. 4D, the same chip was used as in FIG. 4C, (274.8 RU of PGLYRP1 dimer, (SEQ ID NO: 1). TREM-1 dimer (SEQ ID NO: 8) was diluted to 150 nM in 1×HBS-P running buffer (BR-1006-71, GE Healthcare, Piscataway, N.J., USA) and injected both before and after repeated injections of peptidoglycan (depicted in FIG. B). Data is reference subtracted vs. an activated and blocked (with ethanolamine) reference surface and the signal of a buffer blank injection is also subtracted. TREM-1 shows clear binding to immobilized PGLYRP1. Soluble TREM-1 dimer binds similarly to immobilized PGLYRP1 alone or to PGLYRP1 that has been loaded with PGN. Overall binding of surface immobilized TREM-1 receptor by soluble PGLYRP1 and PGN consists of a modest affinity for the PGLYRP1:TREM-1 complex enhanced by an avidity effect from the highly crosslinked PGLYRP1.

Example 9: Activation of TREM-1-Response by Recombinant PGLYRP1

To test if recombinant PGLYRP1 can activate a TREM-1 response, the BWZ/hTREM-1 reporter cell line was seeded into black 96 well plates and stimulated with recombinant human PGLYRP1 (Cat. no. 2590-PG-050, R&D Systems: Minneapolis, Minn.) in the presence or absence of 10 μg/ml PGN. TREM-1 activation was read out after 24 hours of culture using the BetaGlo reagent (Cat. no. E4720, Promega Madison, Wis., USA) and luminescence measured using a TopCount Luminescence counter from Perkin Elmer. As shown in FIG. 5A, stimulation of the TREM-1 reporter cell line with PGLYRP1 induced a dose-dependent activation of TREM-1 in the presence of PGN. Several different PGN were tested, including PGN-EC (from E. coli), PGN-SA (from S. aureus) and PGN-BS (from B. subtilis) (Invivogen, San Diego, Calif., USA), and were all able to fascilitate the PGLYRP1-induced TREM-1 response.

The response induced by recombinant PGLYRP1 could be inhibited by addition of a recombinant TREM-1-Fc-fusion protein (SEQ ID NO: 2) (FIG. 5B) or by addition of polyclonal anti-PGLYRP1 antibody (Cat. no. AF-2590, R&D Systems: Minneapolis, Minn., USA) confirming that PGLYRP1 is the TREM-1 ligand and that soluble TREM-1 and anti-PGLYRP1 are potentially useful as TREM-1 antagonists.

Example 10: TNFalpha Release from M1 Macrophages Stimulated by PGLYRP1

Monocytes were differentiated into M1 macrophages and stimulated with PGLYRP1 complex resulting in TNFalpha release in two different donors.

Those skilled in the art will recognize the value of establishing a freezer bank collection of primary cells from multiple donors thus providing for convenient replication of experiments. In vitro derived macrophages were produced from peripheral blood monocytes as follows. Negatively enriched monocytes were isolated from a peripheral blood “leukopak” obtained from Research Blood Components (Brighton, Mass., USA) using a Rosette Sep kit (cat. no. Cat. no. 15068 Stem Cell Technologies, Vancouver BC, Canada) following the manufacturer's instructions. Isolated monocytes were suspended in 10% DMSO/FBS aliquots of 50 e6 cell/ml and gradually cooled to −80 C. To produce macrophage cells, one or more frozen vials of monocytes were rapidly thawed in a 37 C water bath, diluted to 10 ml with growth media [RPMI 1640 (Cat. no. 72400-047, Life Technologies, Carlsbad Calif., USA)) with 10% FBS (Cat. no. 03-600-511, Themo Fisher, Waltham Mass., USA) and centrifuged 5 minutes at 250 g. Cells were suspended to 2 e6 cells/ml in growth media supplemented with 50 ng/ml human MCSF (Cat. no. PHC9501, Life Technologies, Carlsbad Calif., USA), placed into tissue culture treated, petri style tissue culture plates and into a humidified incubator programmed to maintain a “hypoxic” atmosphere of 5% CO2, 2% O2. On the third day in culture, the cells were fed with the addition of an equal volume of growth media supplemented with 50 ng/ml human MCSF. After 6 days in culture the monocytes had differentiated into MO macrophages. M0 cells were further differentiated by changing the media to growth media supplemented with 50 ng/ml human IFNg (Cat. no., PHC4031, Life Technologies, Carlsbad Calif., USA) for M1 macrophages or 40 ng/ml human IL-4 (Cat. no. PHC0045, Life Technologies, Carlsbad Calif., USA) for M2 macrophages and returning to the incubator for an additional 22 hours. On the seventh day, macrophages were suitably differentiated to be used in a bioassay. Briefly, macrophages were recovered from the petri plates by washing with 1×PBS, followed by 5 mM EDTA in PBS. The plates were then returned to 37 C for 30 minutes and cells were “power washed” off the plate using a 10 ml syringe and 22 G needle. Cells were then diluted into growth media, centrifuged at 250 g for 5 minutes after which the cell pellet was suspended to a final concentration of 1 e6/ml.

Macrophage cells prepared as above were used in bioassays where cytokines such as TNF-alpha produced in response to stimulation of the cells with TREM-1 ligand were measured in the conditioned media by ELISA. Such a bioassay was further utilized to measure blockade of TREM-1 ligand stimulation by TREM-1 specific antibodies. TREM ligand or negative controls were prepared at 4× concentrations in growth media and 50 microliters/well were added to 96 well microtiter dishes. Final concentrations of TREM-1 ligand consisted of 7.5 ng/ml recombinant human PGLYRP1 (generated as described in example 5) and 3 μg/ml PGN-BS (Cat. no., tlrl-pgnbs, Invivogen, SanDiego Calif., USA). Cells were cultured under humidified hypoxic conditions as described above for 22 hours after which conditioned media was collected and TNF-alpha levels were measured by ELISA, following manufacturer's instructions (Cat. no. DY210, R&D Systems, Minneapolis Minn., USA).


Donor 1
Donor 2
TNF-α,
TNF-α,
pg/ml
pg/ml
M1 macrophage with:
Avg
SD
Avg
SD
No addition
0
0
22
1
0.4 μg/ml PGN-BS
357
153
764
139
2.0 μg/ml PGN-BS
3086
151
5792
226
5 μg/ml PGLYRP1 + 0.4 μg/ml
7502
384
7819
945
PGN-BS
5 μg/ml PGLYRP1 + 2 μg/ml PGN-BS
31440
1030
40418
1633

This example shows that the TREM-1 ligand PGLYRP1 is able to further increase TNFa release from macrophages from two different donors.

Example 11: Cytokine Release from Synovial Tissue Cells from RA Patients Upon Stimulation with PGLYRP1

Synovial tissue samples were obtained from RA patients during total knee replacement. Single suspension of synovial tissue cells was isolated by a digestion via 4 mg/ml of collagenase (Cat. no. 11088793001, Roche, Mannheim, Germany) and 0.1 mg/ml of DNase (Cat. no. 11284932001, Roche, Mannheim, Germany) for 1 h at 37 degree. Synovial tissue cells at 1×10^5/well in culture medium RPMI (Cat. no. R0883, Sigma Aldrich, St Louis, Mo., USA) +10% FCS (Cat. no. S0115, BioChrom AG, Grand Island, N.Y. 14072, USA) were stimulated with 4 ug/ml of PGLYRP1 and 1 ug/ml of PGN-ECNDi (Cat. no. tlrl-kipgn, Invivogen,San Diego, Calif. 92121, USA). After 24 h incubation, cell supernatants were harvested, and cytokines were measured by either ELISA (TNFa (Cat. no. DY210, R&D Systems, Minneapolis, Minn. 55413 USA), IL-1b (Cat. no. 88-7010-88, eBioscience, San Diego Calif. USA), GM-CSF (Cat. no. 88-7339-88, eBioscience, San Diego Calif. USA) or Flowcytomix (TNFa, IL-1b, MIP-1b, MCP-1, IL-6, and IL-8 (Cat. no. BMS, eBioscience, San Diego Calif. USA). The cytokines were secreted from the synovial tissue cells upon stimulation with the TREM-1 ligand.


Cytokine (pg/ml)
PGN
PGN + PGLYRP1
TNFalpha
623.69
1444.59
IL-1beta
2419.42
3772.74
GM-CSF
181.91
615.91
MIP-1beta
2457.955
4394.725
MCP-1
273.055
471.26
IL-6
2056.94
4189.355
IL-8
2574.56
5509.195

This example shows that cells from synovial tissue from rheumatoid arthritis patients will respond to stimulation by the TREM-1 ligand PGLYRP1 by secreting numerous cytokines.

Example 12: Identification of Anti-PGLYRP1 mAbs that Inhibit TREM-1 Activation

In order to identify monoclonal anti-PGLYRP1 mAbs that can block TREM-1 responses, wtbalb/c mice were immunized with recombinant human PGLYRP1. Primary screening was done by means of direct ELISA on PGLYRP1 protein, and all PGLYRP1-specific hybridoma supernatants were subsequently tested in the BWZ/hTREM-1 reporter cell assay to identify monoclonal anti-PGLYRP1 antibodies capable of inhibiting TREM-1 activation induced by PGN-stimulated neutrophils, as described under example 1. The bioassay was run as follows: 40,000 hTREM-1/BWZ.36 cells/well were plated in a clear bottom, black 96 well plate in the presence of 75 ng/ml PGLYRP1 (SEQ ID NO: 1) with 2.5 pg/ml PGN-ECndi (Cat. no. tlrl-kipgn, Invivogen San Diego, Calif., USA) to provide a sub-maximal positive signal, or alternatively in the presence of a sub-maximal level (1 μg/ml) of plastic adsorbed anti TREM-1 monoclonal antibody (Cat. no. MAB1278 R&D Systems, Minneapolis, Minn., USA) to provide a positive signal. Test antibodies were titered into the assay starting at 10 μg/ml, with 5 serial 2-fold dilutions. The assay was incubated overnight at 37 C, and developed with Beta Glo (Cat. no. E4740,Promega Madison, Wis., USA), as per the Beta Glo protocol, and luminescence was recorded. Data was plotted showing Beta Glo relative luminescent units vs test antibody concentration. Non-neutralizing negative control mIgG1 (Cat. no. MAB002, R&D Systems Minneapolis, Minn., USA) and neutralizing positive control polyclonal goat anti hPGLYRP1 antibody (Cat. no. AF2590, R&D Systems, Minneapolis, Minn., USA) were run on each assay plate. As shown in FIG. 6A, F10, F14, F29, F36, F38, F77, F95 and F105 were identified as being able to block the TREM-1 response to PGLYRP1. FIG. 6B shows other PGLYRP1 antibody hybridoma supernatants able to block the TREM-1 dependent signal. M-hPGRPS-2F5 (SEQ ID NOs: 31-32 and -2F7 (SEQ ID NOs: 35-36) are very efficient blockers. In contrast, testing of commercially available monoclonal anti-PGLYRP1 antibodies in the same assay protocol alongside the antiPGLYRP1 goat polyclonal positive control (Cat. no. AF2590, R&D Systems Minneapolis, Minn., USA) showed that none of these could block TREM-1 activation (FIG. 6C). The anti PGLYRP1 antibodies tested include: 188C424 from Thermo Scientific (6D), 4H230 and 9A319 Mabs from US Biological (FIG. 6D, 6E); all failed to block PGLYRP1 stimulation of the TREM-1 bioassay. Clone 6D653 from Santa Cruz Biotechnology (FIGS. 6F and 6G) had was found to block the assay non-specifically based on the observation that the Mab blocked both PGLYRP1 (6F) and anti-TREM-1 stimulation (6G) while the positive control anti PGLYRP1 polyclonal Ab only blocked PGLYRP1 mediated activation. This non-specific blocking may be due to azide toxicity on the bioassay.

In conclusion, PGLYRP1 monoclonal antibodies have been identified that not only bind PGLYRP1 but also neutralize its TREM-1 signalling activity. The method used to identify these molecules provides a unique advantage over routine methods used to identify PGLYRP1 antibodies, evidenced by the failure of available commercial antibodies to neutralize PGLYRP1.

Example 13: PGLYRP1 Antibodies Block a TREM-1 Specific Signal

PGLYRP1 hybridoma clones were sequenced and recombinantly expressed as a hIgG4 antibody. Two of these mAb 0182 (from 1F36) (SEQ ID 15 and 16) and mAb 0184 (from 1F105) (SEQ ID 23 and 24) were retested in the BWZ/hTREM-1 reporter cell assay as described in example 13. These anti-PGLYRP1 antibodies block the TREM-1 response in the BWZ/hTREM-1 reporter cell assay in a dose-dependent manner.


BWZ.36/hTREM-1 response, Beta Glo RLU
Antibody
Isotype
182
184
ug/ml
Avg
SD
Avg
SD
Avg
SD
0
74636
10004
74636
10004
74636
10004
0.16
70289
13018
81858
3336
60738
5449
0.31
68555
5585
73382
650
59830
2837
0.63
68105
11547
73831
7818
51198
397
1.25
71797
8545
63280
1663
46447
708
2.5
69207
5004
51675
1270
42062
1953
5
76951
901
33641
842
36194
1461
10
83930
8962
20655
1080
25239
407
20
74555
511
11852
464
21333
115
40
72296
8228
7696
306
15693
1861

This example shows that, in contrast to the commercial available antibodies against PGLYRP1 shown in FIG. 6C-G, the PGLYRP1 antibodies disclosed herein are able to block the TREM-1 specific signal in the BWZ/hTREM-1 reporter cell assay.

Example 14: TNF-Alpha Release from M2 Macrophages Stimulated by PGLYRP1

Monocytes were differentiated into M2 macrophages and stimulated with PGLYRP1 complex. Antibodies (10 μg/ml) mAb-0182 and -0184 directed against PGLYRP1 is able to lower the TNF-alpha release. M2 macrophages were differentiated as described in Example 10. Antibodies to be tested were prepared at 4× concentrations in growth media and 50 microliters/well were added. The final step in initiating the bioassay was the addition of 100 microliters/well of M2 macrophage cells prepared as described above. PGLYRP1 monoclonal antibodies (mAb 0182 and mAb 0184) were tested for neutralizing activity on M2 macrophages. Duplicate (unless otherwise noted) test wells were tested under the following conditions: no added stimulation, 7.5 ng/ml PGLYRP1 only, 3 μg/ml PGN-BS (Cat. no. tlrl-pgnbs, Invivogen San Diego, Calif., USA) only (sextuplicates), PGLYRP1 with PGN-BS (sextuplicates), and PGLYRP1 with PGN-BS in the presence of PGLYRP1 antibodies or hIgG4 isotype control antibody titrated in at concentrations between 40 μg/ml and 0.31 μg/ml in 2 fold dilutions.


Donor 1
Donor 2
TNF-α,
TNF-α,
pg/ml
pg/ml
M2 macrophages with:
Avg
SD
Avg
SD
No addition
108
42
68
16
PGLYRP1
167
98
89
33
PGN
424
105
635
156
PGLYRP1 + PGN
1660
198
2168
210
PGLYRP1 + PGN + isotype
1726
182
2483
251
PGLYRP1 + PGN + mAb 0182
1322
173
2014
107
PGLYRP1 + PGN + mAb 0184
1207
168
1948
173

This example illustrates that the TREM-1 ligand PGLYRP1 is able to further increase TNFa release from M2 macrophages from two different donors and that the antibodies disclosed herein are able to decrease such TNFa release. Therefore, these PGLYRP1 antibodies are potentially useful as TREM-1 antagonists.

Example 15: Survey of Binding Interactions Between TREM-1 and PGLYRP1 Related Molecules Confirms Specificity

Having identified TREM-1 as being able to bind to PGLYRP1 and activate TREM-1 in the presence of PGN, we set out to determine whether or not TREM-1 could bind to the other PGLYRP family members. PGLYRP1 was artifically anchored in the cell-membrane through addition, at the N-terminus, of an intracellular (IC) and transmembrane domain (TM) derived from the Type II receptor MDL-1. The latter constructs were denoted Type II PGLYRP1. In Type II 1.0 (SEQ ID NO: 37), the charged amino acid of the native MDL-1 receptor TM is maintained, and efficient expression of this protein, is dependent on co-expression of DAP12. In the Type II 2.0 PGLYRP1 construct (SEQ ID NO: 38), the charged TM residue has been substituted with a neutral amino acid (Lysine to Leucine), enabling the protein to be expressed independently of eg. DAP12, and an epitope tag of DYKDDDDK (SEQ ID NO: 39) was added. Full length hPGLYRP2 (SEQ ID NO: 40), hPGLYRP3 (SEQ ID NO: 41), and hPGLYRP4 (SEQ ID NO: 42]), cDNAs were synthesised as membrane anchored proteins using N terminal fusion of the Type II 2.0 MDL1 N terminus as utilized for PGLYRP1 above. cDNAs were subcloned into a modified pTT5 expression plasmid vector (Zhang J et al. Protein Expression and Purification, Vol. 65, Issue 1, May 2009, pp 77-8), and transfected as described in example 8 into HEK293-6E cells alongside an empty vector negative control (Mock). Cells were assayed by flow cytometry for both surface and intracellular binding of the following probes: goat anti-human PGLYRP1 (PGRPS) (Cat. no. AF2590, R&D Systems, Minneapolis, Minn., USA); Mab anti-human PGLYRP2 (PGRP-L) (Cat. no. MAB0755, Abnova, Walnut, Calif., USA); Mab anti-human PGLYRP3 (PGRP-1a) (Cat. no. MAB0068, Abnova, Walnut, Calif., USA); Mab anti-human PGLYRP3 (PGRP-1a) (Cat. no. ab13901, Abcam, Cambridge Mass., USA); rabbit anti-human PGLYRP3 (PGRP-1a) (Cat. no. 18082-1-AP, Protein Tech, Chicago Ill., USA); goat anti-human PGLYRP4-biotin (PGRP-1b) (Cat. no. BAF3018, R&D Systems, Minneapolis, Minn., USA); goat anti-mouse PGLYRP1 (PGRPS) (Cat. no. AF2696, R&D Systems, Minneapolis, Minn., USA); huTREM-1.Fc dimer (C0099); huTREML1.Fc dimer (CO246); huTREML2.Fc dimer (CO247); huTREM2.Fc dimer (CO248); The binding protocol was conducted as follows, using either cytofix/perm buffer (Cat. no. 51.2090KZ, BD Biosciences, San Jose Calif., USA) for intracellular staining or 2% FBS/PBS for surface staining: cell pellets were resuspended in a 96-well round bottom plate (to begin with: 160,000 cells/well) with 200 μl cytofix/perm buffer for 15 minutes at 22 C, washed twice with 200 μl 1× PermWash Buffer (diluted 10× in DiH20), stained with 50 μl probe diluted to 5 □g/ml in 1× PermWash buffer, incubated for 1 hour at 4 C, cells were then washed with 200 μl 1× PermWash Buffer, a secondary probe was added in 50 μl 1× PermWash buffer, cells were incubated at 4 C for 30 minutes, washed twice with 200 μl 1× PermWash Buffer, pellets were resuspended in 50 μl 1:1 PBS diluted CytoFix (BD:554655, San Jose, Calif.), incubated for 5 minutes at 22 C, 150 μl PBS/2% FBS was added, centrifuged for 5 minutes at 300 g, washed 1× with 200 μl PBS/2% FB Sand resuspended in 100 ∞l PBS/2% FBS. Binding was analysed using FACS on LSRII (BD, San Jose Calif., USA).

As summarised in the table below, TREM-1 probe bound exclusively to hPGLYRP1 and no hTREM family member binding to PGLYRP2, PGLYRP3 or PGLYRP4 was detected.


Type II
Type II
Type II
Type II
Type II
Type II
Mock
PGLYRP1
PGLYRP2
PGLYRP3
PGLYRP4
PGLYRP3 + 4
muPGLYRP1
anti
++++
++
+
++++
huPGLYRP1
(R&D,
AF2590)
anti
++++
n/d
huPGLYRP2
(Abnova,
MAB07755)
anti
n/d
huPGLYRP3
(Abnova,
MAB0068)
anti
n/d
huPGLYRP3
(Abcam,
ab13901)
anti
n/d
huPGLYRP3
(Proteintech,
18082-1-AP)
anti
++++
++++
++++
n/d
huPGLYRP4
(R&D,
BAF3018)
anti
++++
n/d
n/d
n/d
n/d
++++
muPGLYRP1
(R&D,
AF2696)
huTrem1.Fc
++++
(C0099)
huTremL1.Fc
(C0246)
huTremL2.Fc
n/d
(C0247)
huTrem2.Fc
n/d
(C0248)

Human membrane anchored PGLYRP1, PGLYRP2, PGLYRP3 and PGLYRP4 were transiently expressed in HEK293 and probed with both in-house and commercial soluble receptors and antibodies in order to identify new interactions between family members. Binding scores were expressed as “n/d”, “−”, “+” or “++++”. Scores are a ratio of mean florescent intensity (MFI) of probe staining over negative control staining; “−” equals a ratio of <1, “++++” represents a score of >30 and “++” represents approximately 10-15, “+” represents 2-5 with a statistically significant difference (p<0.05). Those skilled in the art may characterize this scoring as negative, bright or dim respectively.

TREM-1 only binds to PGLYRP1 and none of the other PGLYRP members, and vice versa: PGLYRP1 only interacts with TREM-1 and none of the other TREM members.

Example 16: TREM-1 Ligand is Present in RA Synovial Fluid Samples

Rheumatoid arthritis is characterized by metacarpophalageal (MCP) joint inflammation in which activated granulocytes play a significant role. PGYLRP1 was assayed by ELISA and tested in BWZ/hTREM-1 reporter cell assay from synovial fluid drawn from MCP joints of 9 RA patients. Commercially sourced synovial fluid (Asterand, Detroit Mich., USA) was thawed, vortexed and serially diluted in ELISA buffer and run in a PGLYRP1 assay following the manufacturers guidelines (Promega, Madison Wis., USA) Four of 9 patients showed elevated PGLYRP1 levels:


PGLYRP1,
ng/ml
RA SF donor
Avg
SD
1
14
5
2
8
1
3
213
44
4
229
58
5
135
49
6
47
4
7
39
13
8
116
33
9
32
1

The RA synovial fluid samples were subsequently assayed for TREM ligand activity in the BWZ reporter assay as described in example 2. Briefly synovial fluid was thawed, vortexed, and serially diluted, assayed in duplicate +/−10 μg/ml PGNECndi (Invivogen, SanDiego, Calif., USA) with the addition of polyclonal PGLYRP1 antibody (Cat. no. AF2590, Promega, Madison Wis., USA) or a negative control polyclonal. Plastic adhered monoclonal TREM-1 and isotype antibodies (R&D Systems, Minneaopolis Minn., USA) served as positive and negative controls respectively. FIG. 8 illustrates an example of how the synovial fluid from a rheumatoid arthritis patient is able to trigger the BWZ/hTREM-1 reporter cell assay in a PGLYRP1 dependent manner.

Example 17: Assembly of Mammalian Expression Vectors Useful in the Identification and Characterization of the PGLYRP1: TREM 1 Interaction

A.) Construction of pJSV002 hTREM-1-G4Sx3-hTREM-1/Fc6mut (SEQ ID NO: 9)

An Fc receptor binding deficient version of human IgG1 was built by eliminating the first 215 amino acids of human IgG1 comprising the variable and constant one (CH1) domain and making the following amino acid substitutions within the hinge, constant 2 and constant 3 domains: (E216G, C220S, L234A, L235E, G237A, A330S, P331S). This construct was given the name Fc6mut since six mutations were made to modulate binding to Fc receptors while a 7th mutation (E216G) was incorporated to create a ApaI restriction cloning site. These mutations were built into pJSV002 (modified pTT5, Zhang J et al. Protein Expression and Purification, Vol. 65, Issue 1, May 2009, pp 77-8) allow cloning of extracellular domains of receptors 5′ of Fc6mut as EcoRI/ApaI fragments. A cDNA was synthesised with a 5′ EcoRI restriction site, a GCCACC Kozak sequence the CD33 leader sequence followed by the extracellular domain of human TREM-1 (aa17-200) with a interspaced KpnI restriction site and glycine-glycine-glycine-serine spacer repeated three times (G4Sx3) followed by an additional copy of the extracellular domain of human TREM-1 (aa17-200) and an Apa1 site to allow cloning upstream of the Fc6mut. This synthesised DNA was cut with EcoRI and ApaI and ligated into pJSV002 Fc6mut that had also been prepared with an EcoRI/ApaI digestion. This ligation was electroporated into DH10B E. coli and plated onto ampicillin agar plates. Individual clones were grown overnight in 2 mls LB+ampicillin cultures and miniprepped followed by restriction screening with EcoRI/ApaI to find clones with the appropriate 1219 base pair insert. Correct clones were sequenced, and one of the correct clones (#519) was selected for preparation of additional DNA by big prep.

B.) Construction of hTREM-1-COMP-SBP38x2-6His (SEQ ID NO: 10)

To create a pentameric TREM ECD molecule, a C terminal epitope tag was created in the pJSV002 expression vector. A synthetic cDNA encoding the 3′ end of cartilage oligomeric protein (COMP) was fused to two copies of the streptavidin binding protein domain (SBP) followed by a C-terminal 6× His domain, built into pJSV002 such that extracellular domains of receptors could be cloned 5′ as of this fragment, as an EcoRI/KpnI fragment. Subsequently a hTREMcDNA was synthesised containing an EcoRI restriction site followed by GCCACC Kozak sequence and the extracellular domain of human TREM-1 with a C-terminal KpnI site. This EcoRI/KpnI fragment was ligated into pJSV002 COMP-SBP38x2-6His vector described above. This ligation was electroporated into DH10B E. coli (Life Technolohies, Carlsbad Calif., USA) and plated onto ampicillin agar plates. Individual clones were grown overnight in 2 mls LB+ampicillin cultures and miniprepped followed by restriction screening with EcoRI/KpnI to find clones with the appropriate 616 bp insert. Correct clones were sequenced, and one of the correct clones #525 was selected for preparation of additional DNA by big prep. The full length cDNA is listed as SEQ ID NO: 10.

C.) Construction of pJSV002 hCD83-G4Sx3-hCD83/Fc6mut (SEQ ID NO: 11)

hCD83 tetramer has previously been shown to have low binding by FACS analysis when tested against a wide range of cell lines and therefore made an excellent negative control for the IPMS experiment outlined in example 3. To create this molecule, a cDNA was synthesised with a 5′ EcoRI restriction site, a GCCACC Kozak sequence, the CD33 leader sequence followed by the extracellular domain of human CD83 with a interspaced KpnI restriction site and glycine-glycine-glycine-serine spacer repeated three times (G4Sx3) followed by an additional copy of the extracellular domain of human CD83. This cDNA was then cloned upstream of the hFc6mut in the pJSV002 expression vector previously described. The resulting mature protein is listed as SEQ ID NO: 6 and the tandem CD83 extracellular domain cDNA sequence between EcoR1 and Apa1 is shown as SEQ ID NO: 11.

D.) Construction of pJSV002 NCOMP-hDCIR (SEQ ID NO: 12)

As a negative control for the hTREM-1-COMP pentamer used in example 5, hDCIR-COMP was used. A pJSV002 based expression plasmid was created with the following elements: 6×HIS tag followed by two copies of the streptavidin binding protein domain (SBP) fused to the 3′ end of cartilage oligomeric protein (COMP) such that extracellular domains of type 2 receptors could be cloned 3′ of this fragment as an BgIII/BamHI fragments and expressed as pentameric soluble receptors. A PCR fragment was amplified from a synthetic cDNA template to generate a DNA fragment with BgIII and BamHI ends on the 5′ and 3′ ends respectively. This fragment was cut with BgIII and BamHI restriction enzymes followed by band purification. The resulting fragment was ligated into pJSV002 NCOMP that had been previously cut with BgIII and BamHI. The ligation was electroporated into DH10B E. coli and plated onto ampicillin selection agar. Clones were picked, mini-prepped and screened with EcoRI and BamHI and clones with the proper 1.137 kB insert were sequenced. The cDNA coding for the full open reading frame including NCOMP-SBP and DCIR sequence is designated SEQ ID NO: 12 and codes for the previously referred to mature peptide sequence SEQ ID NO: 4.

E.) Construction of pNNC649-hTREM1-hFc6mut Dimer

TREM1-Fc dimer was used in Example 14 to confirm binding to PGLYRP1 and test binding to other PGLYRP family members. A pTT5 based plasmid (Zhang J et al., Protein Expression and Purification, Vol. 65, Issue 1, May 2009, pp. 77-8) pNNC649 was utilized to allow cloning of extracellular domains of receptors in frame and 5′ of Fc6mut. In order to express hTREM1-Fc6mut, a cDNA was synthesised with a 5′ EcoRI restriction site, a GCCACC Kozak sequence and a hCD33 leader sequence followed by the extracellular domain of human TREM-1 (aa17-200) followed by a Kpn1 site. This cDNA was cloned into pNNC549 using restriction enzyme and DNA ligase techniques familiar to those skilled in the art. The cDNA coding for the full open reading frame including CD33 leader, hTREM1 ECD and Fc6mut sequence is designated SEQ ID NO: 43 and codes for the mature peptide sequence SEQ ID NO: 44.

F.) Construction of pNNC649-hTREML1-Fc6mut Dimer

A synthetic cDNA was created with a 5′ EcoRI restriction site, a GCCACC Kozak sequence and a hCD33 leader sequence followed by the extracellular domain of human TREML1 (aa16-162) followed by a Kpn1 site. This cDNA was cloned into pNNC549 vector previously described using restriction enzyme and DNA ligase techniques familiar to those skilled in the art. The cDNA coding for the full open reading frame including CD33 leader, hTREML1 ECD and Fc6mut sequence is designated SEQ ID NO: 45 and codes for the mature peptide sequence SEQ ID NO: 46.

G.) Construction of pNNC649-hTREML2-Fc6mut Dimer

A synthetic cDNA was created with a 5′ EcoRI restriction site, a GCCACC Kozak sequence and a hCD33 leader sequence followed by the extracellular domain of human TREML2 (aa19-268) followed by a Kpn1 site. This cDNA was cloned into pNNC549 vector previously described using restriction enzyme and DNA ligase techniques familiar to those skilled in the art. The cDNA coding for the full open reading frame including CD33 leader, hTREML2 ECD and Fc6mut sequence is designated SEQ ID NO: 47 and codes for the mature peptide sequence SEQ ID NO: 48.

H.) Construction of pNNC649-hTREM2-Fc6mut Dimer

A synthetic cDNA was created with a 5′ EcoRI restriction site, a GCCACC Kozak sequence and a hCD33 leader sequence followed by the extracellular domain of human TREM2 (aa19-174) followed by a Kpn1 site. This cDNA was cloned into pNNC549 vector previously described using restriction enzyme and DNA ligase techniques familiar to those skilled in the art. The cDNA coding for the full open reading frame including CD33 leader, hTREM2 ECD and Fc6mut sequence is designated SEQ ID NO: 49 and codes for the mature peptide sequence SEQ ID NO: 50.

Example 18: A Multimerised PGLYRP1 Activates TREM-1

A counter-structure or ligand for TREM-1 was identified through binding analysis and IP/MS proteomics. The identity of the ligand, PGLYRP1 (or PGRP-S) was subsequently validated through specific blockage.

Interestingly, while binding of the soluble PGLYRP1 can be demonstrated to the TREM-1, activation of the TREM-1 by PGLYRP1 requires the concurrent presence of a scaffolding agent such as Neutrophil Extracellular Traps (NETs) or PGN.

To test if such alternative, and multimerised formats of PGLYRP1 might bind and/or activate TREM-1, a cell-associated PGLYRP1 protein was designed and expressed. Two conceptually distinct PGLYRP1 constructs were tested. In one, a GPI-anchoring sequence motif was added to the C-terminal end of PGLYRP1. In another, PGLYRP1 was artificially anchored in the cell-membrane through addition, at the N-terminus, of an intracellular (IC) and transmembrane domain (TM) derived from the Type II receptor MDL-1. The latter constructs were denoted Type II PGLYRP1. In Type II 1.0 (SEQ ID NO: 37), the charged amino acid of the native MDL-1 receptor TM is maintained, and efficient expression of this protein, is dependent on co-expression of DAP12. In the Type II 2.0 PGLYRP1 construct (SEQ ID NO: 38), the charged TM residue has been substituted with a neutral amino acid (Lysine to Leucine), enabeling the protein to be expressed independently of eg. DAP12. The cDNAs encoding these constructs were transiently expressed in HEK293 6E cells, cells were harvested on day two post-transfection and analyzed for their ability to stimulate the reporter cell line BWZ/hTREM1. Type II PGLYRP1 transfectants were co-incubated with BWZ/hTREM1 reporter cells in the absence of PGN. TREM1 activation was read out after 18 hours using the BetaGlo reagent (Cat. no. E4720, Promega, Madison Wis., USA). The transfectants expressing Type II PGLYRP1 induced activation of TREM-1 in the absence of PGN, to a level 24-fold higher than seen with control cells transfected with empty expression vector. In contrast, the GPI-anchored PGLYRP1, immobilized via the C-terminus of PGLYRP1, did not mediate any TREM-1 activation. Expression of the membrane-bound and immobilized PGLYRP1 protein at the cell-surface was ascertained by flow cytometry using a polyclonal anti-PGLYRP1 antibody (AF2590). Both proteins, Type II PGLYRP1 and GPI-PGLYRP1, were demonstrated to indeed be cell-surface expressed.


Construct
Fold activity
C-GPI PGLYRP1
2.0 ± 0.4 (n = 3)
Type II 1.0 PGLYRP1
 24 ± 3.3 (n = 3)

The table above shows that PGLYRP1 bound to the cell membrane surface via a C-terminal GPI anchor is not as potent in inducing TREM-1 activity as a Type II PGLYRP1 protein bound to the cell membrane via the N-terminal part. This illustrates the importance of a free C-terminal PGLYRP1 part to be able to stimulate TREM-1.

Type II PGLYRP1 activation was further demonstrated to be inhibited specifically by anti-PGLYRP1 antibody. Addition of the polyclonal (Cat. no. AF2590, R&D Systems, Minneapolis Minn., USA) PGLYRP1 antibody at high concentration (1 μg/100 μl assay volume) was thus able to totally inhibit this PGLYRP1 induced activity.


Condition
Activity
Type II 1.0 PGLYRP1
100%
Type II 1.0 PGLYRP1 + Iso Ab
 98%
Type II 1.0 PGLYRP1 + AF2590
 9.7%

Sequences at the very C-terminal domain of PGLYRP1 appear critical to the ability to activate the TREM-1 receptor. Several constructs, which share as a common feature, the modification at the extreme C-terminus of PGLYRP1, have thus been seen to not mediate activation of TREM1/BWZ reporter activity, while the corresponding constructs, which in contrast have been modified at the N-terminus, do exhibit activity.


Construct
Activity
PGLYRP1 Native
+++
PGLYRP1 N-Flag
+++
PGLYRP1 C-Flag
PGLYRP1 N-Fc
+++
PGLYRP1 C-Fc
(+)
Type II 1.0 PGLYRP1
+++
PGLYRP1 C-GPI
(+)

This indicates the importance of a free C-terminal PGLYRP1 part to be able to stimulate TREM-1.

Interpretation and Biological Perspectives

The ability to activate the TREM-1 receptor using either the native PGLYRP1-ligand in the presence of PGN or alternatively, novel PGLYRP1 variants which have been demonstrated to overcome the need for PGN, clearly demonstrates that PGN is not an absolute co-factor requirement for TREM-1 activation. The common feature of the various molecular PGLYRP1 formats which were demonstrated to confer PGN-independent TREM-1 activation appear to be a high density format leading to the hypothesis that the predominant role of PGN, when acting as co-factor for the native ligand, is to provide a scaffold for multimerisation. In vivo, such a scaffold could be provided by Neutrophil Extracellular Traps (NETs) (Blood 2005, 106: 2551-58) or other naturally occurring matrix structures, such as hyaloronic acid, proteoglycan structures, such as versican, aggrecan, decorin, or fibrin, all of which may multimerise or otherwise present PGLYRP1.

These findings suggest that modification of the C-terminal part of PGLYRP1 reduces TREM-1 activation which in turn indicates that blocking the C-terminal part of PGLYRP1 with an agent, such as an antibody directed against the C-terminal part of PGLYRP1, would decrease the TREM-1 interaction and thereby TREM-1 stimulation.

Example 19: Type II PGLYRP1 is Able to Induce TNFalpha Release in Synovial Tissue Cells from Rheumatoid Arthritis Patients

Synovial tissue samples were obtained from RA patients during total knee replacement. Single suspension of synovial tissue cells was isolated by a digestion via 4 mg/ml of collagenase (Cat. no. 11088793001, Roche, Mannheim, Germany) and 0.1 mg/ml of DNase (Cat. no. 11284932001, Roche, Mannheim, Germany) for 1 h at 37 degree. The synovial tissue cells (1×10^5/well in culture medium RPMI (Cat. no. 22400105, Life Technologies, Carlsbad Calif., USA) +10% FCS (Cat. no. 50115, BioChrom AG, Berlin, Germany) were co-cultured with various doses of HEK cells transiently transfected with type II PGLYRP1 under hypoxic condition. After 24 h incubation, cell supernatants were harvested, and cytokines were measured by TNFa ELISA (Cat. no. DY210, R&D Systems, Minneapolis, Minn., USA).


Type II PGLYRP1 (HEK
transfected)/Control
TNF-α (pg/ml) release
HEK
1 × 10{circumflex over ( )}5
3 × 10{circumflex over ( )}4
1 × 10{circumflex over ( )}4
3 × 10{circumflex over ( )}3
1 × 10{circumflex over ( )}3
0
IgG4 + Type II
121.17
114.08
95.02
54.56
57.87
33.47
IgG4 + Control
55.65
63.73
57.99
33.78
36.40
36.32

This example shows that the TREM-1 ligand can induce TNF-alpha in a dose-dependent manner in synovial tissue cells from rheumatoid arthritis patients.

Example 20: PGLYRP1 Antibodies Block the TREM-1 Mediated Signal in Neutrophils and Decrease IL-8 Release

Having demonstrated that neutrophils can release PGLYRP1 and that neutrophils also express the TREM-1 receptor, we tested whether neutrophil-derived PGLYRP1 can stimulate neutrophils in an autocrine manner. Isolated neutrophils were stimulated with PGN-SA (Cat. no. tlrl-pgnsa, Invivogen, SanDiego Calif., USA), and the release of IL-8 into the culture medium was measured. A PGLYRP1 antibody, mAb 0184, was able to decrease the PGN-SA-induced IL-8 release. Neutrophils were isolated from human healthy donor whole blood as described in example 3 and resuspended in RPMI/10% FBS. The cells were plated out at 1.5×10 E6 cells/ml, and triplicate test wells were tested under the following conditions: no added stimulation, 10 μg/ml PGN-SA only, or 10 μg/ml PGN-SA in the presence of PGLYRP1 antibody or hIgG4 isotype control antibody at 4 μg/ml. The samples were cultured 24 hours in a 37° C., 5% CO2 incubator. Supernatants were then harvested and analysed for IL-8 using the Bioplex Pro Human Cytokine IL-8 set (Cat. no. 171-B5008M, BioRad, Hercules Calif., USA).


IL-8, pg/ml
Neutrophils stimulated with
Avg
SD
No addition
52
3
PGN-SA
1158
341
PGN-SA + isotype control
1195
144
PGN-SA + mAb 0184
449
50

This example illustrates that IL-8 release from neutrophils induced by stimulation with the bacterially derived PGN-SA can be reduced by anti-PGLYRP1 antibody. The TREM-1 ligand PGLYRP1 is thus an autocrine stimulant of neutrophils, and the PGLYRP1 antibody disclosed herein are potentially useful in down-regulating neutrophil responses.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now be apparent to those of ordinary skill in the art. It is, therefore, to be understood that the appended embodiments are intended to cover all such modifications and changes as fall within the true spirit of the invention.

<160> NUMBER OF SEQ ID NOS: 53

<210> SEQ ID NO: 1

<211> LENGTH: 175

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 1

Gln Glu Thr Glu Asp Pro Ala Cys Cys Ser Pro Ile Val Pro Arg Asn

1 5 10 15

Glu Trp Lys Ala Leu Ala Ser Glu Cys Ala Gln His Leu Ser Leu Pro

20 25 30

Leu Arg Tyr Val Val Val Ser His Thr Ala Gly Ser Ser Cys Asn Thr

35 40 45

Pro Ala Ser Cys Gln Gln Gln Ala Arg Asn Val Gln His Tyr His Met

50 55 60

Lys Thr Leu Gly Trp Cys Asp Val Gly Tyr Asn Phe Leu Ile Gly Glu

65 70 75 80

Asp Gly Leu Val Tyr Glu Gly Arg Gly Trp Asn Phe Thr Gly Ala His

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Ser Gly His Leu Trp Asn Pro Met Ser Ile Gly Ile Ser Phe Met Gly

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Asn Tyr Met Asp Arg Val Pro Thr Pro Gln Ala Ile Arg Ala Ala Gln

115 120 125

Gly Leu Leu Ala Cys Gly Val Ala Gln Gly Ala Leu Arg Ser Asn Tyr

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Val Leu Lys Gly His Arg Asp Val Gln Arg Thr Leu Ser Pro Gly Asn

145 150 155 160

Gln Leu Tyr His Leu Ile Gln Asn Trp Pro His Tyr Arg Ser Pro

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<210> SEQ ID NO: 2

<211> LENGTH: 617

<212> TYPE: PRT

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: TREM ECD+Fc6mut

<400> SEQENCE: 2

Glu Leu Arg Ala Ala Thr Lys Leu Thr Glu Glu Lys Tyr Glu Leu Lys

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Glu Gly Gln Thr Leu Asp Val Lys Cys Asp Tyr Thr Leu Glu Lys Phe

20 25 30

Ala Ser Ser Gln Lys Ala Trp Gln Ile Ile Arg Asp Gly Glu Met Pro

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Lys Thr Leu Ala Cys Thr Glu Arg Pro Ser Lys Asn Ser His Pro Val

50 55 60

Gln Val Gly Arg Ile Ile Leu Glu Asp Tyr His Asp His Gly Leu Leu

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Arg Val Arg Met Val Asn Leu Gln Val Glu Asp Ser Gly Leu Tyr Gln

85 90 95

Cys Val Ile Tyr Gln Pro Pro Lys Glu Pro His Met Leu Phe Asp Arg

100 105 110

Ile Arg Leu Val Val Thr Lys Gly Phe Ser Gly Thr Pro Gly Ser Asn

115 120 125

Glu Asn Ser Thr Gln Asn Val Tyr Lys Ile Pro Pro Thr Thr Thr Lys

130 135 140

Ala Leu Cys Pro Leu Tyr Thr Ser Pro Arg Thr Val Thr Gln Ala Pro

145 150 155 160

Pro Lys Ser Thr Ala Asp Val Ser Thr Pro Asp Ser Glu Ile Asn Leu

165 170 175

Thr Asn Val Thr Asp Ile Ile Arg Gly Thr Gly Gly Gly Gly Ser Gly

180 185 190

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Arg Ala Ala Thr Lys

195 200 205

Leu Thr Glu Glu Lys Tyr Glu Leu Lys Glu Gly Gln Thr Leu Asp Val

210 215 220

Lys Cys Asp Tyr Thr Leu Glu Lys Phe Ala Ser Ser Gln Lys Ala Trp

225 230 235 240

Gln Ile Ile Arg Asp Gly Glu Met Pro Lys Thr Leu Ala Cys Thr Glu

245 250 255

Arg Pro Ser Lys Asn Ser His Pro Val Gln Val Gly Arg Ile Ile Leu

260 265 270

Glu Asp Tyr His Asp His Gly Leu Leu Arg Val Arg Met Val Asn Leu

275 280 285

Gln Val Glu Asp Ser Gly Leu Tyr Gln Cys Val Ile Tyr Gln Pro Pro

290 295 300

Lys Glu Pro His Met Leu Phe Asp Arg Ile Arg Leu Val Val Thr Lys

305 310 315 320

Gly Phe Ser Gly Thr Pro Gly Ser Asn Glu Asn Ser Thr Gln Asn Val

325 330 335

Tyr Lys Ile Pro Pro Thr Thr Thr Lys Ala Leu Cys Pro Leu Tyr Thr

340 345 350

Ser Pro Arg Thr Val Thr Gln Ala Pro Pro Lys Ser Thr Ala Asp Val

355 360 365

Ser Thr Pro Asp Ser Glu Ile Asn Leu Thr Asn Val Thr Asp Ile Ile

370 375 380

Arg Gly Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

385 390 395 400

Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

405 410 415

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

420 425 430

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

435 440 445

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

450 455 460

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

465 470 475 480

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

485 490 495

Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

500 505 510

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

515 520 525

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

530 535 540

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

545 550 555 560

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

565 570 575

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

580 585 590

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

595 600 605

Lys Ser Leu Ser Leu Ser Pro Gly Lys

610 615

<210> SEQ ID NO: 3

<211> LENGTH: 228

<212> TYPE: PRT

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: Fc5mut

<400> SEQENCE: 3

Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu

1 5 10 15

Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Pro Gly Lys

225

<210> SEQ ID NO: 4

<211> LENGTH: 358

<212> TYPE: PRT

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hDCIR ECD COMP for pentamer

<400> SEQENCE: 4

His His His His His His Glu Asp Leu Tyr Phe Gln Ser Met Asp Glu

1 5 10 15

Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly Leu Ala Gly

20 25 30

Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro Gln Gly Gln

35 40 45

Arg Glu Pro Gly Ser Gly Met Asp Glu Lys Thr Thr Gly Trp Arg Gly

50 55 60

Gly His Val Val Glu Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala

65 70 75 80

Arg Leu Glu His His Pro Gln Gly Gln Arg Glu Pro Gly Gly Gly Ser

85 90 95

Gly Gly Gly Ser Gly Gly Gly Ser Gly Asp Leu Ala Pro Gln Met Leu

100 105 110

Arg Glu Leu Gln Glu Thr Asn Ala Ala Leu Gln Asp Val Arg Glu Leu

115 120 125

Leu Arg Gln Gln Val Lys Glu Ile Thr Phe Leu Lys Asn Thr Val Met

130 135 140

Glu Cys Asp Ala Cys Gly Met Gln Pro Ala Arg Thr Pro Gly Leu Ser

145 150 155 160

Val Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Leu Glu Val

165 170 175

Leu Phe Gln Gly Pro Arg Ser Ile Ala Phe Val Ile Phe Phe Gln Lys

180 185 190

Tyr Ser Gln Leu Leu Glu Lys Lys Thr Thr Lys Glu Leu Val His Thr

195 200 205

Thr Leu Glu Cys Val Lys Lys Asn Met Pro Val Glu Glu Thr Ala Trp

210 215 220

Ser Cys Cys Pro Lys Asn Trp Lys Ser Phe Ser Ser Asn Cys Tyr Phe

225 230 235 240

Ile Ser Thr Glu Ser Ala Ser Trp Gln Asp Ser Glu Lys Asp Cys Ala

245 250 255

Arg Met Glu Ala His Leu Leu Val Ile Asn Thr Gln Glu Glu Gln Asp

260 265 270

Phe Ile Phe Gln Asn Leu Gln Glu Glu Ser Ala Tyr Phe Val Gly Leu

275 280 285

Ser Asp Pro Glu Gly Gln Arg His Trp Gln Trp Val Asp Gln Thr Pro

290 295 300

Tyr Asn Glu Ser Ser Thr Phe Trp His Pro Arg Glu Pro Ser Asp Pro

305 310 315 320

Asn Glu Arg Cys Val Val Leu Asn Phe Arg Lys Ser Pro Lys Arg Trp

325 330 335

Gly Trp Asn Asp Val Asn Cys Leu Gly Pro Gln Arg Ser Val Cys Glu

340 345 350

Met Met Lys Ile His Leu

355

<210> SEQ ID NO: 5

<211> LENGTH: 367

<212> TYPE: PRT

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hTREM-1 ECD COMP for pentamer

<400> SEQENCE: 5

Glu Leu Arg Ala Ala Thr Lys Leu Thr Glu Glu Lys Tyr Glu Leu Lys

1 5 10 15

Glu Gly Gln Thr Leu Asp Val Lys Cys Asp Tyr Thr Leu Glu Lys Phe

20 25 30

Ala Ser Ser Gln Lys Ala Trp Gln Ile Ile Arg Asp Gly Glu Met Pro

35 40 45

Lys Thr Leu Ala Cys Thr Glu Arg Pro Ser Lys Asn Ser His Pro Val

50 55 60

Gln Val Gly Arg Ile Ile Leu Glu Asp Tyr His Asp His Gly Leu Leu

65 70 75 80

Arg Val Arg Met Val Asn Leu Gln Val Glu Asp Ser Gly Leu Tyr Gln

85 90 95

Cys Val Ile Tyr Gln Pro Pro Lys Glu Pro His Met Leu Phe Asp Arg

100 105 110

Ile Arg Leu Val Val Thr Lys Gly Phe Ser Gly Thr Pro Gly Ser Asn

115 120 125

Glu Asn Ser Thr Gln Asn Val Tyr Lys Ile Pro Pro Thr Thr Thr Lys

130 135 140

Ala Leu Cys Pro Leu Tyr Thr Ser Pro Arg Thr Val Thr Gln Ala Pro

145 150 155 160

Pro Lys Ser Thr Ala Asp Val Ser Thr Pro Asp Ser Glu Ile Asn Leu

165 170 175

Thr Asn Val Thr Asp Ile Ile Arg Gly Thr Leu Glu Val Leu Phe Gln

180 185 190

Gly Pro Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Asp

195 200 205

Leu Ala Pro Gln Met Leu Arg Glu Leu Gln Glu Thr Asn Ala Ala Leu

210 215 220

Gln Asp Val Arg Glu Leu Leu Arg Gln Gln Val Lys Glu Ile Thr Phe

225 230 235 240

Leu Lys Asn Thr Val Met Glu Cys Asp Ala Cys Gly Met Gln Pro Ala

245 250 255

Arg Thr Pro Gly Leu Ser Val Gly Gly Gly Ser Gly Gly Gly Ser Gly

260 265 270

Gly Gly Ser Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val

275 280 285

Val Glu Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu

290 295 300

His His Pro Gln Gly Gln Arg Glu Pro Gly Ser Gly Met Asp Glu Lys

305 310 315 320

Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly Leu Ala Gly Glu

325 330 335

Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro Gln Gly Gln Arg

340 345 350

Glu Pro Glu Asp Leu Tyr Phe Gln Ser His His His His His His

355 360 365

<210> SEQ ID NO: 6

<211> LENGTH: 505

<212> TYPE: PRT

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hCD83 ECD Fc

<400> SEQENCE: 6

Thr Pro Glu Val Lys Val Ala Cys Ser Glu Asp Val Asp Leu Pro Cys

1 5 10 15

Thr Ala Pro Trp Asp Pro Gln Val Pro Tyr Thr Val Ser Trp Val Lys

20 25 30

Leu Leu Glu Gly Gly Glu Glu Arg Met Glu Thr Pro Gln Glu Asp His

35 40 45

Leu Arg Gly Gln His Tyr His Gln Lys Gly Gln Asn Gly Ser Phe Asp

50 55 60

Ala Pro Asn Glu Arg Pro Tyr Ser Leu Lys Ile Arg Asn Thr Thr Ser

65 70 75 80

Cys Asn Ser Gly Thr Tyr Arg Cys Thr Leu Gln Asp Pro Asp Gly Gln

85 90 95

Arg Asn Leu Ser Gly Lys Val Ile Leu Arg Val Thr Gly Cys Pro Ala

100 105 110

Gln Arg Lys Glu Glu Thr Phe Lys Lys Tyr Arg Ala Glu Gly Thr Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro

130 135 140

Glu Val Lys Val Ala Cys Ser Glu Asp Val Asp Leu Pro Cys Thr Ala

145 150 155 160

Pro Trp Asp Pro Gln Val Pro Tyr Thr Val Ser Trp Val Lys Leu Leu

165 170 175

Glu Gly Gly Glu Glu Arg Met Glu Thr Pro Gln Glu Asp His Leu Arg

180 185 190

Gly Gln His Tyr His Gln Lys Gly Gln Asn Gly Ser Phe Asp Ala Pro

195 200 205

Asn Glu Arg Pro Tyr Ser Leu Lys Ile Arg Asn Thr Thr Ser Cys Asn

210 215 220

Ser Gly Thr Tyr Arg Cys Thr Leu Gln Asp Pro Asp Gly Gln Arg Asn

225 230 235 240

Leu Ser Gly Lys Val Ile Leu Arg Val Thr Gly Cys Pro Ala Gln Arg

245 250 255

Lys Glu Glu Thr Phe Lys Lys Tyr Arg Ala Glu Gly Ala Leu Ala Gly

260 265 270

Thr Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

275 280 285

Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

290 295 300

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

305 310 315 320

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

325 330 335

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

340 345 350

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

355 360 365

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

370 375 380

Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

385 390 395 400

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

405 410 415

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

420 425 430

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

435 440 445

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

450 455 460

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

465 470 475 480

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

485 490 495

Lys Ser Leu Ser Leu Ser Pro Gly Lys

500 505

<210> SEQ ID NO: 7

<211> LENGTH: 684

<212> TYPE: DNA

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hPGYRP1-GPI

<400> SEQENCE: 7

gaattcgcca ccatgtcccg ccgctctatg ctgcttgcct gggctctccc cagcctcctt 60

cgactcggag cggctcagga gacagaagac ccggcctgct gcagccccat agtgccccgg 120

aacgagtgga aggccctggc atcagagtgc gcccagcacc tgagcctgcc cttacgctat 180

gtggtggtat cgcacacggc gggcagcagc tgcaacaccc ccgcctcgtg ccagcagcag 240

gcccggaatg tgcagcacta ccacatgaag acactgggct ggtgcgacgt gggctacaac 300

ttcctgattg gagaagacgg gctcgtatac gagggccgtg gctggaactt cacgggtgcc 360

cactcaggtc acttatggaa ccccatgtcc attggcatca gcttcatggg caactacatg 420

gatcgggtgc ccacacccca ggccatccgg gcagcccagg gtctactggc ctgcggtgtg 480

gctcagggag ccctgaggtc caactatgtg ctcaaaggac accgggatgt gcagcgtaca 540

ctctctccag gcaaccagct ctaccacctc atccagaatt ggccacacta ccgctccccc 600

tcctcatcga caactacgac cacaactacg accctacttc tcctgctact tctgctccta 660

cttctgctcc tactttgact cgag 684

<210> SEQ ID NO: 8

<211> LENGTH: 180

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 8

Ala Thr Lys Leu Thr Glu Glu Lys Tyr Glu Leu Lys Glu Gly Gln Thr

1 5 10 15

Leu Asp Val Lys Cys Asp Tyr Thr Leu Glu Lys Phe Ala Ser Ser Gln

20 25 30

Lys Ala Trp Gln Ile Ile Arg Asp Gly Glu Met Pro Lys Thr Leu Ala

35 40 45

Cys Thr Glu Arg Pro Ser Lys Asn Ser His Pro Val Gln Val Gly Arg

50 55 60

Ile Ile Leu Glu Asp Tyr His Asp His Gly Leu Leu Arg Val Arg Met

65 70 75 80

Val Asn Leu Gln Val Glu Asp Ser Gly Leu Tyr Gln Cys Val Ile Tyr

85 90 95

Gln Pro Pro Lys Glu Pro His Met Leu Phe Asp Arg Ile Arg Leu Val

100 105 110

Val Thr Lys Gly Phe Ser Gly Thr Pro Gly Ser Asn Glu Asn Ser Thr

115 120 125

Gln Asn Val Tyr Lys Ile Pro Pro Thr Thr Thr Lys Ala Leu Cys Pro

130 135 140

Leu Tyr Thr Ser Pro Arg Thr Val Thr Gln Ala Pro Pro Lys Ser Thr

145 150 155 160

Ala Asp Val Ser Thr Pro Asp Ser Glu Ile Asn Leu Thr Asn Val Thr

165 170 175

Asp Ile Ile Arg

180

<210> SEQ ID NO: 9

<211> LENGTH: 1221

<212> TYPE: DNA

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hTREM ECD x2

<400> SEQENCE: 9

gaattcgcaa ctatgcctct gctgctgctg ctgcctctgc tgtgggctgg cgcactggct 60

gaactgaggg ccgctaccaa actgaccgaa gaaaagtacg agctgaaaga agggcagacc 120

ctggacgtga agtgcgatta tacactggaa aagttcgcaa gctcccagaa agcctggcag 180

atcattagag acggagagat gcccaagact ctggcttgta ccgaacgccc ttcaaaaaac 240

agccacccag tgcaggtcgg ccgaatcatt ctggaggact accacgatca tgggctgctg 300

cgggtgagaa tggtcaatct gcaggtggag gactccggcc tgtaccagtg cgtcatctat 360

cagcccccta aggaaccaca tatgctgttc gataggattc gcctggtggt cactaaaggc 420

ttttctggga cccccggaag taacgagaac agcacccaga acgtgtacaa gatcccaccc 480

accacaacta aggccctgtg ccccctgtat acatctcctc gaaccgtgac acaggcccct 540

ccaaagagta ccgctgacgt gagcacaccc gattccgaga ttaacctgac aaatgtgact 600

gacatcatta ggggtaccgg aggaggagga tccggaggag gaggaagcgg aggcggggga 660

tccgaactgc gcgccgctac caagctgaca gaggaaaaat atgagctgaa agaaggccag 720

actctggacg tgaaatgcga ttataccctg gagaagtttg cttctagtca gaaagcatgg 780

cagatcattc gcgatgggga gatgcctaag acactggcct gtactgaacg gccctccaaa 840

aactctcacc ctgtgcaggt cggaagaatc attctggaag actatcacga tcatggcctg 900

ctgcgagtgc ggatggtgaa tctgcaggtc gaggacagtg gactgtatca atgcgtcatc 960

tatcaacccc ctaaggaacc tcatatgctg ttcgatagaa ttaggctggt ggtcacaaaa 1020

ggctttagcg ggactccagg atctaacgag aatagtactc agaacgtgta caaaattcct 1080

cctactacca ccaaggccct gtgcccactg tatacaagcc cacgaactgt gacccaggca 1140

cctccaaaga gcactgcaga tgtgagcact ccagatagcg aaatcaacct gaccaatgtg 1200

acagatatta ttagggggcc c 1221

<210> SEQ ID NO: 10

<211> LENGTH: 1170

<212> TYPE: DNA

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hTREM COMP

<400> SEQENCE: 10

gaattcgcaa ctatgcctct gctgctgctg ctgcctctgc tgtgggctgg cgcactggct 60

gaactgaggg ccgctaccaa actgaccgaa gaaaagtacg agctgaaaga agggcagacc 120

ctggacgtga agtgcgatta tacactggaa aagttcgcaa gctcccagaa agcctggcag 180

atcattagag acggagagat gcccaagact ctggcttgta ccgaacgccc ttcaaaaaac 240

agccacccag tgcaggtcgg ccgaatcatt ctggaggact accacgatca tgggctgctg 300

cgggtgagaa tggtcaatct gcaggtggag gactccggcc tgtaccagtg cgtcatctat 360

cagcccccta aggaaccaca tatgctgttc gataggattc gcctggtggt cactaaaggc 420

ttttctggga cccccggaag taacgagaac agcacccaga acgtgtacaa gatcccaccc 480

accacaacta aggccctgtg ccccctgtat acatctcctc gaaccgtgac acaggcccct 540

ccaaagagta ccgctgacgt gagcacaccc gattccgaga ttaacctgac aaatgtgact 600

gacatcatta ggggtaccct ggaggtgctg ttccagggac caggaggagg aagcggagga 660

ggcagcggcg ggggatctgg ggacctggcc cctcagatgc tgagggagct gcaggaaacc 720

aacgccgctc tgcaggatgt gcgggagctg ctgagacagc aggtgaagga aatcacattt 780

ctgaaaaata ctgtgatgga gtgcgacgct tgtggaatgc agccagctag gacacctgga 840

ctgagcgtgg gaggaggaag tggaggagga tcaggaggag gaagcatgga tgagaagacc 900

acaggatgga gaggaggaca cgtggtggaa ggactggctg gagagctgga acagctgagg 960

gctagactgg agcaccatcc acagggacag agggagccag ggtccggaat ggacgaaaaa 1020

actaccggat ggaggggagg acacgtggtg gagggcctgg ccggcgaact ggagcagctg 1080

agagctcgcc tggaacacca tcctcagggc cagagagagc cagaggacct gtacttccag 1140

tctcaccatc accatcacca ttaaggatcc 1170

<210> SEQ ID NO: 11

<211> LENGTH: 867

<212> TYPE: DNA

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hCD83 for tetramer

<400> SEQENCE: 11

gaattcgcca ccatgcctct gctgctgctg ctgccactgc tgtgggctgg cgctctggct 60

accccagagg tgaaggtggc ttgctccgaa gacgtggatc tgccttgtac agccccctgg 120

gaccctcagg tgccatacac cgtgagctgg gtgaaactgc tggagggcgg ggaggaacgg 180

atggagacac cacaggaaga ccacctgaga gggcagcact atcatcagaa ggggcagaac 240

ggatctttcg atgctcccaa tgaacggcct tacagtctga aaatcagaaa caccacaagc 300

tgcaattccg gcacatatag gtgtactctg caggaccctg atggacagcg caacctgagc 360

ggcaaagtga tcctgcgggt gacaggctgc ccagctcaga gaaaggagga aacttttaag 420

aagtaccggg ccgagggtac cggaggcggg ggatccggag gaggaggaag cggaggagga 480

ggatccactc ctgaagtgaa ggtggcttgc agtgaagacg tggatctgcc ctgtaccgcc 540

ccctgggatc ctcaggtgcc atacacagtg tcttgggtga agctgctgga gggaggcgag 600

gaacggatgg agacccctca ggaagaccat ctgagaggcc agcattacca tcagaagggc 660

cagaacgggt cattcgatgc cccaaatgaa aggccctaca gcctgaaaat ccgcaacact 720

acctcttgca atagtggaac ctataggtgt acactgcagg accccgatgg gcagcgcaat 780

ctgtccggca aagtgatcct gagggtgact ggctgtcctg ctcagcgcaa agaggaaacc 840

tttaagaaat atagggccga ggggccc 867

<210> SEQ ID NO: 12

<211> LENGTH: 1143

<212> TYPE: DNA

<213> ORGANISM: Artificial

<220> FEATURE:

<223> OTHER INFORMATION: hDCIR COMP

<400> SEQENCE: 12

gaattcgcca ccatgccact gctgctgctg ctgccactgc tgtgggctgg agctctggct 60

caccatcacc atcaccatga ggacctgtac ttccagtcca tggatgaaaa gaccacagga 120

tggaggggag gacacgtggt ggagggactg gctggagagc tggaacagct gagggctaga 180

ctggaacacc atcctcaggg ccagagggag ccagggtctg gaatggacga aaaaactacc 240

ggctggagag gcggccatgt cgtcgaaggc ctggccggcg aactggagca gctgagagct 300

aggctggagc accatccaca gggacagagg gaacctggag gaggcagcgg aggaggctcc 360

ggaggaggct ctggcgacct ggccccacag atgctgagag agctgcagga aaccaacgcc 420

gctctgcagg atgtgcggga gctgctgaga cagcaggtga aggaaatcac attcctgaaa 480

aatactgtga tggagtgcga tgcttgtgga atgcagccag ctaggacacc tggactgagc 540

gtgggaggag gcagtggagg aggctcagga ggaggcagcc tggaggtgct gtttcagggc 600

cccagatcta ttgcttttgt cattttcttt caaaaatatt ctcagcttct tgaaaaaaag 660

actacaaaag agctggttca tacaacattg gagtgtgtga aaaaaaatat gcccgtggaa 720

gagacagcct ggagctgttg cccaaagaat tggaagtcat ttagttccaa ctgctacttt 780

atttctactg aatcagcatc ttggcaagac agtgagaagg actgtgctag aatggaggct 840

cacctgctgg tgataaacac tcaagaagag caggatttca tcttccagaa tctgcaagaa 900

gaatctgctt attttgtggg gctctcagat ccagaaggtc agcgacattg gcaatgggtt 960

gatcagacac catacaatga aagttccaca ttctggcatc cacgtgagcc cagtgatccc 1020

aatgagcgct gcgttgtgct aaattttcgt aaatcaccca aaagatgggg ctggaatgat 1080

gttaattgtc ttggtcctca aaggtcagtt tgtgagatga tgaagatcca cttatgagga 1140

tcc 1143

<210> SEQ ID NO: 13

<211> LENGTH: 426

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 13

aagcttgccg ccaccatgcc tctgctgctg ctgctgcctc tgctgtgggc cggggctctg 60

gctcaggtcc agctgcagca gcctggggcc gaactggtcc gaccaggggc atctgtgaaa 120

ctgagttgca aggcctctgg atacagtttc acctcatatt ggatgaactg ggtcaaacag 180

cgaccaggac agggactgga gtggatcggc atgattcacc ctagcgactc cgaaacaaga 240

ctgaatcaga agtttaaaga caaggctacc ctgacagtgg ataagagctc ctctacagca 300

tacatgcagc tgagttcacc cactagcgag gactccgccg tctactattg tgctagggat 360

tactctgact atgatgggtt cgcctattgg ggacagggca ctctggtgac cgtcagcgct 420

gctagc 426

<210> SEQ ID NO: 14

<211> LENGTH: 390

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 14

aagcttgccg ccaccatgcc tctgctgctg ctgctgcctc tgctgtgggc tggggctctg 60

gctgatattg tgatgactca gtcacccgct accctgtccg tgacaccagg ggaccgggtg 120

agcctgtcct gcagagcctc tcagagtatc tcagattacc tgcactggta tcagcagaag 180

tctcatgaga gtcccaggct gctgatcaag tacgccagcc agtccatctc tggcattcct 240

tcccggttca gtggctcagg gagcggatcc gactttactc tgtctatcaa cagcgtcgag 300

ccagaagatg tgggagtcta ctattgtcag aatggccaca gcttccccct gacctttggc 360

accgggacaa agctggaact gaaacgtacg 390

<210> SEQ ID NO: 15

<211> LENGTH: 446

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 15

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Ser Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> SEQ ID NO: 16

<211> LENGTH: 214

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 16

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

85 90 95

Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> SEQ ID NO: 17

<211> LENGTH: 429

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 17

aagcttgccg ccaccatgcc tctgctgctg ctgctgcctc tgctgtgggc cggagccctg 60

gccgaaatcc agctgcagca gtcaggacct gaactggtga aacccggggc ctcagtgaaa 120

gtcagctgca tggcttcagt gtacagcttc accgactaca acatgtattg ggtcaaacag 180

tcccggggga agtctctgga gtggatcgga tacattgacc cttataacgg cgatacatcc 240

tataatcaga agttcaaagg caaggcaact ctgaccgtgg acaagagctc caacacagcc 300

tttatgcacc tgaattccct gacttctgaa gatagtgctg tctactattg tatcagagga 360

gactacggca atccctttta cctggattat tggggccagg ggaccacact gaccgtgtct 420

agtgctagc 429

<210> SEQ ID NO: 18

<211> LENGTH: 387

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 18

aagcttgccg ccaccatgcc cctgctgctg ctgctgcctc tgctgtgggc cggcgcactg 60

gctcagattg tcctgattca gtctcccgcc atcgtgtccg cttcccctgg ggagaaggtg 120

accatgacat gcagcgtgag ctcctctgtc aactacatgt attggtacca gcagaagagc 180

ggaacatccc ccaaacggtg gatctatgac acttctaaac tgccaagtgg agtcccagca 240

agattcagcg gatccggatc tggaactagt tactcactga ccattagttc aatggaggcc 300

gaagatgccg ctacatacta ttgtcagcag tggacatcta acccccctac ttttggcagc 360

gggaccaatc tggaaatcaa gcgtacg 387

<210> SEQ ID NO: 19

<211> LENGTH: 447

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 19

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Met Ala Ser Val Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Met Tyr Trp Val Lys Gln Ser Arg Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Phe

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ile Arg Gly Asp Tyr Gly Asn Pro Phe Tyr Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> SEQ ID NO: 20

<211> LENGTH: 213

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 20

Gln Ile Val Leu Ile Gln Ser Pro Ala Ile Val Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Val Ser Ser Ser Val Asn Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Pro Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Ser Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> SEQ ID NO: 21

<211> LENGTH: 426

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 21

aagcttgccg ccaccatgcc cctgctgctg ctgctgcctc tgctgtgggc cggagccctg 60

gccgaagtcc agctgcagca gtctggcgct gagtttgtga agccaggggc aagcgtgaag 120

ctgtcctgca ccgcctctgg attcaacatc aaagacacat acattcactg ggtcaatcag 180

cggcccgagc agggactgga atggatcggc agaattgacc ccgccaacga cgatactaag 240

tacgatccta attttcaggg caaagctacc atcacatccg acactagctc caacaccgca 300

tatgtgcagc tgtctagtct gacaagcgag gatactgccg tctactattg tgcttcaagc 360

gacaattccg attcttggtt cgcctattgg ggccagggga cactggtgag tgtctcagct 420

gctagc 426

<210> SEQ ID NO: 22

<211> LENGTH: 387

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 22

aagcttgccg ccaccatgcc cctgctgctg ctgctgcctc tgctgtgggc tggagccctg 60

gcccagattg tcctgactca gtcccccgct attatgtccg cttcaccagg ggagaaggtg 120

accatcacat gcagtgtgag ctcctctgtc aacttcatga attggtacca gcagaagctg 180

ggaagttcac ccaaactgtg gatctacgac accagcaaac tggcaccagg agtccctgca 240

cggttttcag gaagcggatc cggaacttct tacagtctga ccatcagctc catggagaca 300

gaagatgccg cttcctactt ctgtcaccag tggtctagtt attctctgac atttggcgct 360

gggactaagc tggaactgaa acgtacg 387

<210> SEQ ID NO: 23

<211> LENGTH: 446

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 23

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Phe Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Asn Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Asp Asp Thr Lys Tyr Asp Pro Asn Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Val Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Asp Asn Ser Asp Ser Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Ser Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> SEQ ID NO: 24

<211> LENGTH: 213

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 24

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Asn Phe Met

20 25 30

Asn Trp Tyr Gln Gln Lys Leu Gly Ser Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Thr Glu

65 70 75 80

Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Ser Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> SEQ ID NO: 25

<211> LENGTH: 420

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 25

aagcttgccg ccaccatgcc tctgctgctg ctgctgcctc tgctgtgggc cggggctctg 60

gccgagattc agctgcagca gtctggacct gacctggtga aacccggcac ctctgtgaaa 120

gtcagttgca aggcttctgg gtacagtttc acagactata acatgcactg ggtgaaacag 180

tctcatggga agagcctgga gtggatcgga tacgtcgacc cttatgatgg cgggactagc 240

tccaatcaga agttcaaagg caaggcaact ctgaccgtgg ataaatctag ttcaacagcc 300

tttatgcacc tgaagtcact gactagcgag gactccgccg tgtactattg tgctcgggaa 360

gtcccctact attttgatta ctggggacag ggcaccacac tgacagtgag ctccgctagc 420

<210> SEQ ID NO: 26

<211> LENGTH: 384

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 26

aagcttgccg ccaccatgcc cctgctgctg ctgctgcccc tgctgtgggc cggagctctg 60

gctcagattg tgctgactca gtcccctagt attatgtccg cctcccctgg cgagaaggtg 120

accatgacat gcgtcgccag ctcctctgtg acatacatgt attggtacca gcagaaaagc 180

ggaacctccc cacaggacgg cttcatgaca cacccactgg caagcggagt cccagcacgg 240

tttatcggag gaggatctgg cacaagttat tcactgacta ttagtaacat ggaggccgaa 300

gatgccgcta cttactattg tccccattgg aacactaatc cccctacctt cggctctggg 360

accaagctgg aaatcaatcg tacg 384

<210> SEQ ID NO: 27

<211> LENGTH: 444

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 27

Glu Ile Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Val Asp Pro Tyr Asp Gly Gly Thr Ser Ser Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> SEQ ID NO: 28

<211> LENGTH: 212

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 28

Gln Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Val Ala Ser Ser Ser Val Thr Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Gln Asp Gly Phe Met

35 40 45

Thr His Pro Leu Ala Ser Gly Val Pro Ala Arg Phe Ile Gly Gly Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Asn Met Glu Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Pro His Trp Asn Thr Asn Pro Pro Thr Phe

85 90 95

Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

Arg Gly Glu Cys

210

<210> SEQ ID NO: 29

<211> LENGTH: 360

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 29

gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt ctctttcagt gactattaca tgtattgggt tcgccagact 120

ccggaaaaga ggctggagtg ggtcgcagcc attagtgatg atagtactta cacctactat 180

ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caacctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagagggggg 300

tatggtaacc tctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360

<210> SEQ ID NO: 30

<211> LENGTH: 327

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 30

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc 60

atgacctgca ctgccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag 120

ccaggatcct cccccaaact ctggatttat agcacatcca acctggcttc tggagtccca 180

gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240

gctgaagatg ctgccactta ttactgccac cagtatcatc gttccccatt cacgttcggc 300

tcggggacaa agttggaaat aaaacgg 327

<210> SEQ ID NO: 31

<211> LENGTH: 120

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 31

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ala Ile Ser Asp Asp Ser Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Gly Asn Leu Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> SEQ ID NO: 32

<211> LENGTH: 109

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 32

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro

85 90 95

Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> SEQ ID NO: 33

<211> LENGTH: 360

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 33

gaggtccagc tgcagcagtc tggacctgag gtggtaaagc ctgggacttc agtgaagatg 60

tcctgcaagg cttctggata cacattcact aactatgtta tgcactgggt gaggcagaag 120

cctgggcagg gccttgaatg ggttggatgg attaatccct tcaatgatgg tacaaattat 180

aatgagaact tcaaaaacaa ggccacactg acttcagaca aatcctccag cacagcctac 240

atggagctca gcagcctgac ctctgaggac tctgcggtct attactgtgc aagatccggt 300

tttattacta cacttataga agactactgg ggccaaggca ccactctcac agtctcctca 360

<210> SEQ ID NO: 34

<211> LENGTH: 321

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 34

aacattgtga tgacccaatc tcccaaatac atgtccatgt cagtaggaga gagggtcacc 60

ttgacctgca aggccagtga gagtgtggga agttttgtat cctggtatca acagaaagca 120

gaccagtctc ctaacctact gatatacggg gcatccaacc ggtacactgg ggtccccgat 180

cgcttcacag gcagtggatc tgcaagagat ttcactctga ccatcagtag tgtgcaggct 240

aaagaccttg cagaatatta ctgtggacag tattacaccc atcccacgtt cggtgctggg 300

accaagctgg agctgaaacg g 321

<210> SEQ ID NO: 35

<211> LENGTH: 120

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 35

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Met His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Pro Phe Asn Asp Gly Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Asn Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Phe Ile Thr Thr Leu Ile Glu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> SEQ ID NO: 36

<211> LENGTH: 107

<212> TYPE: PRT

<213> ORGANISM: Mus musculus

<400> SEQENCE: 36

Asn Ile Val Met Thr Gln Ser Pro Lys Tyr Met Ser Met Ser Val Gly

1 5 10 15

Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Ser Val Gly Ser Phe

20 25 30

Val Ser Trp Tyr Gln Gln Lys Ala Asp Gln Ser Pro Asn Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Ala Arg Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Lys Asp Leu Ala Glu Tyr Tyr Cys Gly Gln Tyr Tyr Thr His Pro Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> SEQ ID NO: 37

<211> LENGTH: 200

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Type II 1.0 PGLYRP1

<400> SEQENCE: 37

Met Asn Trp His Met Ile Ile Ser Gly Leu Ile Val Val Val Leu Lys

1 5 10 15

Val Val Gly Met Thr Leu Phe Leu Leu Gln Glu Thr Glu Asp Pro Ala

20 25 30

Cys Cys Ser Pro Ile Val Pro Arg Asn Glu Trp Lys Ala Leu Ala Ser

35 40 45

Glu Cys Ala Gln His Leu Ser Leu Pro Leu Arg Tyr Val Val Val Ser

50 55 60

His Thr Ala Gly Ser Ser Cys Asn Thr Pro Ala Ser Cys Gln Gln Gln

65 70 75 80

Ala Arg Asn Val Gln His Tyr His Met Lys Thr Leu Gly Trp Cys Asp

85 90 95

Val Gly Tyr Asn Phe Leu Ile Gly Glu Asp Gly Leu Val Tyr Glu Gly

100 105 110

Arg Gly Trp Asn Phe Thr Gly Ala His Ser Gly His Leu Trp Asn Pro

115 120 125

Met Ser Ile Gly Ile Ser Phe Met Gly Asn Tyr Met Asp Arg Val Pro

130 135 140

Thr Pro Gln Ala Ile Arg Ala Ala Gln Gly Leu Leu Ala Cys Gly Val

145 150 155 160

Ala Gln Gly Ala Leu Arg Ser Asn Tyr Val Leu Lys Gly His Arg Asp

165 170 175

Val Gln Arg Thr Leu Ser Pro Gly Asn Gln Leu Tyr His Leu Ile Gln

180 185 190

Asn Trp Pro His Tyr Arg Ser Pro

195 200

<210> SEQ ID NO: 38

<211> LENGTH: 210

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Type II 2.0 PGLYRP1

<400> SEQENCE: 38

Met Asn Trp His Met Ile Ile Ser Gly Leu Ile Val Val Val Leu Leu

1 5 10 15

Val Val Gly Met Thr Leu Phe Leu Leu Asp Tyr Lys Asp Asp Asp Asp

20 25 30

Lys Gly Ser Gln Glu Thr Glu Asp Pro Ala Cys Cys Ser Pro Ile Val

35 40 45

Pro Arg Asn Glu Trp Lys Ala Leu Ala Ser Glu Cys Ala Gln His Leu

50 55 60

Ser Leu Pro Leu Arg Tyr Val Val Val Ser His Thr Ala Gly Ser Ser

65 70 75 80

Cys Asn Thr Pro Ala Ser Cys Gln Gln Gln Ala Arg Asn Val Gln His

85 90 95

Tyr His Met Lys Thr Leu Gly Trp Cys Asp Val Gly Tyr Asn Phe Leu

100 105 110

Ile Gly Glu Asp Gly Leu Val Tyr Glu Gly Arg Gly Trp Asn Phe Thr

115 120 125

Gly Ala His Ser Gly His Leu Trp Asn Pro Met Ser Ile Gly Ile Ser

130 135 140

Phe Met Gly Asn Tyr Met Asp Arg Val Pro Thr Pro Gln Ala Ile Arg

145 150 155 160

Ala Ala Gln Gly Leu Leu Ala Cys Gly Val Ala Gln Gly Ala Leu Arg

165 170 175

Ser Asn Tyr Val Leu Lys Gly His Arg Asp Val Gln Arg Thr Leu Ser

180 185 190

Pro Gly Asn Gln Leu Tyr His Leu Ile Gln Asn Trp Pro His Tyr Arg

195 200 205

Ser Pro

210

<210> SEQ ID NO: 39

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Epitope tag

<400> SEQENCE: 39

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> SEQ ID NO: 40

<211> LENGTH: 589

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 40

Met Asn Trp His Met Ile Ile Ser Gly Leu Ile Val Val Val Leu Leu

1 5 10 15

Val Val Gly Met Thr Leu Phe Leu Leu Asp Tyr Lys Asp Asp Asp Asp

20 25 30

Asp Lys Ser Leu Pro Leu Leu Met Asp Ser Val Ile Gln Ala Leu Ala

35 40 45

Glu Leu Glu Gln Lys Val Pro Ala Ala Lys Thr Arg His Thr Ala Ser

50 55 60

Ala Trp Leu Met Ser Ala Pro Asn Ser Gly Pro His Asn Arg Leu Tyr

65 70 75 80

His Phe Leu Leu Gly Ala Trp Ser Leu Asn Ala Thr Glu Leu Asp Pro

85 90 95

Cys Pro Leu Ser Pro Glu Leu Leu Gly Leu Thr Lys Glu Val Ala Arg

100 105 110

His Asp Val Arg Glu Gly Lys Glu Tyr Gly Val Val Leu Ala Pro Asp

115 120 125

Gly Ser Thr Val Ala Val Glu Pro Leu Leu Ala Gly Leu Glu Ala Gly

130 135 140

Leu Gln Gly Arg Arg Val Ile Asn Leu Pro Leu Asp Ser Met Ala Ala

145 150 155 160

Pro Trp Glu Thr Gly Asp Thr Phe Pro Asp Val Val Ala Ile Ala Pro

165 170 175

Asp Val Arg Ala Thr Ser Ser Pro Gly Leu Arg Asp Gly Ser Pro Asp

180 185 190

Val Thr Thr Ala Asp Ile Gly Ala Asn Thr Pro Asp Ala Thr Lys Gly

195 200 205

Cys Pro Asp Val Gln Ala Ser Leu Pro Asp Ala Lys Ala Lys Ser Pro

210 215 220

Pro Thr Met Val Asp Ser Leu Leu Ala Val Thr Leu Ala Gly Asn Leu

225 230 235 240

Gly Leu Thr Phe Leu Arg Gly Ser Gln Thr Gln Ser His Pro Asp Leu

245 250 255

Gly Thr Glu Gly Cys Trp Asp Gln Leu Ser Ala Pro Arg Thr Phe Thr

260 265 270

Leu Leu Asp Pro Lys Ala Ser Leu Leu Thr Met Ala Phe Leu Asn Gly

275 280 285

Ala Leu Asp Gly Val Ile Leu Gly Asp Tyr Leu Ser Arg Thr Pro Glu

290 295 300

Pro Arg Pro Ser Leu Ser His Leu Leu Ser Gln Tyr Tyr Gly Ala Gly

305 310 315 320

Val Ala Arg Asp Pro Gly Phe Arg Ser Asn Phe Arg Arg Gln Asn Gly

325 330 335

Ala Ala Leu Thr Ser Ala Ser Ile Leu Ala Gln Gln Val Trp Gly Thr

340 345 350

Leu Val Leu Leu Gln Arg Leu Glu Pro Val His Leu Gln Leu Gln Cys

355 360 365

Met Ser Gln Glu Gln Leu Ala Gln Val Ala Ala Asn Ala Thr Lys Glu

370 375 380

Phe Thr Glu Ala Phe Leu Gly Cys Pro Ala Ile His Pro Arg Cys Arg

385 390 395 400

Trp Gly Ala Ala Pro Tyr Arg Gly Arg Pro Lys Leu Leu Gln Leu Pro

405 410 415

Leu Gly Phe Leu Tyr Val His His Thr Tyr Val Pro Ala Pro Pro Cys

420 425 430

Thr Asp Phe Thr Arg Cys Ala Ala Asn Met Arg Ser Met Gln Arg Tyr

435 440 445

His Gln Asp Thr Gln Gly Trp Gly Asp Ile Gly Tyr Ser Phe Val Val

450 455 460

Gly Ser Asp Gly Tyr Val Tyr Glu Gly Arg Gly Trp His Trp Val Gly

465 470 475 480

Ala His Thr Leu Gly His Asn Ser Arg Gly Phe Gly Val Ala Ile Val

485 490 495

Gly Asn Tyr Thr Ala Ala Leu Pro Thr Glu Ala Ala Leu Arg Thr Val

500 505 510

Arg Asp Thr Leu Pro Ser Cys Ala Val Arg Ala Gly Leu Leu Arg Pro

515 520 525

Asp Tyr Ala Leu Leu Gly His Arg Gln Leu Val Arg Thr Asp Cys Pro

530 535 540

Gly Asp Ala Leu Phe Asp Leu Leu Arg Thr Trp Pro His Phe Thr Ala

545 550 555 560

Thr Val Lys Pro Arg Pro Ala Arg Ser Val Ser Lys Arg Ser Arg Arg

565 570 575

Glu Pro Pro Pro Arg Thr Leu Pro Ala Thr Asp Leu Gln

580 585

<210> SEQ ID NO: 41

<211> LENGTH: 355

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 41

Met Asn Trp His Met Ile Ile Ser Gly Leu Ile Val Val Val Leu Leu

1 5 10 15

Val Val Gly Met Thr Leu Phe Leu Leu Asp Tyr Lys Asp Asp Asp Asp

20 25 30

Asp Lys Trp Asp Thr Pro Thr Ile Val Ser Arg Lys Glu Trp Gly Ala

35 40 45

Arg Pro Leu Ala Cys Arg Ala Leu Leu Thr Leu Pro Val Ala Tyr Ile

50 55 60

Ile Thr Gln Leu Pro Gly Met Gln Cys Gln Gln Gln Ser Val Cys Ser

65 70 75 80

Gln Met Leu Arg Gly Leu Gln Ser His Ser Val Tyr Thr Ile Gly Trp

85 90 95

Cys Asp Val Ala Tyr Asn Phe Leu Val Gly Asp Asp Gly Arg Val Tyr

100 105 110

Glu Gly Val Gly Trp Asn Ile Gln Gly Leu His Thr Gln Gly Tyr Asn

115 120 125

Asn Ile Ser Leu Gly Ile Ala Phe Phe Gly Asn Lys Ile Gly Ser Ser

130 135 140

Pro Ser Pro Ala Ala Leu Ser Ala Ala Glu Gly Leu Ile Ser Tyr Ala

145 150 155 160

Ile Gln Lys Gly His Leu Ser Pro Arg Tyr Ile Gln Pro Leu Leu Leu

165 170 175

Lys Glu Glu Thr Cys Leu Asp Pro Gln His Pro Val Met Pro Arg Lys

180 185 190

Val Cys Pro Asn Ile Ile Lys Arg Ser Ala Trp Glu Ala Arg Glu Thr

195 200 205

His Cys Pro Lys Met Asn Leu Pro Ala Lys Tyr Val Ile Ile Ile His

210 215 220

Thr Ala Gly Thr Ser Cys Thr Val Ser Thr Asp Cys Gln Thr Val Val

225 230 235 240

Arg Asn Ile Gln Ser Phe His Met Asp Thr Arg Asn Phe Cys Asp Ile

245 250 255

Gly Tyr His Phe Leu Val Gly Gln Asp Gly Val Tyr Glu Gly Val Gly

260 265 270

Trp His Ile Gln Gly Ser His Thr Tyr Gly Phe Asn Asp Ile Ala Leu

275 280 285

Gly Ile Ala Phe Ile Gly Tyr Phe Val Glu Lys Pro Pro Asn Ala Ala

290 295 300

Ala Leu Glu Ala Ala Gln Asp Leu Ile Gln Cys Ala Val Val Glu Gly

305 310 315 320

Tyr Leu Thr Pro Asn Tyr Leu Leu Met Gly His Ser Asp Val Asn Ile

325 330 335

Leu Ser Pro Gly Gln Ala Leu Tyr Asn Ile Ile Ser Thr Trp Pro His

340 345 350

Phe Lys His

355

<210> SEQ ID NO: 42

<211> LENGTH: 390

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 42

Met Asn Trp His Met Ile Ile Ser Gly Leu Ile Val Val Val Leu Leu

1 5 10 15

Val Val Gly Met Thr Leu Phe Leu Leu Asp Tyr Lys Asp Asp Asp Asp

20 25 30

Asp Lys Asp Ser Ser Trp Asn Lys Thr Gln Ala Lys Gln Val Ser Glu

35 40 45

Gly Leu Gln Tyr Leu Phe Glu Asn Ile Ser Gln Leu Thr Glu Lys Gly

50 55 60

Leu Pro Thr Asp Val Ser Thr Thr Val Ser Arg Lys Ala Trp Gly Ala

65 70 75 80

Glu Ala Val Gly Cys Ser Ile Gln Leu Thr Thr Pro Val Asn Val Leu

85 90 95

Val Ile His His Val Pro Gly Leu Glu Cys His Asp Gln Thr Val Cys

100 105 110

Ser Gln Arg Leu Arg Glu Leu Gln Ala His His Val His Asn Asn Ser

115 120 125

Gly Cys Asp Val Ala Tyr Asn Phe Leu Val Gly Asp Asp Gly Arg Val

130 135 140

Tyr Glu Gly Val Gly Trp Asn Ile Gln Gly Val His Thr Gln Gly Tyr

145 150 155 160

Asn Asn Ile Ser Leu Gly Phe Ala Phe Phe Gly Thr Lys Lys Gly His

165 170 175

Ser Pro Ser Pro Ala Ala Leu Ser Ala Met Glu Asn Leu Ile Thr Tyr

180 185 190

Ala Val Gln Lys Gly His Leu Ser Ser Ser Tyr Val Gln Pro Leu Leu

195 200 205

Gly Lys Gly Glu Asn Cys Leu Ala Pro Arg Gln Lys Thr Ser Leu Lys

210 215 220

Lys Ala Cys Pro Gly Val Val Pro Arg Ser Val Trp Gly Ala Arg Glu

225 230 235 240

Thr His Cys Pro Arg Met Thr Leu Pro Ala Lys Tyr Gly Ile Ile Ile

245 250 255

His Thr Ala Gly Arg Thr Cys Asn Ile Ser Asp Glu Cys Arg Leu Leu

260 265 270

Val Arg Asp Ile Gln Ser Phe Tyr Ile Asp Arg Leu Lys Ser Cys Asp

275 280 285

Ile Gly Tyr Asn Phe Leu Val Gly Gln Asp Gly Ala Ile Tyr Glu Gly

290 295 300

Val Gly Trp Asn Val Gln Gly Ser Ser Thr Pro Gly Tyr Asp Asp Ile

305 310 315 320

Ala Leu Gly Ile Thr Phe Met Gly Thr Phe Thr Gly Ile Pro Pro Asn

325 330 335

Ala Ala Ala Leu Glu Ala Ala Gln Asp Leu Ile Gln Cys Ala Met Val

340 345 350

Lys Gly Tyr Leu Thr Pro Asn Tyr Leu Leu Val Gly His Ser Asp Val

355 360 365

Ala Arg Thr Leu Ser Pro Gly Gln Ala Leu Tyr Asn Ile Ile Ser Thr

370 375 380

Trp Pro His Phe Lys His

385 390

<210> SEQ ID NO: 43

<211> LENGTH: 1317

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTrem1 ECD(aa17-200)-Fc6mut

<400> SEQENCE: 43

gaattcgcaa ctatgcctct gctgctgctg ctgcctctgc tgtgggctgg cgcactggct 60

gaactgaggg ccgctaccaa actgaccgaa gaaaagtacg agctgaaaga agggcagacc 120

ctggacgtga agtgcgatta tacactggaa aagttcgcaa gctcccagaa agcctggcag 180

atcattagag acggagagat gcccaagact ctggcttgta ccgaacgccc ttcaaaaaac 240

agccacccag tgcaggtcgg ccgaatcatt ctggaggact accacgatca tgggctgctg 300

cgggtgagaa tggtcaatct gcaggtggag gactccggcc tgtaccagtg cgtcatctat 360

cagcccccta aggaaccaca tatgctgttc gataggattc gcctggtggt cactaaaggc 420

ttttctggga cccccggaag taacgagaac agcacccaga acgtgtacaa gatcccaccc 480

accacaacta aggccctgtg ccccctgtat acatctcctc gaaccgtgac acaggcccct 540

ccaaagagta ccgctgacgt gagcacaccc gattccgaga ttaacctgac aaatgtgact 600

gacatcatta ggggtaccga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc 660

ccagcacctg aagccgaggg ggcaccgtca gtcttcctct tccccccaaa acccaaggac 720

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 780

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 840

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 900

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 960

tcctccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1020

accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1080

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1140

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctatagcaag 1200

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1260

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtccccggg taaatga 1317

<210> SEQ ID NO: 44

<211> LENGTH: 434

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTrem1 ECD(aa17-200)-Fc6mut

<400> SEQENCE: 44

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Glu Leu Arg Ala Ala Thr Lys Leu Thr Glu Glu Lys Tyr Glu Leu Lys

20 25 30

Glu Gly Gln Thr Leu Asp Val Lys Cys Asp Tyr Thr Leu Glu Lys Phe

35 40 45

Ala Ser Ser Gln Lys Ala Trp Gln Ile Ile Arg Asp Gly Glu Met Pro

50 55 60

Lys Thr Leu Ala Cys Thr Glu Arg Pro Ser Lys Asn Ser His Pro Val

65 70 75 80

Gln Val Gly Arg Ile Ile Leu Glu Asp Tyr His Asp His Gly Leu Leu

85 90 95

Arg Val Arg Met Val Asn Leu Gln Val Glu Asp Ser Gly Leu Tyr Gln

100 105 110

Cys Val Ile Tyr Gln Pro Pro Lys Glu Pro His Met Leu Phe Asp Arg

115 120 125

Ile Arg Leu Val Val Thr Lys Gly Phe Ser Gly Thr Pro Gly Ser Asn

130 135 140

Glu Asn Ser Thr Gln Asn Val Tyr Lys Ile Pro Pro Thr Thr Thr Lys

145 150 155 160

Ala Leu Cys Pro Leu Tyr Thr Ser Pro Arg Thr Val Thr Gln Ala Pro

165 170 175

Pro Lys Ser Thr Ala Asp Val Ser Thr Pro Asp Ser Glu Ile Asn Leu

180 185 190

Thr Asn Val Thr Asp Ile Ile Arg Gly Thr Glu Pro Lys Ser Ser Asp

195 200 205

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala

210 215 220

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

225 230 235 240

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

245 250 255

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

260 265 270

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

275 280 285

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

290 295 300

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu

305 310 315 320

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

325 330 335

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

340 345 350

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

355 360 365

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

370 375 380

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

385 390 395 400

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

405 410 415

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

420 425 430

Gly Lys

<210> SEQ ID NO: 45

<211> LENGTH: 1206

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTremL1 ECD(aa16-162)-Fc6mut

<400> SEQENCE: 45

gaattcgcca ccatgcctct gctgctgctg ctgcctctgc tgtgggctgg cgcactggct 60

cagggcatag ttggcagcct ccctgaggtg ctgcaggcac ccgtgggaag ctccattctg 120

gtgcagtgcc actacaggct ccaggatgtc aaagctcaga aggtgtggtg ccggttcttg 180

ccggaggggt gccagcccct ggtgtcctca gctgtggatc gcagagctcc agcgggcagg 240

cgtacgtttc tcacagacct gggtgggggc ctgctgcagg tggaaatggt taccctgcag 300

gaagaggatg ctggcgagta tggctgcatg gtggatgggg ccagggggcc ccagattttg 360

cacagagtct ctctgaacat actgccccca gaggaagaag aagagaccca taagattggc 420

agtctggctg agaacgcatt ctcagaccct gcaggcagtg ccaacccttt ggaacccagc 480

caggatgaga agagcatccc cggtaccgag cccaaatctt ctgacaaaac tcacacatgc 540

ccaccgtgcc cagcacctga agccgagggg gcaccgtcag tcttcctctt ccccccaaaa 600

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 660

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 720

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 780

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 840

gccctcccat cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 900

caggtgtaca ccctgccccc atcccgggag gagatgacca agaaccaggt cagcctgacc 960

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1020

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1080

tatagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1140

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtccccgggt 1200

aaatga 1206

<210> SEQ ID NO: 46

<211> LENGTH: 397

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTremL1 ECD(aa16-162)-Fc6mut

<400> SEQENCE: 46

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Gln Gly Ile Val Gly Ser Leu Pro Glu Val Leu Gln Ala Pro Val Gly

20 25 30

Ser Ser Ile Leu Val Gln Cys His Tyr Arg Leu Gln Asp Val Lys Ala

35 40 45

Gln Lys Val Trp Cys Arg Phe Leu Pro Glu Gly Cys Gln Pro Leu Val

50 55 60

Ser Ser Ala Val Asp Arg Arg Ala Pro Ala Gly Arg Arg Thr Phe Leu

65 70 75 80

Thr Asp Leu Gly Gly Gly Leu Leu Gln Val Glu Met Val Thr Leu Gln

85 90 95

Glu Glu Asp Ala Gly Glu Tyr Gly Cys Met Val Asp Gly Ala Arg Gly

100 105 110

Pro Gln Ile Leu His Arg Val Ser Leu Asn Ile Leu Pro Pro Glu Glu

115 120 125

Glu Glu Glu Thr His Lys Ile Gly Ser Leu Ala Glu Asn Ala Phe Ser

130 135 140

Asp Pro Ala Gly Ser Ala Asn Pro Leu Glu Pro Ser Gln Asp Glu Lys

145 150 155 160

Ser Ile Pro Gly Thr Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys

165 170 175

Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu

180 185 190

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

195 200 205

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

210 215 220

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

225 230 235 240

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

245 250 255

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

260 265 270

Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

275 280 285

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

290 295 300

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

305 310 315 320

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

325 330 335

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

340 345 350

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

355 360 365

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

370 375 380

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

385 390 395

<210> SEQ ID NO: 47

<211> LENGTH: 1515

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTremL2 ECD(aa19-268)-Fc6mut

<400> SEQENCE: 47

gaattcgcca ccatgcctct gctgctgctg ctgcctctgc tgtgggctgg cgcactggct 60

ggcccctctg ctgacagtgt atacacaaaa gtgaggctcc ttgaagggga gactctgtct 120

gtgcagtgct cctataaggg ctacaaaaac cgcgtggagg gcaaggtttg gtgcaaaatc 180

aggaagaaga agtgtgagcc tggctttgcc cgagtctggg tgaaagggcc ccgctacttg 240

ctgcaggacg atgcccaggc caaggtggtc aacatcacca tggtggccct caagctccag 300

gactcaggcc gatactggtg catgcgcaac acctctggga tcctgtaccc cttgatgggc 360

ttccagctgg atgtgtctcc agctccccaa actgagagga acattccttt cacacatctg 420

gacaacatcc tcaagagtgg aactgtcaca actggccaag cccctacctc aggccctgat 480

gcccctttta ccactggtgt gatggtgttc accccaggac tcatcacctt gcctaggctc 540

ttagcctcca ccagacctgc ctccaagaca ggctacagct tcactgctac cagcaccacc 600

agccagggac ccaggaggac catggggtcc cagacagtga ccgcgtctcc cagcaatgcc 660

agggactcct ctgctggccc agaatccatc tccactaagt ctggggacct cagcaccaga 720

tcgcccacca cagggctctg cctcaccagc agatctctcc tcaacagact accctccatg 780

ccctccatca ggcaccagga tgtttactcc ggtaccgagc ccaaatcttc tgacaaaact 840

cacacatgcc caccgtgccc agcacctgaa gccgaggggg caccgtcagt cttcctcttc 900

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 960

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 1020

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 1080

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 1140

tccaacaaag ccctcccatc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1200

cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 1260

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 1320

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1380

ttcttcctct atagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1440

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 1500

tccccgggta aatga 1515

<210> SEQ ID NO: 48

<211> LENGTH: 500

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTremL2 ECD(aa19-268)-Fc6mut

<400> SEQENCE: 48

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Gly Pro Ser Ala Asp Ser Val Tyr Thr Lys Val Arg Leu Leu Glu Gly

20 25 30

Glu Thr Leu Ser Val Gln Cys Ser Tyr Lys Gly Tyr Lys Asn Arg Val

35 40 45

Glu Gly Lys Val Trp Cys Lys Ile Arg Lys Lys Lys Cys Glu Pro Gly

50 55 60

Phe Ala Arg Val Trp Val Lys Gly Pro Arg Tyr Leu Leu Gln Asp Asp

65 70 75 80

Ala Gln Ala Lys Val Val Asn Ile Thr Met Val Ala Leu Lys Leu Gln

85 90 95

Asp Ser Gly Arg Tyr Trp Cys Met Arg Asn Thr Ser Gly Ile Leu Tyr

100 105 110

Pro Leu Met Gly Phe Gln Leu Asp Val Ser Pro Ala Pro Gln Thr Glu

115 120 125

Arg Asn Ile Pro Phe Thr His Leu Asp Asn Ile Leu Lys Ser Gly Thr

130 135 140

Val Thr Thr Gly Gln Ala Pro Thr Ser Gly Pro Asp Ala Pro Phe Thr

145 150 155 160

Thr Gly Val Met Val Phe Thr Pro Gly Leu Ile Thr Leu Pro Arg Leu

165 170 175

Leu Ala Ser Thr Arg Pro Ala Ser Lys Thr Gly Tyr Ser Phe Thr Ala

180 185 190

Thr Ser Thr Thr Ser Gln Gly Pro Arg Arg Thr Met Gly Ser Gln Thr

195 200 205

Val Thr Ala Ser Pro Ser Asn Ala Arg Asp Ser Ser Ala Gly Pro Glu

210 215 220

Ser Ile Ser Thr Lys Ser Gly Asp Leu Ser Thr Arg Ser Pro Thr Thr

225 230 235 240

Gly Leu Cys Leu Thr Ser Arg Ser Leu Leu Asn Arg Leu Pro Ser Met

245 250 255

Pro Ser Ile Arg His Gln Asp Val Tyr Ser Gly Thr Glu Pro Lys Ser

260 265 270

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu

275 280 285

Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330 335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser

370 375 380

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

385 390 395 400

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

405 410 415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

465 470 475 480

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

485 490 495

Ser Pro Gly Lys

500

<210> SEQ ID NO: 49

<211> LENGTH: 1233

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTrem2 ECD(aa19-174)-Fc6mut

<400> SEQENCE: 49

gaattcgcca ccatgcctct gctgctgctg ctgcctctgc tgtgggctgg cgcactggct 60

cacaacacca cagtgttcca gggcgtggcg ggccagtccc tgcaggtgtc ttgcccctat 120

gactccatga agcactgggg gaggcgcaag gcctggtgcc gccagctggg agagaagggc 180

ccatgccagc gtgtggtcag cacgcacaac ttgtggctgc tgtccttcct gaggaggtgg 240

aatgggagca cagccatcac agacgatacc ctgggtggca ctctcaccat tacgctgcgg 300

aatctacaac cccatgatgc gggtctctac cagtgccaga gcctccatgg cagtgaggct 360

gacaccctca ggaaggtcct ggtggaggtg ctggcagacc ccctggatca ccgggatgct 420

ggagatctct ggttccccgg ggagtctgag agcttcgagg atgcccatgt ggagcacagc 480

atctccagga gcctcttgga aggagaaatc cccttcccac ccacttccgg taccgagccc 540

aaatcttctg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgagggggca 600

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 660

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 720

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 780

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 840

gagtacaagt gcaaggtctc caacaaagcc ctcccatcct ccatcgagaa aaccatctcc 900

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 960

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1020

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1080

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1140

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1200

cagaagagcc tctccctgtc cccgggtaaa tga 1233

<210> SEQ ID NO: 50

<211> LENGTH: 406

<212> TYPE: PRT

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: huCD33-huTrem2 ECD(aa19-174)-Fc6mut

<400> SEQENCE: 50

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

His Asn Thr Thr Val Phe Gln Gly Val Ala Gly Gln Ser Leu Gln Val

20 25 30

Ser Cys Pro Tyr Asp Ser Met Lys His Trp Gly Arg Arg Lys Ala Trp

35 40 45

Cys Arg Gln Leu Gly Glu Lys Gly Pro Cys Gln Arg Val Val Ser Thr

50 55 60

His Asn Leu Trp Leu Leu Ser Phe Leu Arg Arg Trp Asn Gly Ser Thr

65 70 75 80

Ala Ile Thr Asp Asp Thr Leu Gly Gly Thr Leu Thr Ile Thr Leu Arg

85 90 95

Asn Leu Gln Pro His Asp Ala Gly Leu Tyr Gln Cys Gln Ser Leu His

100 105 110

Gly Ser Glu Ala Asp Thr Leu Arg Lys Val Leu Val Glu Val Leu Ala

115 120 125

Asp Pro Leu Asp His Arg Asp Ala Gly Asp Leu Trp Phe Pro Gly Glu

130 135 140

Ser Glu Ser Phe Glu Asp Ala His Val Glu His Ser Ile Ser Arg Ser

145 150 155 160

Leu Leu Glu Gly Glu Ile Pro Phe Pro Pro Thr Ser Gly Thr Glu Pro

165 170 175

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

180 185 190

Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

195 200 205

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

210 215 220

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

225 230 235 240

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

245 250 255

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

260 265 270

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

275 280 285

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

290 295 300

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

305 310 315 320

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

325 330 335

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

340 345 350

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

355 360 365

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

370 375 380

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

385 390 395 400

Ser Leu Ser Pro Gly Lys

405

<210> SEQ ID NO: 51

<211> LENGTH: 29

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Primer

<400> SEQENCE: 51

tagtagggat ccgctggtgc acaggaagg 29

<210> SEQ ID NO: 52

<211> LENGTH: 32

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Primer

<400> SEQENCE: 52

tagtaggcgg ccgcttcgtg ggcctagggt ac 32

<210> SEQ ID NO: 53

<211> LENGTH: 16

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 53

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

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Citation

Patents Cited in This Cited by
Title Current Assignee Application Date Publication Date
A receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof NOVO-NORDISK A/S 28 March 2001 20 September 2002
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