Anti-HLA-G antibodies and methods of use thereof

1. An isolated antibody that binds to human HLA-G, wherein the antibody comprises

A) (a) a VH domain comprising (i) HVR-H1 comprising an amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO:2, and (iii) HVR-H3 comprising an amino acid sequence SEQ ID NO:3; and (b) a VL domain comprising (i) HVR-L1 comprising an amino acid sequence of SEQ ID NO:4; (ii) HVR-L2 comprising an amino acid sequence of SEQ ID NO:5, and (iii) HVR-L3 comprising an amino acid sequence of SEQ ID NO:6; or

B) (a) a VH domain comprising (i) HVR-H1 comprising an amino acid sequence of SEQ ID NO:9, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO:10, and (iii) HVR-H3 comprising an amino acid sequence SEQ ID NO:11; and (b) a VL domain comprising (i) HVR-L1 comprising an amino acid sequence of SEQ ID NO:12; (ii) HVR-L2 comprising an amino acid sequence of SEQ ID NO:13, and (iii) HVR-L3 comprising an amino acid sequence of SEQ ID NO:14; or

C) (a) a VH domain comprising (i) HVR-H1 comprising an amino acid sequence of SEQ ID NO:17, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO:18, and (iii) HVR-H3 comprising an amino acid sequence SEQ ID NO:19; and (b) a VL domain comprising (i) HVR-L1 comprising an amino acid sequence of SEQ ID NO:20; (ii) HVR-L2 comprising an amino acid sequence of SEQ ID NO:21 and (iii) HVR-L3 comprising an amino acid sequence of SEQ ID NO:22.

2. The antibody according to claim 1 , wherein the antibody comprises

A)

i) a VH sequence of SEQ ID NO:7 and a VL sequence of SEQ ID NO:8;

ii) a humanized variant of the VH and VL of the antibody under i); or

iii) a VH sequence of SEQ ID NO:33 and a VL sequence of SEQ ID NO:34; or

B)

a VH sequence of SEQ ID NO:15 and a VL sequence of SEQ ID NO:16; or

C)

a VH sequence of SEQ ID NO:23 and a VL sequence of SEQ ID NO:24.

3. The anti-HLA-G antibody according to claim 1 , wherein the antibody has at least one of the following properties:

a) does not cross-react with a modified human HLA-G ß2M MHC I complex comprising SEQ ID NO:44;

b) does not cross-react with human HLA-A2 ß2M MHC I complex comprising SEQ ID NO:39 and

SEQ ID NO: 37;

c) does not cross-react with a mouse H2Kd ß2M MHC I complex comprising SEQ ID NO:45;

d) does not cross-react with rat RT1A ß2M MHC I complex comprising SEQ ID NO:47;

e) inhibits ILT2 binding to monomeric HLA-G ß2M MHC I complex;

f) inhibits ILT2 binding to trimeric HLA-G ß2M MHC I complex;

g) inhibits ILT2 binding to monomeric HLA-G ß2M MHC I complex by more than 50%;

h) inhibits ILT2 binding to dimeric HLA-G ß2M MHC I complex by more than 50%;

i) inhibits ILT2 binding to trimeric HLA-G ß2M MHC I complex by more than 50%;

j) inhibits ILT2 binding to JEG3 cells (ATCC No. HTB36);

k) binds to HLA-G on JEG3 cells (ATCC No. HTB36), and inhibits ILT2 binding to HLA-G on JEG3 cells (ATCC No. HTB36); or

l) inhibits CD8a binding to HLA-G by more than 80%.

4. The antibody according to claim 1 , wherein the antibody is of IgG1 isotype.

5. The antibody according to claim 4 , wherein the antibody is of IgG1 isotype with mutations L234A, L235A and P329G (numbering according to the EU index of Kabat).

6. A pharmaceutical formulation comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier.

7. A method for selecting anti-HLA-G antibodies according to claim 1 , comprising:

a) determining the binding of anti-HLA-G antibodies to human HLA-G ß2M MHC I complex comprising SEQ ID NO: 43 by a Surface Plasmon Resonance assay;

b) determining the inhibition of ILT2 binding to at least one of monomeric, dimeric, or trimeric HLA-G ß2M MHC I complex by the respective anti-HLA-G antibodies;

c) (i) selecting anti-HLAG antibodies which inhibit ILT2 binding to monomeric HLA-G ß2M MHC I complex by more than 50% or by more than 80% when compared to the binding without antibody, or (ii) selecting anti-HLA-G antibodies which inhibit ILT2 binding to at least one of monomeric, dimeric, or trimeric HLA-G ß2M MHC I complex by more than 50% or by more than 70% when compared to the binding without antibody; and wherein the selecting anti-HLA-G antibodies restore HLA-G specific suppressed immune response by monocytes co-cultured with JEG-3 cells (ATCC HTB36).

8. The method of claim 7 , wherein restoring HLA-G specific suppressed immune response by monocytes co-cultures with JEG-3 cells (ATCC HTB36) comprises suppressing tumor necrosis factor (TNF) alpha release from the monocytes.

9. A method for selecting anti-HLA-G antibodies of claim 4 , comprising:

a) determining the binding of anti-HLA-G antibodies to JEG3 cells (ATCC No. HTB36) in a flow cytometry assay;

b) determining the inhibition of ILT2 binding to JEG3 cells (ATCC No. HTB36) by the respective anti-HLA-G antibodies a flow cytometry assay; and

c) selecting anti-HLA-G antibodies which bind to JEG3 (ATCC No. HTB36) cells, and which inhibit ILT2 binding to JEG3 cells (ATCC No. HTB36) by more than 50% or by more than 80% when compared to the binding without antibody.

10. The method of claim 9 , wherein the flow cytometry assay is fluorescence-activated cell sorting (FACS) assay.

11. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the antibody of claim 1 .