NOVEL IL-17B ANTIBODIES

Abstract

The invention relates to novel IL-17B antagonist antibodies and their use in the diagnosis or treatment of IL-17B mediated diseases.

Claims

1. An isolated IL-17B antibody which binds the sequence of the human IL-17B as set forth in SEQ ID NO: 3.

2. The antibody of claim 1 which further binds a sequence selected from the group consisting of SEQ NOs: 4 to 6.

3. The antibody of claim 1 which comprises: a Kabat heavy chain complementarity determining region-1 (VH-CDR1) amino acid sequence which is identical to the VH-CDR1 of the VH region comprising the amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 8, (b) SEQ ID NO:24, (c) SEQ ID NO:40, and (d) SEQ ID NO: 56, and a Kabat heavy chain complementarity determining region-2 (VH-CDR2) amino acid sequence which is identical to the VH-CDR2 of the VH region comprising the amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 8, (b) SEQ ID NO:24, (c) SEQ ID NO:40, and (d) SEQ ID NO: 56, and a Kabat heavy chain complementarity determining region-3 (VH-CDR3) amino acid sequence which is identical to the VH-CDR3 of the VH region comprising the amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 8, (b) SEQ ID NO:24, (c) SEQ ID NO:40, and (d) SEQ ID NO: 56, and a Kabat light chain complementarity determining region-1 (VL-CDR1) amino acid sequence which is identical to the VL-CDR1 of the VL region comprising the amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 9, (b) SEQ ID NO:25, (c) SEQ ID NO:41, and (d) SEQ ID NO: 57, and a Kabat light chain complementarity determining region-2 (VL-CDR2) amino acid sequence which is identical to the VL-CDR2 of the VL region comprising the amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 9, (b) SEQ ID NO: 25, (c) SEQ ID NO: 41, and (d) SEQ ID NO: 57, and a Kabat light chain complementarity determining region-3 (VL-CDR3) amino acid sequence which is identical to the VL-CDR3 of the VL region comprising the amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 9; (b) SEQ ID NO: 25; (c) SEQ ID NO: 41; and (d) SEQ ID NO: 57.

4. The antibody of claim 1 which comprises: a Kabat heavy chain complementarity determining region-1 (VH-CDR1) comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 12, (b) SEQ ID NO: 28, (c) SEQ ID NO: 44, and (d) SEQ ID NO: 60, a Kabat heavy chain complementarity determining region-2 (VH-CDR2) comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 13, (b) SEQ ID NO: 29, (c) SEQ ID NO: 45, and (d) SEQ ID NO: 61, and a Kabat heavy chain complementarity determining region-3 (VH-CDR3) comprising an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 14, (b) SEQ ID NO: 30, (c) SEQ ID NO: 46, and (d) SEQ ID NO: 62, and a Kabat light chain complementarity determining region-1 (VL-CDR1) comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 15, (b) SEQ ID NO: 31, (c) SEQ ID NO: 47, and (d) SEQ ID NO: 63, and a Kabat light chain complementarity determining region-2 (VL-CDR2) comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 16, (b) SEQ ID NO: 32, (c) SEQ ID NO: 48, and (d) SEQ ID NO: 64, and a Kabat light chain complementarity determining region-3 (VL-CDR3) comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 17, (b) SEQ ID NO: 33, (c) SEQ ID NO: 49, and (d) SEQ ID NO: 65.

5. The antibody of claim 1 which comprises: a VH chain comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 8, (b) SEQ ID NO: 24, (c) SEQ ID NO: 40, and (d) SEQ ID NO: 56, and a VL chain comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 9, (b) SEQ ID NO: 25, (c) SEQ ID NO: 41, and (d) SEQ ID NO: 57.

6. An antibody which binds the same epitope(s) as the antibody (a), (b), (c) and (d) as defined in claim 3.

7. The antibody of claim 1, which comprises a light chain constant region selected from the group consisting of a human kappa constant region and a human lambda constant region.

8. The antibody of claim 1, which comprises a heavy chain constant region or fragment thereof, wherein the heavy chain constant region is a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, a human IgG3 heavy chain constant region, a human IgG4 heavy chain constant region, a human IgM heavy chain constant region, a human IgA1 heavy chain constant region, a human IgA2 heavy chain constant region, a human IgE heavy chain constant region, or a human IgD heavy chain constant region.

9. The antibody of claim 1, wherein said antibody is a chimeric antibody, a human or humanized antibody, and/or a monoclonal antibody.

10. The antibody of claim 1, wherein said antibody is an antagonist of IL-17B.

11. The antibody of claim 1, which is an antibody fragment directed against IL-17B, wherein the antibody fragment is selected from the group consisting of Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, and diabodies.

12. An isolated nucleic acid comprising a sequence encoding the antibody of claim 1.

13. A composition comprising an isolated nucleic acid sequence encoding a VL region and an isolated nucleic acid encoding a VH region of the antibody of claim 1.

14. A vector comprising an isolated nucleic acid claim 12.

15. A host cell comprising an isolated nucleic acid sequence of claim 12.

16. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

17. An in vitro method of diagnosis comprising measuring a level of an IL-17B using the antibody of claim 1.

18. A method of treating an autoimmune disease, a chronic inflammatory disease, or cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the antibody of claim 1.

19. A method of treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the antibody of claim 1, wherein said method increases the effectiveness of a therapeutic agent and/or prevents or treats tumor metastasis.

20. A method of producing an antibody that specifically binds IL-17B, comprising culturing the host cell of claim 15 under conditions suitable for expressing said antibody, and recovering said antibody.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0234] FIG. 1: Binding of various anti-IL-17B antibodies (mouse monoclonal antibodies directed against human IL-17B) on human IL-17B: [0235] (A) Antibody 12F9 [0236] (B) Antibody 13B12 [0237] (C) Antibody 13H5 [0238] (D) Antibody 21H6 [0239] (E) Antibody 18G9

[0240] FIG. 2: Human IL-17B induced IL-8 secretion by HepG2 cells in the presence of various anti-IL-17B antibodies (mouse monoclonal antibodies directed against human IL-17B). The % of inhibition is shown for [0241] (A) Antibody 12F9 [0242] (B) Antibody 13B12 [0243] (C) Antibody 13H5 [0244] (D) Antibody 21H6 [0245] (E) Antibody 18G9
as well as for the corresponding control antibody (isotype IgG2a).

EXAMPLES

A—Experimental Procedures

[0246] I—Binding on Human IL-17B

[0247] The following protocol was implemented: [0248] Dilute human IL-17B (R&D Systems, ref : 1248-IB/CF) at 500 ng/ml in PBS; [0249] Immediately coat a Maxisorp 96-well plate; [0250] Seal the plate and incubate overnight at 4° C.; [0251] Aspirate each well and wash with wash buffer (PBS-Tween 0.05%); repeating the process two times; [0252] Block plate by adding 200 μl of PBS-BSA 1% for 2 hours at room temperature (RT); [0253] Aspirate each well and wash with wash buffer (PBS-Tween 0.05%); repeating the process two times; [0254] Add 100 μl of antibodies in dilution from 100 μg/ml to 0.0001 μg/ml in PBS-Tween 0.05%; [0255] Incubate for 2 hours at RT; [0256] Aspirate each well and wash with wash buffer (PBS-Tween 0.05%); repeating the process two times; [0257] Incubate with 100 μl of Goat anti-mouse HRP (Thermo Scientific, ref: 31437) diluted in PBS-BSA 0.5% for 1 hour at RT; [0258] Aspirate each well and wash with wash buffer; repeating the process two times; [0259] Incubate 15 min at RT with 50 μl of ABTS substrate (Sigma-Aldrich, ref: A3219-100 ML) [0260] Measure DO (Optical Density) at 405 nm

[0261] II—Human IL-17B Induced IL-8 Secretion by HepG2 Cells

[0262] The following protocol was implemented:

[0263] At Day 1: [0264] Prepare antibodies in dilution (4.4×) in MEM 2% SVF; [0265] Prepare human IL-17B (R&D Systems, ref: 1248-IB/CF) at 8.8 μg/ml (4.4×) in MEM 2% SVF; [0266] Mix 25 μl of diluted antibodies with 25 μl of hIL-17B and incubate the mix during 30 min at room temperature (RT); [0267] Prepare polymyxin B (Sigma-Aldrich, ref : P4932-5MU) at 110 μg/ml in MEM 2% SVF; [0268] Prepare HepG2 cells suspension at 400.000 cells/ml after trypsinization; [0269] Mix in 96-well plate 50 μl of the mix antibody/hIL-17B, 50 μl of the cell suspension and 10 μl of polymyxin; [0270] Place the plate for 24 hours at 37° C., 5% CO.sub.2; [0271] Prepare αIL-8 antibody solution (R&D Systems, ref: MAB208) at 4 μg/ml in PBS and coat a Maxisorp 96-well plate; [0272] Seal the plate and incubate overnight at 4° C.;

[0273] At Day 2: [0274] Aspirate each well and wash with wash buffer (PBS-Tween 0.05%); repeating the process two times; [0275] Block plate by adding 200 μl of PBS-BSA 1% for 2 hours at room temperature (RT); [0276] Aspirate each well; [0277] Add 50 μl of PBS-BSA 0.5% and 50 μl of standard or HepG2 pure supernatants; [0278] Incubate for 2 hours at RT; [0279] Aspirate each well and wash with wash buffer (PBS-Tween 0.05%); repeating the process two times; [0280] Incubate with 100 μl of biotinylated αIL-8 diluted (R&D Systems, ref: BAB208) in PBS-BSA 0.5% for 2 hours at RT; [0281] Aspirate each well and wash with wash buffer; repeating the process two times; [0282] Incubate with 100 μl of SA-HRP (R&D Systems, ref: DY998) in PBS-BSA 0.5% for 30 min at RT; [0283] Aspirate each well and wash with wash buffer; repeating the process two times; [0284] Incubate with 100 μl of 1:1 color reagent A+color reagent B (R&D Systems, ref: DY999) for 10 min at RT; [0285] Add 50 μl of Stop solution (B (R&D Systems, ref: DY994); [0286] D.O at 450 nm with correction at 540 nm.

B—Results

[0287] I/Tested Antibodies

TABLE-US-00004 Epitope Sequences Name recognized VH domain VL domain 12F9 QVPLDLVSR VH: SEQ ID NO: 8 VL: SEQ ID NO: 9 (SEQ ID NO: 3) CDR1: SEQ ID NO: 12 = CDR1: SEQ ID NO: 15 = DYFIN RSSQSIVHSNGNTYLE CDR2: SEQ ID NO: 13 = CDR2: SEQ ID NO: 16 = WIFPGSGSTYYHEKFKG KVSNRFS CDR3: SEQ ID NO: 14 = CDR3: SEQ ID NO: 17 TLYGNWYFDV FQGSHVPYT 13B12 KPYARMEEY Not Determined Not Determined (SEQ ID NO: 7) + SQVPVRRR (SEQ ID NO: 4) + PPPPRTG (SEQ ID NO: 5 truncated) 13H5 QVPLDLVSR VH: SEQ ID NO: 24 VL: SEQ ID NO: 25 (SEQ ID NO: 3) + CDR1: SEQ ID NO: 28 = CDR1: SEQ ID NO: 31 = SQVPVRRR TFGMGVG SASSSVSYMY (SEQ ID NO: 4) + CDR2: SEQ ID NO: 29 = CDR2: SEQ ID NO: 32 = PPPPRTGPCRQ HIWWDDDKYYNPALKS DTSNLAS (SEQ ID NO: 5) CDR3: SEQ ID NO: 30 = CDR3: SEQ ID NO: 33 = MNDGYLYY QQWSSYPFT 21H6 QVPLDLVSR VH: SEQ ID NO: 40 VL: SEQ ID NO: 41 (SEQ ID NO: 3) + CDR1: SEQ ID NO: 44 = CDR1: SEQ ID NO: 47 = SQVPVRRR TFGMGVG RASQNISDYLH (SEQ ID NO: 4) + CDR2: SEQ ID NO: 45 = CDR2: SEQ ID NO: 48 = PPPPRTGPCRQ HIWWDDDKYYNPALKG YTSQSIS (SEQ ID NO: 5) CDR3: SEQ ID NO: 46 = CDR3: SEQ ID NO: 49 = IEDALDY QNGHSFPFT 18G9 QVPLDLVSR VH: SEQ ID NO: 56 VL: SEQ ID NO: 57 (SEQ ID NO: 3) + CDR1: SEQ ID NO: 60 = CDR1: SEQ ID NO: 63 = SQVPVRRR TSGMGVG KASQSVDYDGDSYMN (SEQ ID NO: 4) + CDR2: SEQ ID NO: 61 = CDR2: SEQ ID NO: 64 = PPPPRTGPCRQ HIWWDDDKYYNPSLKS AASNLES (SEQ ID NO: 5) + CDR3: SEQ ID NO: 62 = CDR3: SEQ ID NO: 65 = CEVNLQLWMS RTQGYFDY QQSNEDPLT (SEQ ID NO: 6)

[0288] II/Binding

[0289] The binding data are shown on FIG. 1. Despite the fact that they recognize different sequences on the human IL-17B cytokine, they are all able to bind IL17B with EC.sub.50 values ranging from 2.28 ng/ml to 43.69 ng/ml.

[0290] III/Neutralizing Activity

[0291] The data reflecting the antagonist activity of each of said antibodies are shown on FIG. 2.

[0292] Interestingly, antibody 13B12 which is not able to bind the sequence QVPLDLVSR (SEQ ID NO: 3 corresponding to residues 44 to 52 of the human IL-17B of sequence SEQ ID NO: 1), but sequence KPYARMEEY (SEQ ID NO: 7) instead, displays no inhibitory effect. This reveals that the sequence QVPLDLVSR would be of high relevance for the neutralizing activity.

[0293] This is confirmed by the data obtained with antibody 12F9 which is merely able to bind said sequence on the human IL-17B and which shows a weak but inhibitory effect.

[0294] Lower EC.sub.50 values are observed with the 3 other antibodies which are further able to bind sequences SQVPVRRR (SEQ ID NO: 4 corresponding to residues 145 to 152 of the human IL-17B) and PPPPRTGPCRQ (SEQ ID NO: 5 corresponding to residues 155 to 165 of the human IL-17B) (and further CEVNLQLWMS (SEQ ID NO: 6) for 18G9). It is to be noted that based on in silico modelization, region 145-165, encompassing SEQ ID NO: 4 and SEQ ID NO: 5, could be involved in the interaction between I1-17B and its receptor.

[0295] In conclusion, the results show that the sequence QVPLDLVSR (SEQ ID NO: 3) of human IL-17B is a key sequence for its activity and then a target for antagonist antibodies. Further targeting region 145-165 would also be important for getting a better inhibition.

REFERENCES

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