ANTI-TREM-1 ANTIBODIES

Abstract

Novel anti-TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) antibodies and antigen-binding fragments thereof. Also, fusion proteins that include these anti-TREM-1 antibodies and antigen-binding fragments thereof, as well as the therapeutic uses thereof for treating of a disease, such as an inflammatory or autoimmune disease, a cardiovascular disease, a cancer, or an infectious disease.

Claims

1-14. (canceled)

15. An isolated anti-TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) antibody or an antigen-binding fragment thereof, wherein: a) the variable region of the heavy chain (VH) of said isolated anti-TREM-1 antibody or antigen-binding fragment thereof comprises the three following complementary-determining regions (CDRs): V.sub.H-CDR1: NTYIH (SEQ ID NO: 1); V.sub.H-CDR2: RIDPAX.sub.1GX.sub.2TKYX.sub.3PKVX.sub.4G (SEQ ID NO: 2), wherein X.sub.1 is Asn (N) or Gly (G), X.sub.2 is Asn (N) or Arg (R), X.sub.3 is Ala (A), Asp (D), or Ser (S), X.sub.4 is Gln (Q) or Lys (K); and V.sub.H-CDR3: HX.sub.5GX.sub.6TMDY (SEQ ID NO: 3), wherein X.sub.5 is Tyr (Y) or Arg (R), X.sub.6 is Ser (S) or Gly (G); b) the variable region of the light chain (VL) of said isolated anti-TREM-1 antibody or antigen-binding fragment thereof comprises the three following CDRs: V.sub.L-CDR1: RASX.sub.7SVX.sub.8NYGISFX.sub.9N (SEQ ID NO: 4), wherein X.sub.7 is Glu (E) or Gln (Q), X.sub.8 is Asp (D) or Ser (S), X.sub.9 is Met (M) or Len (L); and V.sub.L-CDR2: AAX.sub.10X.sub.11X.sub.12X.sub.13X.sub.14 (SEQ ID NO: 5), wherein X.sub.10 is Ser (S) or Glu (E), X.sub.11 is Asn (N) or Tyr (Y), X.sub.12 is Gln (Q) or Arg (R), X.sub.13 is Gly (G), Ala (A), or Lys (K), X.sub.14 is Ser (S) or Arg (R); and V.sub.L-CDR3: QQSX.sub.15X.sub.16X.sub.17PX.sub.18T (SEQ ID NO: 6), wherein X.sub.18 is Lys (K), Arg (R), or Ser (S), X.sub.16 is Glu (E), His (H), or Asn (N), X.sub.17 is Val (V) or Phe (F), X.sub.18 is Trp (W) or Tyr (Y).

16. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein said isolated anti-TREM-1 antibody or antigen-binding fragment thereof comprises the following CDRs: V.sub.H-CDR1: NTYIH (SEQ ID NO: 1), V.sub.H-CDR2: RIDPAGGRTKYDPKVKG (SEQ ID NO: 7), V.sub.H-CDR3: HYGGTMDY (SEQ ID NO: 8), V.sub.L-CDR1: RASESVDNYGISFLN (SEQ ID NO: 9), V.sub.L-CDR2: AAEYRGR (SEQ ID NO: 10), and V.sub.L-CDR3: QQSRHVPYT (SEQ ID NO: 11); or V.sub.H-CDR1: NTYIH (SEQ ID NO: 1), V.sub.H-CDR2: RIDPAGGRTKYSPKVQG (SEQ ID NO: 12), V.sub.H-CDR3: HRGGTMDY (SEQ ID NO: 13), V.sub.L-CDR1: RASQSVSNYGISFLN (SEQ ID NO: 14), V.sub.L-CDR2: AASYQKR (SEQ ID NO: 15), and V.sub.L-CDR3: QQSSNFPWT (SEQ ID NO: 16); or V.sub.H-CDR1: NTYIH (SEQ ID NO: 1), V.sub.H-CDR2: RIDPAGGRTKYAPKVKG (SEQ ID NO: 17), V.sub.H-CDR3: HRGGTMDY (SEQ ID NO: 13), V.sub.L-CDR1: RASQSVSNYGISFLN (SEQ ID NO: 14), V.sub.L-CDR2: AAEYRGR (SEQ ID NO: 10), and V.sub.L-CDR3: QQSSNVPYT (SEQ ID NO: 18); or V.sub.H-CDR1: NTYIH (SEQ ID NO: 1), V.sub.H-CDR2: RIDPAGGRTKYAPKVQG (SEQ ID NO: 19), V.sub.H-CDR3: HYGGTMDY (SEQ ID NO: 8), V.sub.L-CDR1: RASQSVSNYGISFLN (SEQ ID NO: 14), V.sub.L-CDR2: AAEYQGR (SEQ ID NO: 20), and V.sub.L-CDR3: QQSSNVPYT (SEQ ID NO: 18); or V.sub.H-CDR1: NTYIH (SEQ ID NO: 1), V.sub.H-CDR2: RIDPAGGRTKYAPKVKG (SEQ ID NO: 17), V.sub.H-CDR3: HRGGTMDY (SEQ ID NO: 13), V.sub.L-CDR1: RASQSVSNYGISFLN (SEQ ID NO: 14), V.sub.L-CDR2: AAEYRAR (SEQ ID NO: 21), and V.sub.L-CDR3: QQSSNVPYT (SEQ ID NO: 18); or V.sub.H-CDR1: NTYIH (SEQ ID NO: 1), V.sub.H-CDR2: RIDPANGNTKYAPKVQG (SEQ ID NO: 22), V.sub.H-CDR3: HYGSTMDY (SEQ ID NO: 23), V.sub.L-CDR1: RASESVDNYGISFMN (SEQ ID NO: 24), V.sub.L-CDR2: AASNQGS (SEQ ID NO: 25), and V.sub.L-CDR3: QQSKEVPWT (SEQ ID NO: 26).

17. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein said isolated anti-TREM-1 antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain (VH) having a sequence as set forth in any one of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, or a sequence having at least 80% identity with any one of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.

18. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein said isolated anti-TREM-1 antibody or antigen-binding fragment thereof comprises a variable region of the light chain (VL) having a sequence as set forth in any one of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, or a sequence having at least 80% identity with any one of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, and SEQ ID NO: 38.

19. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein said isolated anti-TREM-1 antibody or antigen-binding fragment thereof comprises a variable region of the heavy chain (VH) having a sequence as set forth in SEQ ID NO: 27, or a sequence having at least 80% identity with SEQ ID NO: 27, and a variable region of the light chain (VL) having a sequence as set forth in SEQ ID NO: 33, or a sequence having at least 80% identity with SEQ ID NO: 33.

20. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein said antibody is a monoclonal antibody.

21. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein said antibody is a humanized antibody or a human antibody.

22. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein said antibody or antigen-binding fragment thereof is monovalent.

23. The isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, wherein the antigen-binding fragment is a Fab, a Fv, or a scFv.

24. A fusion protein comprising the anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15.

25. A nucleic acid encoding the anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, or a fusion protein comprising said anti-TREM-1 antibody or antigen-binding fragment thereof.

26. A pharmaceutical composition comprising the isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, or a fusion protein comprising said anti-TREM-1 antibody or antigen-binding fragment thereof, and at least one pharmaceutically acceptable excipient.

27. A medicament comprising the isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, or a fusion protein comprising said anti-TREM-1 antibody or antigen-binding fragment thereof.

28. A method for treating a disease selected from an inflammatory or autoimmune disease, a cardiovascular disease, a cancer, and an infectious disease in a subject in need thereof, said method comprising administering to the subject the isolated anti-TREM-1 antibody or antigen-binding fragment thereof according to claim 15, or a fusion protein comprising said anti-TREM-1 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising said anti-TREM-1 antibody or antigen-binding fragment thereof or said fusion protein.

29. The method according to claim 28, wherein said inflammatory or autoimmune disease is selected from an inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome, fibrosis, pulmonary fibrosis, liver fibrosis, non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, lupus nephritis, vasculitis, systemic inflammatory response syndrome (SIRS), sepsis, septic shock, type I diabetes, Grave's disease, multiple sclerosis, autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft versus host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergy, and asthma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0535] FIG. 1 is a histogram showing the effects of an anti-TREM-1 antibody (INO-10 hIgG1) or Fab (INO-10 Fab) on neutrophil intracellular reactive oxygen species (ROS) production. Human primary neutrophils were incubated for 2 h in resting conditions (NS) or stimulated with LPS (100 ng/mL) in presence or absence of INO-10 IgG1 or INO-10 Fab at the indicated concentrations. *p<0.05, **p<0.01, ***p<0.001 versus LPS alone as determined by a parametric t-test.

[0536] FIG. 2 is a graph showing the expression of TREM-1 (assessed by flow cytometry) on U937 cells and on U937 cells pre-treated with vitamin D3 (U937-vitD3) to induce an up-regulation of TREM-1.

[0537] FIG. 3 is a graph showing the binding (assessed by flow cytometry) of anti-TREM-1 Fab INO-10F and a negative control INO-10F-0 (0.01-10 pg/mL) on U937 cells pre-treated with vitamin D3 to induce an up-regulation of TREM-1.

[0538] FIG. 4A-C are a set of graphs showing the effects of anti-TREM-1 Fab INO-10F on the production of cytokines (IL-6, IL-10 and IL-1?) by U937 cells pre-treated with vitamin D3 (U937-vitD3). The concentrations of IL-6 (FIG. 4A), IL-10 (FIG. 4B) and IL-1? (FIG. 4C) in supernatants were determined after a 24-hour stimulation of the U937-vitD3 cells in resting conditions or PP-activated conditions (stimulation with the PP complex corresponding to PGLYRP1 complexed with peptidoglycan) in presence of INO-10F or control (INO-10F-0Ctrl) at the indicated concentrations. *p<0.05, **p<0.01, ***p<0.001 versus PP alone as determined by a parametric t-test.

[0539] FIGS. 5A-C are a set of graphs showing TREM-1 expression (FIG. 5A), CD14 expression (FIG. 5B) and TLR4 expression (FIG. 5C) on THP-1 cells and THP-1 cells pre-treated with vitamin D3 to induce an up-regulation of TREM-1. The expression of TREM-1, CD14 and TLR4 was assessed by flow cytometry and compared to isotype control.

[0540] FIG. 6 is a graph showing the binding (assessed by flow cytometry) of anti-TREM-1 Fab INO-10F (0.01-10 pg/mL) on THP-1 cells and on THP-1 cells pre-treated with vitamin D3 (THP-1-vitD3).

[0541] FIGS. 7A-B show the effects of anti-TREM-1 Fab INO-10F on the activation of NF-?B in THP-1 Blue cells and THP-1-vitD3 Blue cells (i.e., THP-1 Blue cells pre-treated with vitamin D3 to induce an up-regulation of TREM-1). FIG. 7A is a histogram showing the activation of NF-?B in THP-1 Blue and THP-1-vitD3 Blue cells assessed by determining the activity of SEAP (measured at 650 nm) either in the presence of INO-10F at the indicated concentrations (0.1-10 pg/mL) in resting conditions (resting+INO-10F) for 6 h or in the presence of INO-10F at the indicated concentrations (0.1-10 ?g/mL) and LPS (LPS+INO-10F) for 6 h. *p<0.05, **p<0.01, versus LPS alone as determined by a parametric t-test. FIG. 7B is a graph showing the kinetics of NF-?B activation upon LPS priming (100 ng/mL) in presence of INO-10F at the indicated concentrations (0.1-10 ?g/mL). *p<0.05, **p<0.01 versus LPS alone as determined by a two-way ANOVA test.

[0542] FIG. 8 is a histogram showing the effects of anti-TREM-1 Fab INO-10F on the production of IL-8 by THP-1 cells pre-treated with vitamin D3 to induce an up-regulation of TREM-1 (THP-1-vitD3). The concentration of IL-8 in supernatants was assessed after a 24-hour stimulation of THP-1-vitD3 cells with LPS or no stimulation (NS) in presence of INO-10F at the indicated concentrations (0, 0.1 or 10 ?g/mL). *p<0.05, **p<0.01, ***p<0.001 versus LPS alone as determined by parametric t-test.

[0543] FIG. 9 is a graph showing TREM-1 expression (assessed by flow cytometry) on neutrophils at the indicated times. The human primary neutrophils were either cultured in resting conditions, stimulated with LPS for 3 h, or stimulated with LPS for 24 h. The expression of TREM-1 is compared to isotype control.

[0544] FIG. 10 is a graph showing the binding of INO-10F (assessed by flow cytometry) on freshly isolated human primary neutrophils at different concentrations (from 0.000001 to 10 ?g/mL).

[0545] FIG. 11 is a graph showing the reactive oxygen species (ROS) release by human primary neutrophils upon a 2 h incubation in the presence of INO-10F at the indicated concentrations (from 10.sup.?11 to 10.sup.1 ?g/mL) either with LPS (black squares) or in resting conditions (grey circles).

[0546] FIGS. 12A-B show the effects of anti-TREM-1 Fab INO-10F on reactive oxygen species (ROS) release by neutrophils. FIG. 12A is a histogram showing ROS release by human primary neutrophils upon a 2 h incubation with INO-10F at the indicated concentrations (0-10 ?g/mL) either in resting conditions (NS) or stimulated with the PP complex corresponding to PGLYRP1 complexed with peptidoglycan (PP). FIG. 12B is a graph showing the binding percentage (black circles) of INO-10F at the indicated concentrations (0-10 ?g/mL) on human primary neutrophils and the ROS release percentage (grey squares) by human primary neutrophils after PP stimulation in the presence of INO-10F at the indicated concentrations (0-10 ?g/mL).

[0547] FIG. 13 is a graph showing the effects of anti-TREM-1 Fab INO-10F on the production of IL-6 by neutrophils. The concentration of IL-6 in neutrophil supernatants was assessed after a 6-hour and a 24-hour stimulation with INO-10F at the indicated concentrations (0, 0.1 or 10 ?g/mL) either with LPS or in resting conditions.

[0548] FIGS. 14A-E are a set of histograms showing the effect of anti-TREM-1 Fab INO-10F on cytokine plasma concentration following a 24-hour whole blood stimulation assay. INO-10F was added at the indicated concentrations (0-10 ?g/mL) either in resting conditions (NS) or with LPS. As a positive control, a stimulation with a known TREM-1 inhibitor (peptide LR12) was carried out with LPS. After 24 h, the expression of the following cytokines were assessed: IL-1? (FIG. 14A), IL-10 (FIG. 14B), TNF-? (FIG. 14C), IL-6 (FIG. 14D), and IL-8 (FIG. 14E). *p<0.05, **p<0.01, ***p<0.001 versus LPS alone as determined by a parametric t-test.

[0549] FIG. 15 is a box plot showing IL-8 plasma concentrations following a 24-hour whole blood stimulation assay. INO-10F was added to whole blood from 14 healthy volunteers at the indicated concentrations (0-10 ?g/mL) either in resting conditions (NS) or with LPS. As a positive control, a stimulation with a known TREM-1 inhibitor (peptide LR12) was carried out with LPS. After 24 h, the plasma concentration of IL-8 was assessed. *p<0.05, **p<0.01, ***p<0.001 versus LPS alone as determined by a paired non-parametric t-test.

[0550] FIGS. 16A-G are a set of box plots showing the effect of anti-TREM-1 Fabs INO-10F and HSA-INO-10F on the plasma concentration of human cytokines in transgenic BRGSF-his (humanized immune system) mice suffering from endotoxemia induced by LPS. BRGSF-his mice were administered either PBS (control) or LPS by intraperitoneal injection. The BRGSF-his mice which received LPS first received a pre-treatment by intraperitoneal injection (30 min prior to LPS), consisting either of vehicle (LPS), INO-10F (LPS+10F?10 ?g/mL), or a fusion protein between HSA and INO-10F (LPS+HSA-10F?10 ?g/mL). Blood samples were collected 8 hours following LPS injection and the plasma concentrations of the following human cytokine/chemokine were assessed: CCL2 (FIG. 16A), IL-1? (FIG. 16B), IL-10 (FIG. 16C), IL-6 (FIG. 16D), and IL-8 (FIG. 16E), IP-10 (FIG. 16F), and TNF-? (FIG. 16G). p-values were calculated according to a non-parametric t-test between indicated conditions versus LPS alone.

[0551] FIGS. 17A-B is a set of graphs showing the binding (assessed by flow cytometry) of anti-TREM-1 Fab INO-10F and of anti-TREM-1 Fab variants INO-10F-0 (F0), INO-10F-1 (F1), INO-10F-2 (F2), INO-10F-3 (F3), INO-10F-4 (F4), INO-10F-5 (F5), and INO-10F-6 (F6) on U937 cells (FIG. 17A) and on U937-vitD3 cells, i.e., U937 cells pre-treated with vitamin D3 to induce an up-regulation of TREM-1 (FIG. 17B).

[0552] FIG. 18 is a histogram showing the effects of anti-TREM-1 Fab variants INO-10F-0 (F0), INO-10F-1 (F1), INO-10F-2 (F2), INO-10F-3 (F3), INO-10F-4 (F4), INO-10F-5 (F5), and INO-10F-6 (F6) on the production of IL-6 by U937 cells pre-treated with vitamin D3. The concentrations of IL-6 were determined in the supernatants after a 24-hour stimulation of the U937-vitD3 cells in resting conditions (NS) or PP-activated conditions (stimulation with the PP complex corresponding to PGLYRP1 complexed with peptidoglycan) in presence of the anti-TREM-1 Fab variants at the indicated concentrations (0-10 ?g/mL). As a positive control, a stimulation with a known TREM-1 inhibitor (peptide LR12) was carried out in PP-activated conditions (LR12). *p<0.05, **p<0.01, ***p<0.001, ****p<0.001 versus PP alone.

[0553] FIG. 19 is a graph comparing the binding (assessed by flow cytometry) of anti-TREM-1 Fab variants INO-10F-3 (F3) and INO-10F-0 (F0) at the indicated concentrations (0.001 to 10 ?g/mL) on freshly isolated primary neutrophils.

[0554] FIG. 20 is a histogram showing the effects of anti-TREM-1 Fab variants INO-10F-0 (F0), INO-10F-1 (F1), INO-10F-2 (F2), INO-10F-3 (F3), INO-10F-4 (F4), INO-10F-5 (F5), and INO-10F-6 (F6) on the production of IL-8 by human primary neutrophils. The concentrations of IL-8 were determined in the supernatants after a 24-hour stimulation of the neutrophils in resting conditions (NS) or LPS-activated conditions in presence of the anti-TREM-1 Fab variants at the indicated concentrations (0-10 ?g/mL). As a positive control, a stimulation with a known TREM-1 inhibitor (peptide LR12) was carried out in LPS-activated conditions (LR12). *p<0.05, **p<0.01, ***p<0.001, ****p<0.001 versus LPS alone.

[0555] FIGS. 21A-H are a set of graphs showing the effect of anti-TREM-1 Fab INO-10F and of anti-TREM-1 Fab variants on neutrophil intracellular reactive oxygen species (ROS) production. Human primary neutrophils were stimulated for 2 h with LPS (100 ng/mL) in presence of INO-10 Fab (FIG. 21A) or anti-TREM-1 Fab variants INO-10F-0 or F0 (FIG. 21B), INO-10F-1 or F1 (FIG. 21C), INO-10F-2 or F2 (FIG. 21D), INO-10F-3 or F3 (FIG. 21E), INO-10F-4 or F4 (FIG. 21F), INO-10F-5 or F5 (FIG. 21G), and INO-10F-6 or F6 (FIG. 21H) at the indicated concentrations (0.001-10 ?g/mL).

[0556] FIGS. 22A-C are a set of graphs showing the effects of an anti-TREM-1 Fab variant (INO-10F-3 coupled with HSA or F3-HSA) on neutrophil intracellular reactive oxygen species (ROS) production. Human primary neutrophils were stimulated for 2 h with LPS (100 ng/mL) (FIG. 22A) or with the PP complex corresponding to PGLYRP1 (5 ?g/mL) complexed with peptidoglycan corresponding to PGN (10 ?g/mL) (PP) (FIG. 22B) or only with peptidoglycan (PGN10 ?g/mL) (FIG. 22C) in presence of F3-HSA at the indicated concentrations (0.02-20 ?g/mL). *p<0.05, **p<0.01, ***p<0.001 versus LPS alone (FIG. 22A), PP alone (FIG. 22B) or PGN alone (FIG. 22C) as determined by parametric ANOVA-test.

[0557] FIGS. 23A-B are a set of box plot showing the effect of an anti-TREM-1 Fab variant (INO-10F-3 coupled with HSA or F3-HSA) on cytokine plasma concentration following a 24-hour whole blood stimulation assay after lysis of red blood cells from 5 healthy donors. F3-HSA or an isotype control (CTLR) was added at the indicated concentrations (0-20 ?g/mL) either in resting conditions (NS) or with the PP complex corresponding to PGLYRP1 (5 ?g/mL) complexed with peptidoglycan corresponding to PGN (10 ?g/mL) (PP) or only with peptidoglycan (PGN10 ?g/mL). After 24 h, the expression of the following cytokines were assessed: IL-8 (FIG. 23A) and TNF-? (FIG. 23B). *p<0.05, **p<0.01, ***p<0.001 versus PP alone or PGN alone as determined by a non-parametric t-test.

[0558] FIGS. 24A-C are a set of histograms showing the effect of an anti-TREM-1 Fab variant (INO-10F-3 coupled with HSA or F3-HSA) on cytokine plasma concentration following a 24-hour cynomolgus whole blood stimulation assay after lysis of red blood cells. F3-HSA or an isotype control (CTRL) was added at the indicated concentrations (0-20 ?g/mL) either with the PP complex corresponding to PGLYRP1 (5 ?g/mL) complexed with peptidoglycan corresponding to PGN (10 ?g/mL) (PP) or only with peptidoglycan (PGN20 ?g/mL). After 24 h, the expression of the following cytokines were assessed: IL-8 (FIG. 24A), TNF-? (FIG. 24B) and IL-6 (FIG. 24C). *p<0.05, **p<0.01, ***p<0.001 versus PGN alone as determined by a non-parametric t-test.

EXAMPLES

[0559] The present invention is further illustrated by the following examples.

Example

Materials and Methods

Anti-hTREM-1 Antibody/Fab-Fragments Production

[0560] Novel anti-human TREM-1 (anti-hTREM1) murine antibodies were obtained by immunizing mice with a recombinant hTREM-1 protein. The sequences of the anti-hTREM-1 murine antibodies and Fab fragments were obtained by sequencing of hybridomas and sequence analysis (Diaclone, France). Recombinant chimeric anti-hTREM-1 antibodies (human IgG1 or hIgG1) and Fab fragments were then produced. Sequences from the variable regions were sub-cloned in a pQMCF-1.2 expression vector and the coding regions were verified by sequencing. CHOEBNALT85 1E9 cells (Icosagen) were then transfected with the pQMCF-1.2 expression vector in CHO TF medium (Xell AG) for 96 hours using R007 transfection reagent (Icosagen). Transfection was verified by PCR. Expression was checked by Coomassie staining, and secretion by Endpoint Coomassie staining in order to estimate the productivity. Then purification steps were performed using capture with HiTrap MabSelect SuRe for hIgG1 or HisTrap Excel for Fab fragments (both from GE Healthcare). Finally, a gel filtration was performed with Superdex 200 Increase 10/300 GL (GE Healthcare) and recovered proteins were filtered at 0.22 ?m (Ultra Capsule GF, Merck Millipore). At the end of the process, chimeric hIgG1 or Fab fragments must meet the following acceptance criteria: concentration 1 mg/mL, purity >90% and endotoxin level under 0.1 EU/mg of protein. The purified hIgG1 and Fab fragments were kept in the following buffer: histidine-Tween buffer [20 mM histidine, 150 mM NaCl, 0.02% Tween-80, pH6.0].

Cell Isolation, Culture and Stimulation

[0561] U937 cells: cells of the human myelomonocytic cell line U937 (Culture Collections, Public Health England No 85011440) were cultured in RPMI 1640 medium containing GlutaMAX and supplemented with 10% Fetal Calf Serum or FCS (Thermo Fisher Scientific), 25 mM HEPES, 100 U/mL penicillin and streptomycin (all from Thermo Fisher Scientific). For some experiments, when indicated, U937 cells were cultured in the same conditions supplemented with 100 nM of 1,25-dihydroxyvitamin D3 also referred to as vitamin D3 or vitD3 (Sigma-Aldrich, USA) to induce an up-regulation of TREM-1.

[0562] THP-1 blue cells: the human THP1-Blue cell line is derived from the human THP-1 monocytic cell line by stable transfection of an NF-?B-inducible SEAP (secreted embryonic alkaline phosphatase) reporter construct (InvivoGen, France). Indeed, these cells report the activation of the NF-?B transcription factor. THP1-Blue cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FBS (Fetal Bovine Serum), 2 mM L-glutamine, 25 mM HEPES, 100 ?g/mL of normocin, and 100 U/mL of penicillin and streptomycin. For some experiments, when indicated, THP1-Blue cells were cultured in the same conditions supplemented with 100 nM of 1,25-dihydroxyvitamin D3 (vitD3) to induce an up-regulation of TREM-1.

[0563] TREM-1, TLR4, and CD14 expression on U937 cells, THP1 cells, or human primary neutrophils was assessed by flow cytometry. Cells were incubated for 10 min at 4? C. in the dark with anti-TREM1-APC, anti-CD14-PE, or anti-TLR4-FITC antibodies, or corresponding isotype controls (Miltenyi-Biotec, Germany), then washed and data were collected by flow cytometry (C6 Accuri, BD, USA). Flow cytometry data were analyzed using FlowJo software (TreeStar, USA).

[0564] Primary cells: primary human neutrophils were isolated from the peripheral blood of healthy donors by immunomagnetic negative cell sorting with EasySep? Human Monocyte/Neutrophil Isolation Kits (StemCell, Canada) following the manufacturer's instructions. Purity was assessed by flow cytometry. Cells were suspended in RPMI 1640 medium containing GlutaMAX and supplemented with 10% FCS, 25 mM HEPES, 100 U/ml penicillin and streptomycin (all from Thermo Fisher Scientific) before stimulation. Human primary neutrophils were incubated in resting conditions (also referred to as non-stimulating conditions or NS), or with 100 ng/mL LPS from E. coli serotype 0127:B8 (Sigma-Aldrich), or with PP complex also referred to as PPx (corresponding to PGLYRP1 (peptidoglycan recognition protein 1) at 5 ?g/mL complexed with 10 ?g/mL of peptidoglycan, respectively from Biotechne, U K and Invivogen, France), or with peptidoglycan (PGN) alone (10 ?g/mL), with or without anti-TREM-1 modulators (hIgG1 or Fab), at indicated times and concentrations. When indicated, the neutrophils were incubated with the clinical stage TREM-1 inhibitory peptide LR12 (a TLT-1 peptide having an amino acid as set forth in SEQ ID NO: 61-LQEEDAGEYGCM) at 100 ?g/mL.

Binding to Human and Cynomolgus TREM-1

[0565] Cells: U937 cells or primary neutrophils were centrifuged for 5 minutes at 300 g and the pellets were resuspended to 1?10.sup.6 cells/mL. Tested molecules (hIgG1 or Fab) were diluted at different concentrations (from 0.0001 to 20 ?g/mL) in FACS buffer (1?PBS, 0.5% BSA, 2.5 mM EDTA). The cells were incubated for 30 minutes at 4? C. in presence of the tested molecules (hIgG1 or Fab), and then centrifuged for 5 minutes at 300 g. The supernatants were removed and a 1?PBS wash was performed. The cells were washed again and centrifuged for 5 minutes at 300 g and the pellets were recovered in FACS Buffer. Then, the secondary antibody (1:200, allophycocyanin (APC) AffiniPure F(ab)2 fragment goat anti-human IgG (H+L) (Jackson ImmunoResearch, USA) was added to the cell suspension. After 30 minutes of incubation at 4? C., the cells were washed with 1?PBS and centrifuged at 300 g for 5 minutes. Finally, the cells were resuspended in FACS buffer and analyzed by flow cytometry (C6 Accuri, BD, USA) in order to quantify the binding of the tested molecules (hIgG1 or Fab) to the cells. Finally, flow cytometry data were analyzed using FlowJo software.

[0566] Surface plasmon resonance (SPR): in order to evaluate the interaction kinetics for TREM-1 antibodies or Fabs to hTREM-1 (human TREM-1) and cTREM-1 (cynomolgus monkey TREM-1), surface plasmon resonance (SPR) assays were carried out (Biacore? T200, GE Healthcare Biosciences). The anti-human Fc antibodies (Cytiva) were immobilized on CM5 sensor chip in order to evaluate IgG1 affinities and Fabs were directly immobilized on the chip. Immobilization experiments were performed at 25? C. using HBS-EP+1? running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). The anti-human Fc antibodies or Fabs were diluted in 10 mM sodium acetate at acidic pH before the immobilization procedure using amine coupling on the dextran matrix of the sensor chips. The surface was activated using a solution of 100 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodimide hydrochloride or EDC and 400 mM N-hydroxysulfosuccinimide or NHS (EDC/NHS) (Liu Y, Wilson WD. Methods Mol Biol. 2010; 613:1-23). Following these injections, ethanolamine was injected to deactivate the surface. The immobilization wizard was used to obtain several thousand of immobilized RU (resonance units). The immobilization level was chosen in order to have a proper covering of the sensor chip surface. Preliminary manual runs were performed in order to optimize the capture conditions and to obtain similar capture levels for all the human antibodies. Binding of hTREM-1 or cTREM-1 proteins was conducted by injecting analyte over all flow cells. The human and cynomolgus TREM-1 proteins were diluted into running buffer (HBS-EP+1?) at concentrations of 0.1 nM, 0.5 nM, 2.5 nM, 10 nM and 40 nM, or 0.5 nM, 2 nM, 10 nM, 40 nM and 200 nM, respectively. The concentrations were evaluated using the Single Cycle Kinetics method. This approach consists of a sequential injection of increasing concentration of the analyte, with a single regeneration step using a magnesium chloride buffer (Cytiva) at the end of the cycle. Binding affinity of TREM-1 antibodies or Fabs with hTREM-1 or cTREM-1 was quantified by determination of the equilibrium dissociation constant (K.sub.D) determined by measurement of the kinetics of complex formation and dissociation. The rate constants corresponding to the association and the dissociation of a monovalent complex such as k.sub.a (association rate) and k.sub.d (dissociation rate) were retrieved by fitting data to 1:1 Langmuir model using the Biacore T200 Evaluation Software, version 3.1 (GE Healthcare). K.sub.D is related to k.sub.a and k.sub.d through the equation K.sub.D=k.sub.d/k.sub.a.

Reactive Oxygen Species (ROS) Production

[0567] Quantification of intracellular ROS production was assessed using cell-permeable DCFDA (2,7-dichlorofluorescein diacetate), a chemically reduced form of fluorescein used as an indicator of the presence of ROS in cells (Thermo Fisher Scientific). Upon cleavage of the acetate groups by intracellular esterases and oxidation, the nonfluorescent DCFDA is converted to the highly fluorescent 2,7-dichlorofluorescein (DCF). For example, human primary neutrophils were incubated 2 hours at 37? C. 5% CO.sub.2 with 5 ?M of DCFDA, in presence of the tested molecules (hIgG1 or Fab) with or without 100 ng/mL LPS, or PP complex (corresponding to PGLYRP1 (peptidoglycan recognition protein 1) at 5 ?g/mL complexed with 10 ?g/mL of peptidoglycan, respectively from Biotechne, U K and Invivogen, France), or peptidoglycan (PGN) alone (10 ?g/mL). Data were acquired using flow cytometry (C6 Accuri, BD, USA) or a Fluorometer (Varioskan Lux, ThermoScientific). Results are expressed as mean fluorescence intensity (MFI) or relative fluorescence unit (RFU).

THP-1 Quanti-Blue Assay: NF-?B Cell Line Reporter

[0568] The QUANTI-Blue assay (InvivoGen) is a colorimetric enzymatic test for determining the activity of SEAP. This test is used on THP1-Blue cells which contain the SEAP reporter gene inducible by NF-?B. Using this test, the activation of NF-?B can be assessed by determining the activity of SEAP (measured at 650 nm). After 48 hours of culture of THP-1 blue cells with 100 nM of 1,25-dihydroxyvitamin D3 (vitD3), the Quanti-Blue assay was performed. In a 96-well microplate, the cells (1?10.sup.5 cells/well) were incubated in the presence or absence of the tested molecule (hIgG1 or Fab) at the indicated concentrations (0.1-1-10 ?g/mL) and LPS (0.1 ?g/mL) at 37? C., 5% CO.sub.2 between 1 and 10 hour(s). Subsequently, the cells were centrifuged for 5 minutes at 300 g and the supernatants were collected. In a new transparent 96-well microplate, the cell supernatants were mixed with the Quanti-Blue reagent (1:10) and incubated at 37? C., 5% CO.sub.2 for 30 minutes. Finally, the optical density was measured at 650 nm with a microplate reader (Varioskan Lux, ThermoScientific).

Whole Blood Assay

[0569] Using stimulation assays of whole blood obtained from healthy human donors, inflammatory cytokines levels (IL-1?, TNF-?, IL-6, IL-8, and IL-10) were assessed. The tested molecules (hIgG1 or Fab) were first diluted to different concentrations (0.1-1-10 ?g/mL or as indicated) and added to the wells of 12-well plates in presence or absence of LPS (0.1 ?g/mL, InvivoGen, France), or in presence of PP complex (corresponding to PGLYRP1 at 5 ?g/mL complexed with 10 ?g/mL of peptidoglycan, respectively from Biotechne, U K and Invivogen, France) or only with peptidoglycan also referred to PGN (Invivogen, France). Subsequently, whole blood (after the lysis of red blood cells with ammonium chloride (Stemcell, France)) was added to the wells and incubated for 24 hours at 37? C., 5% CO.sub.2. Then, samples were centrifuged for 10 minutes at 300 g in order to recover the plasma in which the IL-8 level was assessed using the Quantikine ELISA Human IL-8/CXCL8 kit according to the manufacturer's instructions (R&D Systems) or using Ella technology (Protein Simple, UK), an automated immunoassay system. The samples were added in Single Plex or Multiplex cartridges (Protein Simple, UK) in order to evaluate levels of the 5 cytokines (IL-1?, TNF-?, IL-6, IL-8, and IL-10) in a single assay.

[0570] Alternatively, whole blood from cynomolgus was used. Using stimulation assays of whole blood obtained from healthy cynomolgus donors (Macaca fascicularis), inflammatory cytokines levels (IL-8, TNF-?, IL-6) were assessed. The tested molecules (hIgG1 or Fab) were first diluted to different concentrations (0.2-2-20 ?g/mL) and added to the wells of 24-well plates in presence of PP complex (or PPx) (corresponding to PGLYRP1 at 5 ?g/mL complexed with 10 ?g/mL of peptidoglycan, respectively from Biotechne, U K and Invivogen, France) or only with peptidoglycan also referred to PGN (Invivogen, France). Subsequently, whole blood (after the lysis of red blood cells with ammonium chloride (Stemcell, France)) was added to the wells and incubated for 24 hours at 37? C., 5% CO.sub.2. Then, samples were centrifuged for 10 minutes at 300 g in order to recover the plasma in which the TNF-?, IL-6 and IL-8 level was assessed using Ella technology (Protein Simple, UK), an automated immunoassay system. The samples were added in Single Plex or Multiplex cartridges (Protein Simple, UK) in order to evaluate levels of the 3 cytokines (TNF-?, IL-6 and IL-8) in a single assay.

U937-VitD3 Stimulation

[0571] U937 cells were cultured in RPMI 1640 GlutaMAX medium supplemented with 10% FCS, 25 mM HEPES, 100 U/ml penicillin and streptomycin in presence of 100 nM of 1,25-dihydroxyvitamin D3 (vitD3) for 48 hours to induce an up-regulation of TREM-1. Then, cells were recovered and plated (1?10.sup.5 cells/well) in the presence or absence of the tested molecule (hIgG1 or Fab) at the indicated concentrations (0.1-1-10 ?g/mL) and LPS (0.1 ?g/mL) at 37? C., 5% CO.sub.2 for 24 hours. Subsequently, the cells were centrifuged for 5 minutes at 300 g and the supernatants were collected. Finally, the concentrations of inflammatory cytokines levels (IL-1?, IL-6 and IL-10) in the supernatants were assessed using Ella technology (Protein Simple, UK).

Neutrophil Stimulation

[0572] Primary human neutrophils were isolated from the blood of healthy donors as previously described and plated at 1?10.sup.6 cells/mL. Then, cells were incubated in the presence or absence of the tested molecule (hIgG1 or Fab) at the indicated concentrations (0.1-10 ?g/mL) and LPS (0.1 ?g/mL) at 37? C. 5%, CO.sub.2 for 24 h. Subsequently, the cells were centrifuged for 5 minutes at 300 g and the supernatants were collected. Finally, the concentrations of IL-6 or IL-8 in the supernatants were assessed using the Quantikine ELISA Human IL-6 or IL-8 kit according to the manufacturer's instructions (R&D Systems, France) or Ella technology (Protein Simple, UK).

Humanized Immune System (his-) Mice

[0573] BRGSF mice from GenOway (France) are BALB/c mice displaying the Rag2?/?Il2rg?/?SirpaNODFlk2+/? genotype. His (humanized immune system) mice were generated as follows: briefly, newborn mice (<5 days of age) were transplanted with approximately 1?10.sup.5 human hematopoietic progenitor cells (hHPC) CD34.sup.+ obtained from umbilical cord by intra-hepatic injection after a sub-lethal irradiation. In order to boost the myeloid immune system, all mice received 4 intra-peritoneal (i.p.) injections of 10 ?g of recombinant human hFLT3-L/Fc every two days before experiments.

Human Experimental Endotoxemia

[0574] His-mice were subjected to LPS challenge to evaluate the in vivo immunomodulatory effect of anti-TREM-1 INO-10 Fab fragment (INO-10F). In brief, one day following Flt3-ligand (FLT3L) boost, his-mice were administered with a single intraperitoneal (i.p.) dose of 10 mg/kg of either PBS, INO-10F, or a fusion protein with an extended half-life comprising INO-10F coupled with human serum albumin also known as HSA (INO-10F-HSA), followed 30 min later by an i.p. injection of 8 mg/kg of LPS (lipopolysaccharides; E. coli serotype 0127:B8, batch L3129, Sigma Chemical, St Louis, France). Concentrations were adjusted as to inject the same volumes in each group of mice. After 8 hours, blood samples were collected by intracardiac punction and were harvested in EDTA-tubes. Plasma was obtained by centrifugation of the whole blood (300 g, 10 min) and stored at ?80? C. The plasma levels of cytokines (CCL-2, IL-1?, IL-10, IL-6, IL-8, IP-10, and TNF-?) were determined using the simple Plex cartridges, run by Ella technology (Protein Simple, UK).

Results

INO-10F Efficiently Blocks TREM-1 Activation in Human Primary Neutrophils.

[0575] A total of 51 anti-hTREM-1 unique sequences were obtained and produced as human IgG1 chimeric antibodies (hIgG1) and corresponding Fab fragments (or in short Fabs). These constructs were screened for their ability to bind human TREM-1. After validation of their interaction with human TREM-1, all constructs were screened for their ability to decrease the release of reactive oxygen species (ROS) by human primary neutrophils following the activation of the neutrophils with lipopolysaccharides (LPS). Indeed, activation of TREM-1 on neutrophils (which express TREM-1 at their surface) through their incubation with LPS notably leads to ROS production by the neutrophils. The ability of the tested constructs to decrease ROS production by neutrophils activated with LPS thus reflects their ability to inhibit TREM-1. One lead was identified, the so-called INO-10F, an anti-hTREM-1 Fab fragment. As shown on FIG. 1, INO-10F was able to significantly decrease ROS release by neutrophils at 1 ?g/mL and 10 ?g/mL. INO-10F was thus able to inhibit TREM-1 activation. Based on data obtained from functional screening, INO-10F was identified as the best lead compound.

INO-10F Binding to Human TREM-1 by Flow Cytometry and Inhibition of TREM-1 Activation on U937-vitD3 Cells

[0576] Incubation of U937 cells with vitamin D3 (1,25-dihydroxyvitamin D3) was associated with an increase in TREM-1 expression at the membrane as compared to TREM-1 expression at the membrane of untreated U937 control cells (FIG. 2). Using flow cytometry, INO-10F was shown to bind human TREM-1 in a dose-dependent manner on U937-vitD3 cells (i.e., U937 cells pre-treated with vitamin D3), with a 50% binding reached at approximately 0.2 ?g/mL (FIG. 3). As expected, the negative control INO-10F-0 did not show any binding to human TREM-1 on U937-vitD3 cells (FIG. 3). Interestingly, INO-10F was also shown to inhibit TREM-1 activation in a dose-dependent manner. The activation of TREM-1 on myeloid cells (which express TREM-1 at their surface) through the induction of an inflammatory response, for example with a PGLYRP-1:PGN complex (or PP), notably leads to cytokine/chemokine expression and secretion by said cells. As shown on FIG. 4, incubation of increasing concentrations of INO-10F with U937-vitD3 cells was associated with a decrease in the release of interleukin-6 also known as IL-6 (FIG. 4A), interleukin-10 also known as IL-10 (FIG. 4B), and interleukin-1? also known as IL-1? (FIG. 4C) induced by a 24-hour stimulation of the U937-vitD3 cells with the PGLYRP-1:PGN complex (PPx or PP). In that assay, the maximum effect of INO-10F was achieved between 1 and 10 ?g/mL and 50% of inhibition was reached at approximately 0.1 ?g/mL.

INO-10F Binding to Human TREM-1 by Flow Cytometry and Inhibition of TREM-1 Activation on THP-1 Blue-vitD3 Cells

[0577] Incubation of THP-1 cells with vitamin D3 was associated with an increase in TREM-1 expression (FIG. 5A), as well as an increase in human CD14 or hCD14 (FIG. 5B) and a decrease in human Toll Like Receptor 4 or hTLR4 (FIG. 5C) as compared to TREM-1 expression at the membrane of untreated THP-1 cells. Using flow cytometry, INO-10F was shown to bind human TREM-1 expressed in THP-1-vitD3 cells (THP-1 cells pre-treated with vitamin D3) in a dose-dependent manner, with a 50% binding reached at 0.014 ?g/mL (FIG. 6). As expected, there was little binding of INO-10F to untreated THP-1 control cells (FIG. 6).

[0578] In order to evaluate the activity of INO-10F, untreated THP-1 Blue cells or THP-1 Blue cells pretreated with vitamin D3 for 48 hours were incubated with increasing doses of INO-10F in presence or absence of LPS (100 ng/mL). The activation of TREM-1 on myeloid cells such as monocytes (which express TREM-1 at their surface) through the induction of an inflammatory response, for example with LPS, notably leads to NF-?B activation in said cells. After 6 hours, NF-?B activation was assessed using Quanti-Blue reagent. As reflected through the inhibition of NF-?B activation shown on FIG. 7A, INO-10F was able to inhibit TREM-1 only on cells overexpressing TREM-1, with an effect between 0.1 and 10 ?g/mL. INO-10F had no effect on LPS-activated na?ve THP-1 Blue cells (i.e., THP-1 Blue cells which were not pre-treated with vitamin D3). A second set of experiments further confirmed that INO-10F was able to limit the LPS-induced activation of NF-kB in a time- and dose-dependent manner in THP-1 Blue cells pre-treated with vitamin D3. INO-10F inhibited NF-?B activation between 6 and 10 hours with a highest effect at 10 hours depending on the dose (FIG. 7B).

[0579] Then, the IL-8 production of THP-1 Blue cells was assessed after their pre-treatment with vitamin D3 and their stimulation for 24 hours with LPS (100 ng/mL) in presence of increasing concentrations of INO-10F (0, 0.1 and 10 ?g/mL). INO-10F decreased, in a concentration dependent manner, the release of IL-8 induced by LPS stimulation with a maximum effect reached at 10 ?g/mL (FIG. 8). This result confirms that INO-10F is able to inhibit TREM-1 in THP-1 Blue cells pre-treated with vitamin D3.

INO-10F Binding to Human TREM-1 by Flow Cytometry and Inhibition of TREM-1 Activation on Primary Neutrophils and in Whole Blood

[0580] Human primary neutrophils express high levels of TREM-1 at the membrane under physiological conditions and do not up-regulate its expression upon LPS stimulation. Indeed, as shown on FIG. 9, the expression of TREM-1 at the membrane of human primary neutrophils is similar in resting conditions and after stimulation with LPS, either for 3 hours or for 24 hours. INO-10F was able to bind human TREM-1 in a concentration-dependent manner on freshly isolated human neutrophils with a 50% binding reached between 0.01 and 0.1 ?g/mL, at about 0.023 ?g/mL (FIG. 10). Then, the ability of INO-10F to inhibit TREM-1 activation on neutrophils was evaluated through the assessment of their release of ROS upon LPS stimulation. As expected, INO-10F alone did not induce any activation of TREM-1 as observed through the absence of ROS production in resting conditions (FIG. 11). ROS were produced by the human primary neutrophils upon LPS stimulation, and INO-10F decreased said production of ROS by 50% at a concentration of about 4.6 ?g/mL, confirming its ability to inhibit TREM-1 (FIG. 11). A similar experiment was conducted, using the PGLYRP-1:PGN complex (PPx or PP) to induce ROS production in human primary neutrophils by direct activation of TREM-1. INO-10F also decreased ROS production by neutrophils with a maximum effect reached at 1 ?g/mL (FIG. 12A) which corresponds to the maximum binding concentration to human TREM-1 on human primary neutrophils (FIG. 12B).

[0581] Finally, IL-6 secretion by human primary neutrophils was assessed after incubation of the neutrophils during 0, 6 and 24 h in presence of INO-10F (0.1 or 10 ?g/mL) either with LPS (100 mg/mL) or in resting conditions. As shown on FIG. 13, INO-10F reduced the release of IL-6 induced by LPS in a dose- and time-dependent manner.

[0582] To further confirm the immunomodulatory properties of INO-10F through its inhibition of TREM-1, the effect of INO-10F was assessed in a human whole blood cytokine assay stimulation. As detailed above, whole blood obtained from healthy human donors was incubated for 24 hours at 37? C., 5% CO.sub.2 in presence of LPS (100 ng/mL) and either INO-10F or a positive control (i.e., peptide LR12 known to inhibit TREM-1). The plasma was recovered and the plasma levels of several cytokines were measured. As shown on FIG. 14, in this assay, INO-10F reduced, in a dose-dependent manner, the release of several cytokines. Indeed, INO-10F limited the LPS-induced release of IL-1? (FIG. 14A), of IL-10 (FIG. 14B), of TNF-? (FIG. 14C), of IL-6 (FIG. 14D), and of IL-8 (FIG. 14E). The effect of INO-10F on IL-8 plasma concentration was also assessed in vitro after LPS stimulation of whole blood samples collected from 14 healthy volunteers. As shown on FIG. 15, INO-10F induced a dose-dependent decrease (from 0.01 to 10 ?g/mL) of IL-8 plasma concentration. A decrease in the LPS-induced production of IL-8 was also observed with the peptide LR12 (positive control). In the whole blood assays, INO-10F did not induce any significant production of IL-8 or any other studied cytokines in unstimulated condition (i.e., in the absence of LPS).

Blocking Human TREM-1 in a BRGS-F Mouse Endotoxemia Model Reduces Immune-Inflammatory Response.

[0583] The immuno-modulatory effects of INO-10F were assessed in vivo in transgenic BRGSF mice with a humanized immune system in which endotoxemia was induced by intraperitoneal (i.p.) administration of LPS (8 mg/kg). The mice were randomly divided into four treatment groups to receive either an i.p. administration of PBS (control) alone or LPS with either vehicle, INO-10F, or a fusion protein comprising INO-10F. Indeed, among the mice that were administered LPS, the LPS group received a vehicle as treatment, the LPS+10F group received an i.p. administration of 10 ?g/mL of INO-10F, and the LPS+HSA-10F group received an i.p. administration of 10 ?g/mL of a format of INO-10F with an extended half-life consisting of a fusion protein between human serum albumin (HSA) and INO-10F (1? F.). Mice were pre-treated with vehicle, INO-10F or HSA-INO-10F (HSA-10F) for 30 minutes, and then administered with LPS to induce endotoxemia. Blood samples were collected 8 hours following LPS injection, and human cytokine/chemokine concentrations were quantified in the plasma (CCL-2, IL-1?, IL-10, IL-6, IL-8, IP-10, and TNF-?). LPS markedly increased the release of the human inflammatory cytokines/chemokines as compared to the control group (CTRL). Interestingly, as shown on FIG. 16, both INO-10F and INO-10F-HSA were able to modulate the secretion of circulating human inflammatory cytokines chemokine ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 or MCP1 (FIG. 16A), interleukin-1? or IL-1? (FIG. 16B), interleukin-10 or IL-10 (FIG. 16C), interleukin-6 or IL-6 (FIG. 16D), interleukin-8 or IL-8 (FIG. 16E), interferon gamma-induced protein 10 (IP-10) also known as C-X-C motif chemokine ligand 10 or CXCL10 (FIG. 16F), and tumor necrosis factor alpha or TNF-? or TNFa (FIG. 16G), with a more pronounced effect for the HSA-INO-10F fusion protein having an extended half-life. These results confirm the immunomodulatory effect of anti-TREM-1 INO-10F Fab fragment in vivo.

Binding of Optimized INO-10F Variants to TREM-1

[0584] In order to improve INO-10F binding properties and activity, humanized variants of the anti-TREM-1 INO-10 antibody and corresponding humanized variants of the anti-TREM-1 INO-10F Fab fragment were generated. The humanized variants of the anti-TREM-1 INO-10 antibody were named INO-10-2, INO-10-3, INO-10-4, INO-10-5, and INO-10-6 and the humanized variants of the anti-TREM-1 INO-10F Fab fragment were named INO-10F-2 (F2), INO-10F-3 (F3), INO-10F-4 (F4), INO-10F-5 (F5), and INO-10F-6 (F6). Two additional humanized variants were used as controls: INO-10F-0 (F0), and INO-10F-1 (F1), the humanized anti-TREM-1 Fab fragment with CDRs most similar to INO-10F (CDRs are identical except for one amino acid difference in V.sub.H-CDR2). First, the binding affinity constant, association rate and dissociation rate were determined using surface plasmon resonance (SPR) assays. Fab fragments were immobilized on the surface of a CM5 sensor chip followed by injection of increasing concentration of recombinant human TREM-1 or cynomolgus monkeyTREM-1. Results are shown in Table 1 below. No binding to TREM-1 (either hTREM-1 or cTREM-1) was observed with the Fab fragment INO-10F-0 (F0). Fab fragments INO-10F-1 (F1) was only able to bind hTREM-1, with an affinity similar to that of Fab fragment INO-10F. Fab fragments INO-10F-2 to INO-10F-6 (F2 to F6) displayed higher affinities than Fab fragment INO-10F.

TABLE-US-00002 TABLE 1 Binding constants k.sub.a or k.sub.on (association rate), k.sub.d or k.sub.off (dissociation rate) and K.sub.D (equilibrium dissociation constant) for the interaction of human and cynomolgus TREM-1 with different anti-TREM-1 monoclonal antibody Fab fragments Affinity constants to Affinity constants to Human TREM-1 cynomolgus TREM-1 Samples Replicates K.sub.D k.sub.on k.sub.off Replicates K.sub.D K.sub.on k.sub.off INO-10F 1 7.71E?09 1.38E+05 1.06E?03 1 5.79E?8 3.57E04 2.07E?3 2 7.89E?09 1.37E+05 1.08E?03 2 6.66E?8 2.21E4 1.47E?3 Average 7.80E?09 1.38E+05 1.07E?03 Average 6.22E?8 2.89E4 1.77E?3 INO-10- 1 5.76E?09 1.89E+05 1.09E?03 1 No response observed F1 2 7.81E?09 1.58E+05 1.24E?03 2 Average 6.79E?09 1.74E+05 1.17E?03 Average INO-10- 1 1.29E?09 2.29E+05 2.97E?04 1 2.80E?08 3.11E+04 8.69E?04 F2 2 1.75E?09 1.83E+05 3.21E?04 2 2.85E?08 3.16E+04 9.00E?04 Average 1.52E?09 2.06E+05 3.09E?04 Average 2.83E?08 3.14E+04 8.85E?04 INO-10- 1 2.61E?10 2.03E+05 5.31E?05 1 7.60E?09 3.16E+04 2.40E?04 F3 2 3.40E?10 2.00E+05 6.82E?05 2 7.68E?09 3.19E+04 2.45E?04 Average 3.01E?10 2.02E+05 6.07E?05 Average 7.64E?09 3.18E+04 2.43E?04 INO-10- 1 9.61E?10 2.17E+05 2.09E?04 1 2.48E?08 3.10E+04 7.68E?04 F4 2 1.07E?09 2.09E+05 2.24E?04 2 2.44E?08 3.17E+04 7.74E?04 Average 1.02E?09 2.13E+05 2.17E?04 Average 2.46E?08 3.14E+04 7.71E?04 INO-10- 1 8.84E?10 2.04E+05 1.80E?04 1 1.58E?08 3.42E+04 5.39E?04 F5 2 5.95E?10 2.00E+05 1.19E?04 2 1.57E?08 3.47E+04 5.45E?04 Average 7.40E?10 2.02E+05 1.50E?04 Average 1.58E?08 3.45E+04 5.42E?04 INO-10- 1 3.89E?09 1.24E+05 4.81E?04 1 6.01E?08 2.01E+04 1.21E?03 F6 2 4.03E?09 1.16E+05 4.67E?04 2 5.34E?08 2.49E+04 1.33E?03 Average 3.96E?09 1.20E+05 4.74E?04 Average 5.68E?08 2.25E+04 1.27E?03

Optimized INO-10F Variants are Able to Inhibit TREM-1 on U937 Cells and Primary Cells

[0585] The binding of the optimized anti-TREM-1 Fab fragments to human TREM-1 expressed on U937 pre-treated with vitamin D3 (U937-vitD3 cells) and on the untreated control U937 cells was next evaluated. As expected, no binding was observed on U937 cells (FIG. 17A). As shown on FIG. 17B, on U937-vitD3 cells, INO-10F-F0 showed a weak binding at 10 ?g/mL, INO-10F showed a similar binding profile as previously obtained (see FIG. 3). A clear shift toward a better affinity was observed for the optimized variants INO-10F-1 to INO-10F-6, corroborating the SPR data (FIG. 17B).

[0586] In accordance with the results obtained with INO-10F, INO-10F-1 (which is the humanized Fab fragment the most similar to INO-10F, with identical CDRs except for one amino acid difference in V.sub.H-CDR2) was able to decrease the release of IL-6 by U937-vitD3 cells induced by stimulation with the PGLYRP-1:PGN complex (PP complex) with an approximately 50% inhibition (IC50) achieved at 0.5 ?g/mL. INO-10F-0, which showed a decreased affinity to TREM-1 as compared to INO-10F, was associated with a limited decrease of IL-6 release observed only at 10 ?g/mL. The improved affinity of INO-10F-2 (F2) to INO-10F-6 (F6) to TREM-1 translated into a shift toward a decrease in the dose required for them to induce a 50% inhibition (IC50) of IL-6 release. In particular, INO-10F-3 (F3) displayed an IC50 around 0.05 ?g/mL. The peptide LR12, a known inhibitor of TREM-1, was used as a positive control (FIG. 18). Binding of INO-10F-3 (F3) was confirmed on TREM-1 expressed by freshly isolated human neutrophils with a 50% of binding reached at about 0.03 ?g/mL (FIG. 19).

[0587] Next, a neutrophil LPS-stimulation assay was conducted to assess the ability of the optimized anti-TREM-1 Fab fragments to decrease the release of IL-8 by human primary neutrophils after stimulation with LPS for 24 hours. As shown on FIG. 20, INO-10F-2 (F2) and INO-10F-3 (F3) showed good significant inhibitory properties between 0.1 and 10 ?g/mL. INO-10F-4 (F4) and INO-10F-6 (F6) were also able to significantly decrease IL-8 release when added at 1 ?g/mL or 10 ?g/mL. INO-10F-1 (F1) and INO-10F-5 (E5) were only able to significantly decrease IL-8 release when added at 10 ?g/mL. Finally, a neutrophil LPS-stimulation assay was also conducted to assess the ability of the optimized anti-TREM-1 Fab fragments to decrease the ROS production induced after LPS stimulation of human primary neutrophils for 24 hours. As shown on FIGS. 21A-H, INO-10F-2 (F2) and INO-10F-3 (F3) showed the best inhibitory profile of ROS production by LPS-activated neutrophils. Indeed, INO-10F-2 (F2) inhibited ROS production from 28% at 0.01 ?g/mL to 44% at 10 ?g/mL (FIG. 21D), and INO-10F-3 (F3) was able to inhibit ROS release from 21% at 0.01 ?g/mL to 52% at 10 ?g/ml (FIG. 21E). INO-10F-4 (F4) and INO-10F-6 (F6) were also able to inhibit ROS production by LPS-activated neutrophils when added at 1 ?g/mL or 10 ?g/mL. INO-10F-1 (F1) and INO-10F-5 (F5) were only able to inhibit ROS production by LPS-activated neutrophils when added at 10 ?g/mL. As expected, INO-10F-0 (F0) did not inhibit ROS production by LPS-activated neutrophils.

An INO-10F Variant Coupled to HSA is Able to Inhibit TREM-1 on Primary Cells and in Whole Blood Assay

[0588] A fusion protein consisting of the optimized Fab fragment INO-10F-3 (or F3) coupled with human serum albumin also known as HSA was generated. The inhibitory effect of said fusion protein, referred as F3-HSA, was evaluated on the ROS production by human primary neutrophils (FIG. 22). Human primary neutrophils were thus stimulated for 2 h with LPS (100 ng/mL), or with the PP complex corresponding to PGLYRP1 (5 ?g/mL) complexed with peptidoglycan (10 ?g/mL) (PP), or with peptidoglycan only (PGN10 ?g/mL) in presence of F3-HSA at the indicated concentrations (0-20 ?g/mL). F3-HSA was able to inhibit ROS release after stimulation of the neutrophils with LPS (about 50% decrease at about 1 ?g/mLsee FIG. 22A), with PP complex (about 85% decrease at about 1 ?g/mLFIG. 22B), or with PGN alone (about 75% decrease at about 1 ?g/mLFIG. 22C).

[0589] Next, the effect of F3-HSA was assessed on cytokine plasma concentration following a 24-hour whole blood stimulation assay after lysis of red blood cells. F3-HSA or an isotype control (CTLR) were added to whole blood at the indicated concentrations (0-20 ?g/mL) either in resting conditions, or in presence of the PP complex corresponding to PGLYRP1 (5 ?g/mL) complexed with PGN (10 ?g/mL), or in presence of PGN only (10 ?g/mL). After 24 h, the expression of the following cytokines were assessed: IL-8 and TNF-?. As shown on FIG. 23, in this assay, F3-HSA reduced, in a dose-dependent manner, the release of both IL-8 and TNF-?, as compared to the control which did not reduce the cytokine release. Indeed, F3-HSA reduced the release of IL-8 after stimulation either with PP or with PGN alone (FIG. 23A), and the release of TNF-? after stimulation either with PP or with PGN alone (FIG. 23B).

[0590] A similar assay was next conducted with cynomolgus whole blood. F3-HSA or an isotype control (CTLR) were thus added to whole blood obtained from healthy cynomolgus donors (Macaca fascicularis) at the indicated concentrations (0-20 ?g/mL) either in resting conditions, or in presence of the PP complex corresponding to PGLYRP1 (5 ?g/mL) complexed with PGN (10 ?g/mL), or in presence of PGN only (20 ?g/mL). After 24 h, the expression of the following cytokines were assessed: IL-8, TNF-?, and IL-6. As shown on FIG. 24, in this assay, F3-HSA reduced, in a dose-dependent manner, the release of both IL-8, TNF-?, and IL-6, as compared to the control which did not reduce the cytokine release. Indeed, F3-HSA reduced the release of IL-8 after stimulation either with PGN alone or with PP (FIG. 24A), the release of TNF-? after stimulation with PGN alone or with PP (FIG. 24B), and the release of IL-6 after stimulation either with PGN alone or with PP (FIG. 24C).