ANTIBODIES HAVING SPECIFICITY FOR BTLA AND USES THEREOF

Abstract

The present invention relates to antibodies having specificity for BTLA and uses thereof, in particular for the treatment of cancer.

Claims

1. An antibody having specificity for B and T lymphocyte attenuator (BTLA) characterized in that a. it does not block the binding f HVEM to BTLA, and b. it increases the proliferation of Vγ9VδT cells.

2. The antibody of claim 1 which competes for binding to BILA with the 629.3 mAb characterized in that the 629.3 mAb comprises a heavy chain wherein the VH region is identical to SEQ ID NO:1 and a light chain wherein the VL region is identical to SEQ ID NO:2.

3. The antibody of claim 1, which binds to the same epitope as mAb 629.3 comprising a VH region identical to SEQ ID NO:1 and VL region identical to SEQ ID NO:2.

4. The antibody of claim 1 having a heavy chain comprising i) a H-CDR1 having at least 50% of identity with the H-CDRI of the 629.3 mAb, ii) a H-CDR2 having at least 50% of identity with the H-CDR2 of the 629.3 mAb and iii) a H-CDR3 having at least 50% of identity with the H-CDR3 of the 629.3 mAb or a light chain comprising i) a L-CDR1 having at least 50% of identity with the L-CDR 1 of the 629.3 mAb, ii) a L-CDR2 having at least 50% of identity with the L-CDR2 of the 629.3 mAb and iii) a L-CDR3 having at least 50% of identity with the L-CDR3 of 629.3 wherein the H-CDR1 of the 629.3 mAb is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:1 the H-CDR2 of the 6293 mAb is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:1 the H-CDR3 of the 629.3 mAb is defined by the sequence ranging from the amino acid residue at position 98 to the amino acid residue at position 109 in SEQ ID NO:1 the L-CDR I of the 629.3 mAb is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 40 in SEQ ID NO:2 the L-CDR2 of the 629.3 mAb is defined by the sequence ranging from the amino acid residue at position 56 to the amino acid residue at position 62 in SEQ ID NO:2, and the L-CDR3 of the 629.3 mAb is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:2.

5. The antibody of claim 4 comprising a heavy chain comprising i) a H-CDR1 having at least 50% of identity with the H-CDRI of the 629.3 mAb, ii) a H-CDR2 having at least 50% of identity with the H-CDR2 of the 629.3 mAb and iii) a H-CDR3 having at least 50% of identity with the H-CDR3 of the 629.3 rriAb and a light chain comprising i) a L-CDR1 having at least 50% of identity with the L-CDR I of the 629.3 rriAb, ii) a L-CDR2 having at least 50% of identity with the L-CDR2 of the 629.3 mAb and iii) a L-CDR3 having at least 50% of identity with the L-CDR3 of the 629.3 mAb.

6. The antibody of claim 4 comprising a heavy chain having 1) the H-CDR 1 of the 629.3 mAb, ii) the H-CDR2 of the 629.3 mAb and iii) the H-CDR3 of the 629.3 mAb and a light chain having i) the L-CDR1 of the 6293 mAb, ii) the L-CDR2 of the 629.3 mAb and iii) the L-CDR3 of the 629.3 mAb.

7. The antibody of claim 1 which is an antibody comprising a heavy chain wherein the VH region has at least 70% of identity with SEQ ID NO:1 and a light chain wherein the VL region has at least 70% of identity with SEQ ID NO:2.

8. The antibody of claim 1 which is an antibody comprising a heavy chain wherein the VH region is identical to SEQ ID NO:1 and a light chain wherein the VL region is identical to SEQ ID NO:2.

9. An isolated anti-BTLA antibody, wherein said antibody binds to an epitope of the BTLA protein comprising residues VKLEDRQTSNAIKEEKN (SEQ ID NO:5) and PSKDEMASRPWLL (SEQ ID NO:6).

10. A nucleic acid molecule which encodes a heavy chain and/or a light chain of the antibody of claim 9.

11. A host cell comprising the nucleic acid of claim 10.

12. (canceled)

13. A method of treating a cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of the antibody of claim 1.

14. The method of claim 13 wherein the cancer is a haematological malignancy.

15. A method of treating an infectious disease in a subject in need thereof comprising administering the subject with a therapeutically effective amount of the antibody of claim 1.

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

17. The method of claim 14, whereinaid haematological malignancy is lymphoma.

18. A nucleic acid molecule which encodes a heavy chain and/or a light chain of the antibody of claim 1.

19. A host cell comprising the nucleic acid of claim 18.

Description

EXAMPLE

[0090] The new mouse 629.3 mAb mAb having specificity for BTLA was generated and characterized in comparison with different BTLA antibodies, in particular the 7.1 and 8.2 mAbs disclosed in WO2010106051, and the 4C7 mAb disclosed in WO2011014438. T cells (CD3+) among PBMC were stained for BTLA with the anti BTLA mAbs at various concentration (from 0.01 μg/ml to 10μ,g/ml). All the antibodies tested were able to recognize and bind BTLA onto the PBMC, but 629.3 mAb seems to have a higher binding to BTLA than the others mAbs, especially at a low dose (FIG. 1). Then COS transfected with human BTLA were incubated with the anti BTLA mAbs and then with HVEM-Fc before staining with an anti human IgG Fc PE. The percentage of inhibition was determined as 100-maximum of percentage of stained cells. All antibodies except 629.3 were able to block the interaction between HVEM Fc protein and BTLA (onto the cells) in a similar way (FIG. 2). L-BTLA cells were then incubated with 4C7 or 629.3 mAbs and then with the Alexa-647 conjugated mAbs (4C7, 7.1, 8.2; 629.3). The median fluorescence intensity of cells was determined. 629.3 mAb and 4C7 mAb do not cross bind as we observe on FIG. 3. On the other hand, 7.1 and 8.2 antibodies were not able to compete against 4C7 mAb at a high dose (FIG. 3). We can conclude that 629.3 mAb does not cross bind with all the other mAbs tested (4C7, 7.1 and 8.2) and binds a different epitope than those antibodies. Finally, γδT were obtained after a positive sorting of fresh purified PBMC and stained for Cell Trace Violet to follow the proliferation. Cells were incubated 5 days in IL2/Zometa with the different mAbs prior to analysis by flow cytometry. The addition of antibodies in the culture of the γδT lead to an upregulation of their proliferation. Cells incubated with the HVEM 18.10 mAb disclosed in WO2014184360 is able to proliferate more than cells incubated with the mock control (IgG1) (FIG. 4). In the same manner, we observe than all the anti-BTLA antibodies, including the 629.3 mAb were able to induce an increase of the γδT proliferation (FIG. 4).

[0091] In conclusion, we generated a new anti-BTLA antibody that does not inhibit the BTLA/HVEM interaction but still surprisingly inhibits the activation of BTLA.

[0092] Screening method of an antibody binding to the conformational epitope of SEQ ID NO:5 and SEQ ID NO:6

[0093] To screen an antibody which binds to the conformational epitope of SEQ ID NO:5 and SEQ ID NO:6, TK cells transfected with human BTLA are stained with saturing concentration (10 μg/ml) of 629.3 BTLA mAb during 30 minutes at 4° c. After 2 washes, different doses of other BTLA mAbs are tested (30 min at 4° C.) for their competitive potential with 629.3 clone. Only mAbs that do not compete with 629.3 for the same binding site will still be able to recognize BTLA. Data are expressed as mean fluorescence intensity.

REFERENCES

[0094] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.