DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: INVE_001_03US_SeqList_ST25.txt, date recorded: Sep. 3, 2021, file size: ˜45,324 bytes).

TECHNICAL FIELD

The present invention relates to antibodies, or antigen-binding fragments thereof, raised against an immunogenic peptide derived from the α3 domain of human leukocyte antigen G (HLA-G) protein.

TECHNICAL BACKGROUND

In cancer, one major immune escape mechanism is the expression of inhibitory molecules on the cell surface impairing T cell signaling. Many of these inhibitory molecules are considered as Immune Check Points (ICP) and refer to numerous inhibitory pathways first demonstrated to maintain self-tolerance and to modulate the duration and amplitude of physiological immune responses within peripheral tissues to avoid collateral tissue damages.

HLA-G was recently identified as an ICP molecule, which inhibits the effector functions of infiltrating immune cell subsets through the interaction with its specific receptors and is frequently upregulated in tumor cells (Carosella et al., 2015).

Furthermore, HLA-G can also be neo-expressed and/or up-regulated in pathological conditions such as viral infections, auto-immune and inflammatory diseases or after allo-transplantation.

For instance, viruses such as HCMV (Human Cytomegalovirus), HSV-1 (Herpes Virus Simplex), RABV (Rabies Virus), HCV (hepatitis C Virus), IAV (Influenza A Virus) and HIV-1 (Human Immunodeficiency Virus type I) seem to up-regulate the expression of HLA-G to prevent infected cells from being recognized and attacked by CTL and NK cells. HLA-G can also control the CD8 T cell response against HIV-infected cells by directing CD8 cells to apoptosis and by affecting their cytotoxic properties (Tripathi and Agrawal, 2007).

HLA-G is a non-classical MHC class I molecule that was first identified in choriocarcinoma cells. MHC class I antigens comprise the classical antigens HLA-A, HLA-B and HLA-C, which exhibit three extracellular globular domains (α1, α2and α3) associated with β2-microglobulin (β2M), as well as the non-classical antigens HLA-E, HLA-F and HLA-G.

Unlike classical MHC class I molecules, HLA-G is characterized by (i) a limited polymorphism, (ii) a tissue-restricted expression, and (iii) differs as well by its functions.

The eight exon gene coding for HLA-G molecules spans 4.4 kb on chromosome 6 (Geraghty et al., 1987; Ellis et al., 1990). Exons 2, 3 and 4 encode the α1, as and α3extracellular domains, respectively. The primary RNA transcript can be alternatively spliced, resulting in the expression of seven isoforms, four of which are membrane-bound (HLA-G1, HLA-G2, HLA-G3 and HLA-G4), and three are soluble (HLA-G5, HLA-G6 and HLA-G7). HLA-G1 and HLA-G5 are the most prominent isoforms described, in part likely because of limited HLA-G reagents such as antibodies. Their structures are typical of classical HLA class I molecule: a heavy chain composed of three extracellular globular domains non-covalently associated to β2-Microglobulin (β2M) and a peptide, while the other isoforms are shorter, lacking one or two globular domains of the heavy chain, and without β2M association.

The immuno-inhibitory activity of HLA-G takes place through specific binding to three inhibitory receptors: leukocyte immunoglobulin-like receptor B1 (LILRB1/ILT2/CD85j), LILRB2 (ILT4/CD85d) and KIR2DL4 (or CD158d).

Through the interaction with these receptors, and opposite to classical MHC class I molecules, HLA-G acts as a down-regulator of the immune system's main functions, and neither stimulatory functions nor responses directed against allogenic HLA-G have been reported to date (Carosella et al., 2008a).

In a similar manner to other MHC class I molecules, LILRB receptors interact with the α3 domain of HLA-G. The LILRB1 receptor is expressed on B cells, some T cells, some NK cells and on all APCs (monocytes and dendritic cells), whereas LILRB2 expression is restricted to the myeloid lineage and only expressed on monocytes and dendritic cells.

LILRB1 and LILRB2 receptors bind a wide range of classical MHC molecules through the α3 domain/β2M complex or only the α3 domain respectively. Indeed, LILRB1 binds only β2M-associated HLA-G complexes, whereas LILRB2 recognizes both β2M-associated and β2M-free HLA-G molecules as well as truncated α1-α3 domain isoforms.

HLA-G is the ligand of highest affinity for LILRB2 receptor as compared to classical MHC class I molecules. The higher affinity of HLA-G for LILRB1 and LILRB2 receptors is particularly illustrated by the fact that HLA-G displayed at the surface of tumor cells is capable of engaging the LILRB1 and/or LILRB2 receptors even if classical MHC class I molecules are also expressed at their surface. This preferential interaction of HLA-G for LILRB receptors is sufficient to inhibit the cytolytic functions of immune effector cells.

LILRB1 and 2 receptors do not bind the same HLA-G forms and present a higher affinity for HLA-G multimers than monomeric structures (HoWangYin et al., 2012). This higher affinity of LIRB receptors for HLA-G was demonstrated to be related to the presence of aromatic amino acids Phe195 and Tyr197 within the α3 domain of HLA-G that are not present in classical MHC class I molecules.

The relevance of HLA-G expression as an escape mechanism employed by tumor cells to inhibit effector cells has been widely demonstrated (Loustau et al., 2013). Several approaches targeting HLA-G with the goal of mediating tumor cell rejection have been developed (Carosella et al., 2008b; Yan, 2011). Few anti-HLA-G antibodies have been generated, and only one blocking antibody is available (87G) (Blaschitz et al., 2000; Menier et al., 2003). This monoclonal antibody, 87G, interacts with the α1 domain of the heavy chain of HLA-G associated to β2M. Even though it has been described as capable of neutralizing HLA-G, and therefore restoring tumor rejection in vitro and in vivo (Agaugue et al., 2011), its applicability is compromised as HLA-G is frequently expressed as a β2M-free full length molecule or truncated isoforms. These various isoforms can also bind the LILRB2 inhibitory receptor.

HLA-G immunization has been remarkably difficult, yielding few specific antibodies. The reasons for this failure to generate neutralizing antibodies have been elucidated. In kidney-transplanted patients, it was demonstrated that the presence of HLA-G does not favor antibody production (Qiu et al., 2006). Recent in vitro studies have confirmed that HLA-G/LILRB1 interaction impairs B-cell maturation and antibody production in humans (Naji et al., 2014). HLA-G is also known to exert a tolerogenic function in mice, a species commonly used to generate monoclonal antibodies (Favier et al., 2001). HLA-G interacts with the PIR-B receptor, which is expressed in murine B-cells and which is functionally homologous to human LILRB1 and LILRB2 (Liang et al., 2002). HLA-G/PIR-B interaction would lead to B-cell inhibition thus preventing antibody production in mice.

A monoclonal antibody apparently directed to the α3 domain of classical MHC class I molecules, named TP25.99 has been developed (Tanabe et al., 1992). However, others have shown that it does not bind the α3 domain of HLA-G (Desai et al., 2000; Moy et al., 2000). This discrepancy could be explained by the hydrophobic characteristic of HLA-G α3 domain that is unfavorable to antibody generation.

While the international patent application WO2014/072534 proposes a method for generating and developing anti-HLA-G antibodies by DNA immunization with the complete α3 domain of HLA-G, there is still a need for anti-HLA-G antibodies which recognize all HLA-G isoforms and exhibit an improved specificity with respect to HLA-G.

SUMMARY OF THE INVENTION

The inventors have now succeeded in circumventing the difficulties for generating specific anti-HLA-G antibodies, by designing an immunogenic peptide from the α3 domain of HLA-G protein.

The first object of the invention is an isolated peptide consisting of sequence X1-THHPVFDYEATLR-X2 (SEQ ID NO: 49), wherein X1 is absent, Cysteine, Valine, or is a sequence selected from the group consisting of KTHV (SEQ ID NO: 50) or CKTHV (SEQ ID NO: 51), and X2 is absent or Cysteine.

Such a peptide is useful as an immunogen for producing antibodies specific for the α3 domain of HLA-G protein isoforms, preferably monoclonal antibodies.

A further object of the invention is an anti-HLA-G antibody, preferably a monoclonal antibody, which specifically recognizes the peptide as defined herein.

The antibody may be a full-length antibody, an antigen-binding fragment thereof, or a bispecific antibody.

A further object of the invention is a composition comprising such antibody and a pharmaceutically acceptable carrier.

Another subject of the invention is a nucleic acid comprising a nucleotide sequence encoding an antibody heavy chain variable region (VH), an antibody light chain variable region (VL) or both, of the antibody as defined herein.

A vector, preferably an expression vector, comprising said nucleic acid is also provided. A host cell comprising the nucleic acid or the vector is further described.

Methods for producing anti-HLA-G antibodies are further provided. In a preferred embodiment is disclosed a method for producing an anti-HLA-G monoclonal antibody, comprising culturing the host cell comprising said nucleic acid or said vector under conditions allowing for expression of the antibody.

The antibody, nucleic acid or vector expressing said antibody, is particularly useful in treating a cancer or a viral infection.

A further subject of the invention is the use of the anti-HLA-G antibody described herein, in an in vitro diagnostic method for detecting or monitoring HLA-G in a biological sample.

Diagnostic kits comprising said antibody are further encompassed.

