Anti-HLA-g antibodies and use thereof

Abstract

To provide anti-HLA-G antibodies, their preparation, formulations and methods of using the same.SOLUTION: The present invention provides an antibody that binds to human HLA-G, comprising: (a) a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 comprising specific amino acid sequences; and (b) a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 comprising specific amino acid sequences.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to anti-HLA-G antibodies, their preparation, formulations and methods of use. [Background technology]

[0002] Human major histocompatibility complex class I, 6, also known as human leukocyte antigen G (HLA-G), is a protein encoded by the HLA-G gene in humans. HLA-G belongs to the HLA nonclassical class I heavy chain paralogs. This class I molecule is a heterodimer (beta-2 microglobulin) consisting of a heavy chain and a light chain. The heavy chain is membrane anchored, but can also be excreted / secreted.

[0003] The heavy chain consists of three domains: alpha1, alpha2 and alpha3. The alpha1 and alpha2 domains form a peptide-binding groove flanked by two alpha helices. Small peptides (approximately 9-mers) can bind to this groove, as do other MHC I proteins.

[0004] The second chain is beta2 microglobulin which binds to the heavy chain like other MHC I proteins.

[0005] For HLA-G, seven isoforms exist, three secreted and four membrane-bound (schematically depicted in FIG. 1).

[0006] HLA-G can form functionally active complex oligomeric structures (Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729). Disulfide-bonded dimers are formed between Cys42 of two HLA-G molecules. (Shiroishi M et al., J Biol Chem 281 (2006) 10439-10447. Trimeric and tetrameric complexes have also been described, for example, in Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729, Allan DS, et al. J Immunol Methods. 268 (2002) 43-50 and T Gonen-Gross et al., J Immunol 171 (2003) 1343-1351).

[0007] HLA-G is mainly expressed on the cytotrophoblast layer of the placenta. Several tumors (including pancreatic, breast, skin, colorectal, gastric and ovarian) express HLA-G (Lin, A. et al., Mol Med. 21 (2015) 782-791; Amiot, L., et al., Cell Mol Life Sci. 68 (2011) 417-431). Expression has also been reported to be associated with pathological conditions such as inflammatory diseases, GvHD and cancer. HLA-G expression has been reported to be associated with poor prognosis of cancer. Tumor cells escape host immune surveillance by inducing immune tolerance / suppression via HLA-G expression. JPEG2025037987000002.jpg63170

[0008] Because HLA-G shares a high degree of homology (>98%) with other MHC I molecules, it is difficult to generate truly HLA-G-specific antibodies that are free of cross-reactivity to other MHC I molecules.

[0009] Certain antibodies that interact in different ways with HLA-G have been described previously: Tissue Antigens, 55 (2000) 510-518 relates to monoclonal antibodies such as 87G and MEM-G / 9; Neoplasma 50 (2003) 331-338 relates to certain monoclonal antibodies that recognize both intact HLA-G oligomeric complexes (e.g. 87G and MEM-G9) and heavy chains that do not contain HLA-G (e.g. 4H84, MEM-G / 1 and MEM-G / 2); Hum Immunol. 64 (2003) 315-326 relates to several antibodies tested against HLA-G expressing JEG3 tumor cells (e.g. MEM-G / 09 and -G / 13, which react exclusively with native HLA-G1 molecules). MEM-G / 01 recognises denatured HLA-G heavy chains of all isoforms (similar to 4H84 mAb), whereas MEM-G / 04 recognises selectively denatured HLA-G1, -G2 and -G5 isoforms, and Wiendl et al Brain 2003 176-85 relate to different monoclonal HLA-G antibodies such as 87G, 4H84, MEM-G / 9.

[0010] The above publications report antibodies that bind to human HLA-G or to the human HLA-G-β2M MHC complex. However, due to the high polymorphism and high homology of the HLA family, the majority of antibodies lack truly specific HLA-G binding properties and often also bind or cross-react with other HLA family members (either as an MHC complex with β2M or in their β2M-free forms) or simply do not inhibit the binding of the HLA-G β2M MHC complex to its receptors ILT2 and / or ILT4 (considered to be non-antagonistic antibodies).

[0011] WO 2019 / 202040 relates to HLA-G antibodies, including the antibody HLA-G-0090. WO 2019 / 202041 relates to multispecific HLA-G antibodies, including the antibody HLA-G-0090. Summary of the Invention

[0012] The invention described herein is an antibody that binds to human HLA-G, A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) (a) an antibody comprising a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0013] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising: A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or B) An antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7, and a VL domain comprising the amino acid sequence of SEQ ID NO:26.

[0014] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24.

[0015] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26.

[0016] Such antibodies have highly valuable properties such as their binding properties, their high specificity for HLA-G, without cross-reactivity to HLA-A and HLA MHC I complexes from other species. They are able to bind to HLA-G on cells and inhibit the binding of ILT2 and / or ILT4 to HLA-G expressed on these cells. They have been generated from the HLA-G antibody HLA-G-0090.

[0017] The HLA-G antibody HLA-G-0090 described in WO 2019 / 202040 contains a glycosylation site in one of its CDRs (CDR-L1 containing an NSS motif at amino acids 31, 32 and 33 of the light chain (LC)) and therefore its binding properties are affected by N-glycosylation, constituting a liability for its potential developability.

[0018] The homology model of the variable region of HLA-G-0090 showed that the light chain (LC) positions 31-33 are highly solvent accessible. Furthermore, the side chains of N31 and S32 are predicted to point inward toward the CDR-H3, making them likely to be part of the antibody paratope. Indeed, several published antibody-antigen X-ray complex structures have demonstrated that these residues undergo chemical interactions with the antigen. Therefore, there was a high risk that the binding affinity of the antibody would be worsened by introducing mutations at LC positions 31-33. Therefore, we designed various mutants of the antibody HLA-G-0090 with mutations at LC positions 31, 32, and 33, from which most mutants worsened the binding properties or expressibility. Surprisingly, among these various variants of HLAG-0090 in which glycosylation sites have been removed, only two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A were found to no longer show N-glycosylation at the CDR-L1 of the LC (and therefore no Fab glycosylation could be detected), but still show improved binding properties, good expressibility and stability. Since all recently approved pharmaceutical antibody products are produced in mammalian cells, in particular CHO cells (see, for example, Walsh G., Nature Biotech (2018) 1136-1145), providing antibodies (VH and VL, in particular CDRs) that do not have glycosylation sites in the binding region is a valuable advantage, since these antibodies can be easily used for production in mammalian expression systems without the risk of (at least partially) compromising their binding properties by glycosylation.

[0019] In a further embodiment, the HLA-G antibody of the invention comprises an Fc domain of human origin. In one embodiment, the Fc domain is of the IgG isotype, and in one preferred embodiment is of the IgG1 isotype.

[0020] In a further embodiment, the HLA-G antibody of the invention is a bispecific antibody, in particular a bispecific antibody that binds to human HLA-G and human CD3, and which comprises a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3.

[0021] In one embodiment, the HLA-G antibody of the invention is a bispecific antibody that binds to human HLA-G and human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3; The first antigen-binding portion that binds to human HLA-G is A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, a second antigen-binding portion that binds to a T cell activation antigen that binds to human CD3, C) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57; or D) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 64, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 65, or E) A bispecific antibody comprising: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 68, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73.

[0022] One embodiment of the present invention is such a bispecific antibody, The first antigen-binding portion comprises A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or B) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26; The second antigen-binding portion C) a VH domain comprising the amino acid sequence of SEQ ID NO: 58 and a VL domain comprising the amino acid sequence of SEQ ID NO: 59; or D) a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO: 67; or E) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO: 75.

[0023] One embodiment of the present invention is such a bispecific antibody, The first antigen-binding portion comprises A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion It comprises a VH domain comprising the amino acid sequence of SEQ ID NO:58, and a VL domain comprising the amino acid sequence of SEQ ID NO:59. One embodiment of the present invention is such a bispecific antibody, The first antigen-binding portion comprises A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion It comprises a VH domain comprising the amino acid sequence of SEQ ID NO:66, and a VL domain comprising the amino acid sequence of SEQ ID NO:67. One embodiment of the present invention is such a bispecific antibody, The first antigen-binding portion comprises A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion It comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO: 75.

[0024] Such bispecific antibodies binding to human HLA-G and human CD3, in addition to the properties of HLA-G antibodies, exhibit further beneficial properties such as induction of antibody-mediated IFN-gamma secretion by T cells on HLA-G expressing cells, T cell activation in the presence of HLA-G expressing tumor cells, induction of T cell-mediated tumor cell killing on HLA-G expressing cells, and in vivo antitumor efficacy and even tumor regression in different cancer xenograft mouse models.

[0025] The invention provides isolated nucleic acid encoding an antibody or bispecific antibody described herein.

[0026] The invention provides a host cell containing such a nucleic acid.

[0027] The invention provides a method for producing an antibody, comprising culturing a host cell so that the antibody is produced.

[0028] The invention provides such methods of producing an antibody, further comprising recovering the antibody from the host cell.

[0029] The invention provides antibodies produced by such host cells, wherein the host cell is eukaryotic.

[0030] The invention provides pharmaceutical formulations comprising an antibody described herein and a pharma- ceutically acceptable carrier.

[0031] The present invention provides an antibody as described herein for use as a medicament.

[0032] The present invention provides the antibodies described herein for use in the treatment of cancer.

[0033] The invention provides for the use of an antibody as described herein in the manufacture of a medicament. In one embodiment, the medicament is for the treatment of cancer.

[0034] The invention provides a method of treating an individual with cancer, comprising administering to the individual an effective amount of an antibody described herein. [Brief description of the drawings]

[0035] [Figure 1] Different isoforms of HLA-G. [Figure 2A-C]Figure 2A: Schematic of HLA-G molecule associated with β2M: Schematic of HLA-G wt molecule. Figure 2B: Schematic of HLA-G molecule associated with β2M: KIR2DL4 and ILT2 / 4 interactions are extracted from the crystal structure: HLA-G:ILT4 complex structure (PDB code: 2DYP). KIR2DL1 structure was obtained from PDB code 1IM9 (KIR2DL1:HLA-Cw4 complex structure) and placed on HLAG by superposition of HLA-Cw4 and HLAG structures. Figure 2C: Schematic of HLA-G molecule associated with β2M: Schematic of HLA-G chimeric molecule used as counter antigen for identification of specific HLA-G binders. White dots represent surface residues identified as unique to HLA-G. These residues were replaced by HLA consensus sequences in the chimeric molecule. [Figure 2D] Structures of HLA-G molecules associated with specific receptors: HLA-G structures in complex with a given receptor (ILT4 and KIR2DL1, etc.). ILT4 structure (PDB code: 2DYP). The KIR2DL1 structure was taken from PDB code 1IM9 (KIR2DL1:HLA-Cw4 complex structure) and placed on HLA-G by superposition of the HLA-Cw4 and HLA-G structures. The receptor is shown in ribbon representation and HLA-G in molecular surface representation. HLA-G residues that are unique or conserved in other HLA paralogs are colored white and gray, respectively. Unique surface residues were replaced by HLA consensus sequences in the chimeric counter antigen. [Diagram 3] Summary of antibody-antigen binding assay principle - Relative activity concentration (RAC) of HLA-G antibodies for binding to HLA-G [Figure 4A] Mass spectrum of N-glycosylation of HLA-G-0090 shows Fab-glycosylation. [Figure 4B] Mass spectra of N-glycosylation of HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A: Fab-glycosylation not detectable. [Diagram 5]Exemplary FACS staining for anti-HLA-G antibodies HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A (2 μg / ml) on SKOV3 cells (no HLA-G expression), JEG3 cells (expressing HLA-G) and SKOV3-HLA-G cells (SKOV3 cells transfected with HLA-G). [Figure 6] Ability of HLA-G antibodies HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A to modify / inhibit the interaction and binding of recombinant soluble ILT2 (ILT2 Fc domain fusion) to naturally expressed HLA-G on JEG3 tumor cells. JEG3 cells are pre-incubated / pre-treated with HLA-G antibodies such that ILT2 binding to JEG3 cells is inhibited / blocked. Controls were performed with JEG3 cells without HLA-G antibody pre-treatment (ILT2-Fc only) and with isotype antibodies. [Figure 7] Binding of bispecific anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibodies to CD3 expressed on T cells by antibodies P1AF7977, P1AF7978 and P1AF7979. [Figure 8] The bispecific anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibodies P1AF7977, P1AF7978 and P1AF7979 showed binding to HLA-G transfected JEG3 and SKOV3 cells. [Figure 9] Bispecific anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibody mediated / induced IFN-gamma secretion by T cells with antibodies P1AF7977, P1AF7978 and P1AF7979. [Figure 10] The bispecific anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibodies P1AF7977, P1AF7978 and P1AF7979 induced T cell-mediated cytotoxicity / tumor cell killing. [Figure 11]In vivo antitumor efficacy of anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibody P1AF7977 in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G), resulting in tumor regression. [Figure 12] Dose response study with anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibody P1AF7977 in humanized NSG mice bearing human breast cancer PDX tumors (BC004). Strong tumor growth inhibition until tumor regression was observed in mice tested at different doses. [Figure 13] Exemplary configurations of bispecific antigen-binding molecules of the invention. (A,D) Diagrams of "1+1 CrossMab" molecules. (B,E) Diagrams of "2+1 IgG Crossfab" molecules with different order of Crossfab and Fab components ("inverted"). (C,F) Diagrams of "2+1 IgG Crossfab" molecules. (G,K) Diagrams of "1+1 IgG Crossfab" molecules with different order of Crossfab and Fab components ("inverted"). (H,L) Diagrams of "1+1 IgG Crossfab" molecules. (I,M) Diagrams of "2+1 IgG Crossfab" molecules with two CrossFabs. (J,N) Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: optionally introduced oppositely charged amino acids in the CH1 and CL domains. Although Crossfab molecules are shown as comprising swapped VH and VL domains, in embodiments where no charge modifications have been introduced into the CH1 and CL domains, they alternatively comprise swapped CH1 and CL domains. [Figure 14] Induction of T cell activation by the bispecific anti-HLA-G / anti-CD3 antibody P1AF7977 (HLA-G-0090-VL-S32P / CD3 P035) in the presence of SKOV3 HLAG cells. [Figure 15] Relative binding activity (IgG format) of the original optimized CD3 binders, CD3orig and CD3opt (=P035-093 (P035)), to recombinant CD3 measured by SPR under non-stress conditions after 14 days at 40°C, pH 6 or after 14 days at 37°C, pH 7.4. [Figure 16] Binding of the original CD3 binders, CD3orig and CD3opt (=P035-093 (P035)) to Jurkat NFAT cells (IgG format) measured by flow cytometry. Antibody binding to Jurkat NFAT cells was detected using a fluorescently labeled anti-human Fc specific secondary antibody. [Figure 17] Schematic diagram of the CD3 activation assay used in Example 8. [Figure 18] Jurkat NFAT activation (IgG format) by the original optimized CD3 binders, CD3orig and CD3opt (=P035-093(P035)). Jurkat NFAT reporter cells were co-incubated with anti-PGLALA expressing CHO (CHO-PGLALA) cells in the presence of CD3orig or CD3opt (=P035-093(P035)) IgG PGLALA, or CD3opt IgG wt as a negative control. CD3 activation was quantified by measuring luminescence after 24 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] As used herein, the terms "HLA-G", "human HLA-G", "HLAG" refer to HLA-G human major histocompatibility complex, class I, G, also known as human leukocyte antigen G (HLA-G) (exemplary SEQ ID NO: 35). Typically, HLA-G forms an MHC class I complex with β2 microglobulin (B2M or β2m). In one embodiment, HLA-G refers to the MHC class I complex of HLA-G and β2 microglobulin. In a preferred embodiment, HLA-G refers to the cell surface-bound MHC class I complex of HLA-G and β2 microglobulin, also known as HLA-G1 (see FIG. 1 herein and, e.g., Blaschitz et al., Molecular Human Reproduction, 11 (2005) 699-710, especially FIG. 1).

[0037] As used herein, a "binds to human HLA-G", "specifically binds to human HLA-G", "binds to human HLA-G" or "anti-HLA-G" antibody (either monospecific, multispecific or bispecific) or antigen-binding portion has a molecular weight of 5.0×10 -8 K in mol / l D value, in one embodiment 1.0×10 -9 K in mol / l D value, in one embodiment 5.0×10 -8 mol / l~1.0×10 -13 K in mol / l D refers to an antibody / antigen-binding portion that specifically binds to the human HLA-G antigen or its extracellular domain (ECD) with a binding affinity of 0.01 mg / mL. In one embodiment, the antibody binds to the HLA-G β2M MHC I complex comprising SEQ ID NO: 39.

[0038] Binding affinity is determined in standard binding assays, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), using constructs comprising the HLA-G extracellular domain (e.g., its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined in standard binding assays using an exemplary soluble HLA-G comprising an MHC class I complex comprising SEQ ID NO:39.

[0039] HLA-G has a conventional MHC I fold and consists of two chains. Chain 1 consists of three domains, alpha 1, alpha 2, and alpha 3. The alpha 1 and alpha 2 domains form a peptide-binding groove flanked by two alpha helices. Small peptides (approximately 9-mers) can bind to this groove, similar to other MHCI proteins. Chain 2 is beta 2 microglobulin (β2M), which is shared with various other MHCI proteins.

[0040] HLA-G can form functionally active complex oligomeric structures (Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729). Disulfide-bonded dimers are formed between Cys42 of two HLA-G molecules. (Shiroishi M et al., J Biol Chem 281 (2006) 10439-10447. Trimeric and tetrameric complexes have also been described, for example, in Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729, Allan DS, et al. J Immunol Methods. 268 (2002) 43-50 and T Gonen-Gross et al., J Immunol 171 (2003) 1343-1351). HLA-G, unlike most other MHC class I molecules, has several free cysteine ​​residues. Boyson et al., Proc Nat Acad Sci USA, 99:16180 (2002) reported that recombinant soluble HLA-G5 can form disulfide-linked dimers with intermolecular Cys42-Cys42 disulfide bonds. In addition, membrane-bound HLA-G1 can also form disulfide-linked dimers on the cell surface of the JEG3 cell line, which endogenously expresses HLA-G. Disulfide-linked dimeric forms of HLA-G1 and HLA-G5 were also found on the cell surface of trophoblast cells (Apps, R., Tissue Antigens, 68:359 (2006)).

[0041] HLA-G is mainly expressed on the cytotrophoblast layer of the placenta. Several tumors (including pancreatic, breast, skin, colorectal, gastric and ovarian) express HLA-G (Lin, A. et al., Mol Med. 21 (2015) 782-791; Amiot, L., et al., Cell Mol Life Sci. 68 (2011) 417-431). Expression has also been reported to be associated with pathological conditions such as inflammatory diseases, GvHD and cancer. HLA-G expression has been reported to be associated with poor prognosis of cancer. Tumor cells escape host immune surveillance by inducing immune tolerance / suppression via HLA-G expression.

[0042] For HLA-G, seven isoforms exist, three secreted and four membrane-bound (schematically shown in FIG. 1). The most important functional isoforms of HLA-G include b2-microglobulin (β2M)-associated HLA-G1 and HLA-G5. However, the tolerogenic effects of these isoforms are different and depend on the form of the ligand (monomer, dimer) and the affinity of the ligand-receptor interaction.

[0043] HLA-G protein can be produced using standard molecular biology techniques. The nucleic acid sequences of HLA-G isoforms are known in the art. For example, see GENBANK Accession No. AY359818.

[0044] HLA-G isotypes promote signaling through the ILTs, particularly ILT2, ILT4, or a combination thereof.

[0045] ILTs: ILTs represent Ig-type activating and inhibitory receptors involved in the regulation of immune cell activation and control immune cell function (Borges, L., et al., Curr Top Microbial Immunol, 244:123-136 (1999)). ILTs are classified into three groups: (i) inhibitory, which contain a cytoplasmic immunoreceptor tyrosine-dependent inhibitory motif (ITIM) and transmit inhibitory signals (ILT2, ILT3, ILT4, ILT5 and LIR8); (ii) activating, which contain a short cytoplasmic tail and charged amino acid residues in the transmembrane domain (ILT1, ILT7, ILT8 and LIR6alpha) and deliver activation signals via the cytoplasmic immunoreceptor tyrosine-dependent activation motif (ITAM) of the associated common gamma chain of the Fc receptor; and (iii) the soluble molecule ILT6, which lacks a transmembrane domain. Several recent studies have highlighted the immunomodulatory role of ILTs on the surface of antigen-presenting cells (APCs). The best-characterized immunoinhibitory receptors, ILT2, ILT3, and ILT4 receptors, are expressed on a wide range of immune cells, including monocytes, B cells, dendritic cells, plasmacytoid dendritic cells, and subsets of NK cells and T cells. ILT2 is expressed on a subset of T cells and has been shown to inhibit the activation and proliferation of these cells upon ligation (Colonna M. et al., J Immunol. 20011, 66:2514-2521, J Immunol 2000; 165:3742-3755). ILT3 and ILT4 are upregulated by exposure of immature DCs to known immunosuppressants, including IL-10, vitamin D3, or suppressor CD8 T cells (Chang, CC, et al., Nat Immunol, 3:237-243 (2002)). Expression of ILT on DCs is tightly regulated by inflammatory stimuli, cytokines, and growth factors and is downregulated after DC activation (Ju, XS, et al., Gene, 331:159-164 (2004)). Expression of ILT2 and ILT4 receptors is highly regulated by histone acetylation, contributing to tightly regulated gene expression only in the myeloid lineage of cells (Nakajima, H., J Immunol, 171:6611-6620 (2003)).

[0046] Engagement of inhibitory receptors ILT2 and ILT4 can alter the secretion / release profile of cytokines and chemokines of monocytes and inhibit Fc receptor signaling (Colonna, M., et al. J Leukoc Biol, 66:375-381 (1999)). The role and function of ILT3 on DCs has been precisely described by the group of Suciu-Foca (Suciu-Foca, N., Int Immunopharmacol, 5:7-11 (2005)). The ligand of ILT3 is unknown, but ILT4 is known to bind to the third domain of HLA class I molecules (HLA-A, HLA-B, HLA-C and HLA-G) and compete with CD8 for MHC class I binding (Shiroishi, M., Proc Natl Acad Sci USA, 100:8856-8861 (2003)). The preferential ligand of some inhibitory ILT receptors is HLA-G. HLA-G plays a potential role in maternal-fetal tolerance and in mechanisms by which tumor cells escape immune recognition and destruction (Hunt, JS, et al., Faseb J, 19:681-693 (2005)). Regulation of DC function by HLA-G-ILT interactions is most likely an important pathway in DC biology. It has been determined that human monocyte-derived DCs highly expressing ILT2 and ILT4 receptors still maintain a stable tolerogenic-like phenotype (CD80low, CD86low, HLA-DRlow) with the potential to induce T cell anergy when treated with HLA-G and stimulated with allogeneic T cells (Ristich, V., et al., Eur J Immunol, 35:1133-1142 (2005)). Furthermore, HLA-G interaction with DCs highly expressing ILT2 and ILT4 receptors resulted in downregulation of several genes involved in the MHC class II presentation pathway. IFN-amma-inducible lysosomal thiol reductase (GILT), a lysosomal thiol reductase abundantly expressed by professional APCs, was significantly reduced in HLA-G-modified DCs.The repertoire of primed CD4+ T cells may be influenced by DC expression of GILT, since in vivo T cell responses to select antigens were reduced in animals lacking GILT after targeted gene disruption (Marie, M., et al., Science, 294:1361-1365 (2001)). HLA-G / ILT interactions on DCs may interfere with the assembly and transport of MHC class II molecules to the cell surface, resulting in less efficient presentation or expression of structurally abnormal MHC class II molecules. It was determined that HLA-G significantly reduces the transcription of invariant chain (CD74), HLA-DMA, and HLA-DMB genes on human monocyte-derived DCs that highly express ILT inhibitory receptors (Ristich, V., et al; Eur J Immunol 35:1133-1142 (2005)).

[0047] Another receptor for HLA-G is KIR2DL4, as it binds to cells expressing HLA-G (US Patent Application Publication No. 2003232051; Cantoni, C. et al. Eur J Immunol 28 (1998) 1980; Rajagopalan, S. and EOLong. [a welcome errata can be found in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093; Ponte, M. et al. PNAS USA 96 (1999) 5674). KIR2DL4 (also called 2DL4) is a KIR family member (also called CD158d) that shares structural features with both activating and inhibitory receptors (Selvakumar, A. et al. Tissue Antigens 48 (1996) 285). 2DL4 has a cytoplasmic ITIM suggesting an inhibitory function and positively charged amino acids in the transmembrane region, a feature typical of activating KIRs. Unlike other clonally distributed KIRs, 2DL4 is transcribed by all NK cells (Valiante, NM et al. Immunity 7 (1997) 739; Cantoni, C. et al. Eur J Immunol 28 (1998) 1980; Rajagopalan, S. and EOLong. [Published errata can be found in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093).

[0048] HLA-G has also been shown to interact with CD8 on cytotoxic T cells (Sanders et al, J. Exp. Med., 174 (1991), 737-740) and induce CD95-mediated apoptosis in activated CD8-positive cytotoxic T cells (Fournel et al, J. Immun., 164 (2000), 6100-6104). This mechanism of elimination of cytotoxic T cells has been reported as one of the mechanisms of immune evasion and tolerance induction in pregnancy, inflammatory diseases and cancer (Amodio G. et al, Tissue Antigens, 84 (2014), 255-263).

[0049] As used herein, an anti-HLA-G antibody (either monospecific, multispecific or bispecific) or antigen-binding portion that "does not cross-react" or "does not (specifically) bind" to a modified human HLA-G β2M MHC I complex, in which the HLA-G specific amino acids have been replaced by HLA-A consensus amino acids, the complex comprising SEQ ID NO: 40, the mouse H2Kd β2M MHC I complex comprising SEQ ID NO: 41, the rat RT1A β2M MHC I complex comprising SEQ ID NO: 43, and the human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 35 and SEQ ID NO: 33, refers to an anti-HLA-G antibody (either monospecific, multispecific or bispecific) or antigen-binding portion that does not substantially bind to any of these counter antigens. In one embodiment, an anti-HLA-G antibody (either monospecific, multispecific or bispecific) or antigen binding portion that does not "cross-react" or "does not specifically bind" to a modified human HLA-G β2M MHC I complex, in which the HLA-G specific amino acids have been replaced by HLA-A consensus amino acids, wherein the complex comprises SEQ ID NO: 40, the mouse H2Kd β2M MHC I complex comprises SEQ ID NO: 41, the rat RT1A β2M MHC I complex comprises SEQ ID NO: 43, and / or the human HLA-A2 β2M MHC I complex comprises SEQ ID NO: 35 and SEQ ID NO: 33, refers to an anti-HLA-G antibody (either monospecific, multispecific or bispecific) or antigen binding portion that does not show significant binding / interaction, for example in a surface plasmon resonance assay (e.g. as described in Example 2).Binding binding / interactions are determined by standard binding assays, for example surface plasmon resonance technology (BIAcore®, GE-Healthcare, Uppsala, Sweden) using the respective antigen:modified human HLA-G β2M MHC I complex, where the HLA-G specific amino acids have been replaced by HLA-A consensus amino acids, the complex comprising SEQ ID NO: 40, the mouse H2Kd β2M MHC I complex comprising SEQ ID NO: 41, the rat RT1A β2M MHC I complex comprising SEQ ID NO: 43, and / or the human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 35 and SEQ ID NO: 33. The assay set-up and antigen construction / preparation are described in the Examples.

[0050] The term "inhibits ILT2 binding to HLA-G on JEG-3 cells (ATCC HTB36)" refers to inhibition of the binding interaction of (recombinant) ILT2 in the assay described, for example, in Example 5.

[0051] As used herein, "activating T cell antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, that can induce T cell activation upon interaction with an antibody. Specifically, the interaction of an antibody with an activating T cell antigen can induce T cell activation by triggering a cascade of signal transduction in the T cell receptor complex. In a particular embodiment, the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt No. P07766 (version 189), NCBI RefSeq No. NP_000724.1, SEQ ID NO: 88 for human sequence; or UniProt No. Q95LI5 (version 49), NCBI GenBank No. BAB71849.1, SEQ ID NO: 108 for cynomolgus monkey [Macaca fascicularis] sequence).

[0052] "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. CD3 is an exemplary activating T cell antigen. The term "CD3" encompasses "full-length", unprocessed CD3, and any form of CD3 resulting from processing within the cell. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, in particular the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is set forth in UniProt (www.uniprot.org) Accession No. P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO:88. The amino acid sequence of Macaca fascicularis CD3ε is shown in NCBI GenBank No. BAB71849.1. See also SEQ ID NO:89.

[0053] As used herein, "binds human CD3", "specifically binds human CD3", "binds human CD3" or "anti-CD3" antibodies (either monospecific, multispecific or bispecific) or antigen-binding portions refer to anti-antibodies (either monospecific, multispecific or bispecific) or antigen-binding portions that specifically bind to the human CD3 antigen or its extracellular domain (ECD) and show significant binding / interaction in a surface plasmon resonance assay. In one embodiment, D The value is 5.0 x 10 -8 mol / l or less binding affinity, and in one embodiment, D The value is 1.0×10 -9 mol / l or less, and in one embodiment K D The value is 5.0 x 10 -8 mol / l~1.0×10- -13 In one embodiment, the antibody binds to CD3 comprising SEQ ID NO:88.

[0054] Binding affinity is determined in a standard binding assay, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), using a construct comprising the HLA-G extracellular domain (e.g., its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined in a standard binding assay using an exemplary CD3 comprising SEQ ID NO:88.

[0055] Thus, a multispecific or bispecific antibody that binds to human HLA-G and human CD3 and comprises a first antigen-binding moiety that binds to human HLA-G and a second antigen-binding moiety that binds to human CD3 refers to an antibody that binds to a (first) antigen-binding moiety to human HLA-G as described herein and to another (second) antigen-binding moiety to human CD3 as described herein.

[0056] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

[0057] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0058] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0059] An "antibody fragment" is a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0060] The term "bispecific" means that an antibody is capable of specifically binding to at least two distinct antigenic determinants. Typically, a bispecific antibody comprises two antigen binding sites or moieties, each specific for a different antigenic determinant. In certain embodiments, a bispecific antibody is capable of simultaneously binding to two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

[0061] The term "valency" as used herein refers to the presence of a particular number of antigen-binding sites within an antibody, in which case the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-binding site specific for an antigen within the antibody.

[0062] As used herein, the terms "antigen-binding site" and "antigen-binding portion" are interchangeable and refer to a portion of an antibody, i.e., one or more amino acid residues, that provide interaction with an antigen. For example, an antigen-binding portion of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically comprises two antigen-binding sites, and a Fab molecule typically has one antigen-binding site. "Antigen-binding site" and "antigen-binding portion" refer to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding portion can be directed to a portion (e.g., a second antigen-binding portion) that binds to a target site, e.g., a particular type of tumor cell that bears the antigenic determinant. In another embodiment, an antigen-binding portion can activate signaling through its target antigen, e.g., a T-cell receptor complex antigen. Antigen-binding portions include antibodies and fragments thereof as further defined herein. Certain antigen-binding portions include an antigen-binding domain of an antibody, including an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding portion may comprise an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ. In a preferred embodiment, such constant regions are of human origin.

[0063] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virally infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0064] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0065] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. In a preferred embodiment, such subclasses (isotypes) are of human origin. In a preferred embodiment, the antibody of the invention is of the IgG isotype, and in another preferred embodiment, of the IgG1 isotype.

[0066] In one embodiment, the antibody comprises a constant region of human origin. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region comprising human CH1, CH2, CH3 and / or CL domains, in particular an IgG isotype, more particularly an IgG1 isotype. Exemplary sequences of human constant domains are shown in SEQ ID NOs: 47 and 48 (human kappa CL domain and lambda CL domain, respectively), as well as SEQ ID NO: 49 (human IgG1 heavy chain constant domain CH1-CH2-CH3) or SEQ ID NO: 50 (human IgG1 heavy chain constant region with mutations L234A, L235A and P329G). An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0067] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, an antibody produced by a host cell may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, an antibody produced by a host cell may comprise a full-length heavy chain or may comprise a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc domain (or a subunit of an Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as specified herein, which is comprised in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as specified herein, which is comprised in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). Compositions / formulations of the invention, such as pharmaceutical compositions / formulations described herein, comprise a population of antibodies or bispecific antibodies of the invention. The population of antibodies or bispecific antibodies may include molecules with full-length heavy chains and molecules with truncated mutant heavy chains.The population of antibodies or bispecific antibodies may consist of a mixture of molecules with full-length heavy chains and molecules with truncated variant heavy chains, where at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies or bispecific antibodies have truncated variant heavy chains. In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain comprising a subunit of an Fc domain as specified herein comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain comprising a subunit of an Fc domain as specified herein comprising an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention, such compositions comprise antibodies or bispecific antibody populations comprised of molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein, molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat), molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also supra). A "subunit" of an Fc domain, as used herein, refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, capable of stable self-association.For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain. In a preferred embodiment, such an Fc domain is of human origin, in one preferred embodiment of the IgG isotype, in another preferred embodiment of the IgG1 isotype.

[0068] "Framework" or "FR" refers to variable domain residues other than complement determining region (CDR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences generally occur in the following order in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0069] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0070] By "fused" is meant that the components (e.g., a Fab molecule and an Fc domain subunit) are joined either directly by a peptide bond or via one or more peptide linkers.

[0071] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of an immunoglobulin light chain (a "Fab light chain").

[0072] By "crossover" Fab molecule (also called "Crossfab") is meant a Fab molecule in which the variable and constant domains of the Fab heavy and light chains are exchanged (i.e., replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1, in the N-terminal to C-terminal direction) and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in the N-terminal to C-terminal direction). For the sake of clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0073] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a heavy chain (VH-CH1, N-terminal to C-terminal) composed of a heavy chain variable domain and a constant domain, and a light chain (VL-CL, N-terminal to C-terminal) composed of a light chain variable domain and a constant domain. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the host cell regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for the originally transformed cell are included in the invention.

[0074] A "human antibody" is an antibody having an amino acid sequence that corresponds to an antibody produced by a human or a human cell, or to an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0075] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs (e.g., CDRs) correspond to a non-human antibody and all or substantially all of the FRs correspond to a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0076] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Generally, antibodies contain six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3), 26-32 (CDR-H1), 53-55 (CDR-H2), and 96-101 (CDR-H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (CDR-L1), 46-55 (CDR-L2), 89-96 (CDR-L3), 30-35b (CDR-H1), 47-58 (CDR-H2), and 93-101 (CDR-H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b) and / or (c) comprising CDR amino acid residues 24 to 34 (CDR-L1), 50 to 56 (CDR-L2), 89 to 97 (vL3), 31 to 35 (CDR-H1), 50 to 63 (CDR-H2) and 95 to 102 (CDR-H3).

[0077] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0078] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses, etc.), primates (e.g., humans, non-human primates such as monkeys), rabbits, rodents (e.g., mice, rats, etc.), etc. In certain embodiments, the individual or subject is a human.

[0079] An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In one embodiment, the antibody is an isolated antibody. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

[0080] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes nucleic acid molecules that are contained in a cell that originally contained the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0081] "Isolated nucleic acid encoding an anti-HLA-G antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) are present in one or more locations within a host cell.

[0082] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogenous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation, which variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0083] A "modification that promotes the association of a first subunit and a second subunit of an Fc domain" is a peptide backbone manipulation or post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising an Fc domain subunit with an identical polypeptide to form a homodimer. A modification that promotes association, as used herein, includes a separate modification for each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to be associated, which are complementary to each other so as to promote the association of the two Fc domain subunits. For example, a modification that promotes association may change the structure or charge of one or both of the Fc domain subunits to achieve the desired association sterically or electrostatically, respectively. Thus, (hetero)dimerization may occur between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, and may not be identical in the sense that the further components (e.g., antigen-binding moieties) fused to each subunit are not the same. In some embodiments, a modification that promotes association includes an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In certain embodiments, the modifications that promote association comprise distinct amino acid mutations, in particular amino acid substitutions, in each of the two subunits of the Fc domain. Such modifications that promote association of the first and second subunits of the Fc domain play an important role in the heterodimerization of multispecific or bispecific antibodies (see also, for example, the exemplary multispecific anti-HLA-G / anti-CD3 antibodies in A.2 below).

[0084] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0085] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, which may contain information regarding the indications, use, dosage, administration, concomitant therapy, contraindications and / or warnings pertaining to such therapeutic product.

[0086] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment of sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0087] In situations where ALIGN-2 is used for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0088] The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the formulation will be administered.

[0089] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0090] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, remission or palliation of disease symptoms, and improving recovery or prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to delay the progression of disease.

[0091] A "variable region" or "variable domain" is a domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, for example, Kindt, TJ et al. Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), page 91.) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of an antibody that binds the antigen. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0092] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as autonomously replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." I. Compositions and Methods

[0093] In one aspect, the present invention is partially based on the finding that, surprisingly, among the various variants of HLA-G-0090 in which glycosylation sites have been removed, only two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A no longer show glycosylation at CDR-L1 of the LC (and therefore no Fab glycosylation could be detected), but still show improved binding properties, good expressibility and stability. Since all recently approved pharmaceutical antibody products are produced in mammalian cells, in particular CHO cells (see, for example, Walsh G., Nature Biotech (2018) 1136-1145), providing antibodies (VH and VL, in particular CDRs) without glycosylation sites in the binding region is a valuable advantage, since these antibodies can then be easily used for production in mammalian expression systems without the risk of (at least partially) compromising their binding properties by glycosylation. In particular, for antibodies that contain an Fc domain, production of the antibody in a host cell lacking the glycosylation machinery (such as a prokaryotic host cell) does not yield a product of equivalent quality, since the N-glycan attached to amino acid residue ASN297 (numbering according to EU index as per Kabat) in the Fc region is required to maintain solubility and thermal stability and to prevent aggregation of the antibody in aqueous solution (e.g., in pharmaceutical compositions). A.1 Exemplary Anti-HLA-G Antibodies

[0094] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising: A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) (a) an antibody comprising a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0095] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising: A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or B) An antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7, and a VL domain comprising the amino acid sequence of SEQ ID NO:26.

[0096] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24.

[0097] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24.

[0098] In an embodiment, such anti-HLA-G antibodies have improved binding properties in terms of maximum binding (Rmax) and / or binding affinity (KD) compared to a (parent) antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8 (as shown in Example 2). In a further embodiment, the HLA-G antibody of the invention comprises an Fc domain of human origin, which in one embodiment is of the IgG isotype, in one preferred embodiment of the IgG1 isotype. In one embodiment, such an IgG1 isotype Fc domain of human origin comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG") (numbering according to the Kabat EU index).

[0099] In a further embodiment, the HLA-G antibody of the invention comprises a constant region of human origin comprising human CH1, CH2, CH3 and / or CL domains, in particular a constant region of the IgG isotype, more particularly a constant region of the IgG1 isotype.

[0100] In one embodiment, such a constant region of the IgG1 isotype comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG") (numbering according to the Kabat EU index).

[0101] Such Fc domain-containing antibodies may typically be N-glycosylated at position ASN-297 (numbering according to the Kabat EU index) when produced, for example, in eukaryotic cells such as mammalian cells, in particular CHO cells. N-glycosylation at position ASN-297 (numbering according to the EU index (see Kabat)) is a valuable contribution, for example, to high stability, low aggregation tendency and / or good pharmacokinetic and other important quality properties of such antibodies (e.g., Zheng et al, mAbs (2011) 568-576; and Reusch et al, Glycobiology (2015) 1325-133). Thus, such Fc domain-containing antibodies of the invention are easy to prepare for production in mammalian cells, in particular eukaryotic cells such as CHO cells, without the risk of glycosylation in the binding region (which may interfere with its binding properties), but at the same time with the benefits and beneficial quality properties of N-glycosylation at position ASN-297.

[0102] One embodiment of the present invention is an antibody that binds to human HLA-G, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, and having improved binding properties in terms of maximum binding (Rmax) and / or binding affinity (KD) compared to a (parent) antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8 (as shown in Example 2).

[0103] In embodiments, the anti-HLA-G antibody is a) does not cross-react with a modified human HLA-G β2M MHC I complex, in which the HLA-G specific amino acids are replaced by HLA-A consensus amino acids, and the complex comprises SEQ ID NO: 40; and / or b) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 41; and / or c) does not cross-react with rat RT1A β2M MHC I complex containing SEQ ID NO:43. In embodiments, the anti-HLA-G antibody is a) inhibiting ILT2 binding to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36); or b) binds to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36) and inhibits ILT2 binding to JEG-3 cells (ATCC No. HTB36).

[0104] In another aspect, the present invention relates to multispecific antibodies comprising anti-HLA-G antigen binding moieties. These multispecific antibodies (e.g. bispecific antibodies) described herein use selected and improved anti-HLA-G antibodies as first antigen binding moieties / sites. These anti-HLA-G antibodies bind toHLA-G with high specificity and affinity (improved binding properties, no cross-reactivity with other species and human HLA-A consensus sequences) and have the ability to specifically inhibit the binding of ILT2 and / or ILT4 to HLA-G.

[0105] In one embodiment, the present invention relates to multispecific (preferably bispecific) antibodies binding to human HLA-G and human CD3. In one embodiment, the present invention relates to multispecific (preferably bispecific) anti-HLA-G / anti-CD3 antibodies, wherein the multispecific (preferably bispecific) antibodies binding to human HLA-G and human CD3 comprise a first antigen-binding moiety that binds to human HLA-G and a second antigen-binding moiety that binds to human CD3. This bispecific antibody described herein binds to a second antigen-binding moiety / site specific for CD3, in particular CD3 epsilon, and is therefore able to attract CD3-expressing T cells to HLA-G-expressing tumor cells and at the same time inhibit HLA-G-induced immune suppression in the tumor environment by blocking ILT2 / 4 binding to HLA-G. These bispecific anti-HLA-G / anti-CD3 antibodies therefore show strong tumor growth inhibition and tumor regression in vivo. A.2 Exemplary Multispecific Anti-HLA-G / Anti-CD3 Antibodies

[0106] One embodiment of the present invention is a bispecific antibody that binds to human HLA-G and human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3, The first antigen-binding portion that binds to human HLA-G is A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, a second antigen-binding portion that binds to a T cell activation antigen that binds to human CD3, C) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57; or C) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 64, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 65; or D) A bispecific antibody comprising: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 68, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73.

[0107] Another embodiment of the invention is a bispecific antibody that binds to human HLA-G and human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3; The first antigen-binding portion comprises A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or B) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26; The second antigen-binding portion C) a VH domain comprising the amino acid sequence of SEQ ID NO: 58 and a VL domain comprising the amino acid sequence of SEQ ID NO: 59; or D) a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO: 67; or E) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO: 75.

[0108] Another embodiment of the invention is a bispecific antibody that binds to human HLA-G and human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3; The first antigen-binding portion comprises A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion It comprises a VH domain comprising the amino acid sequence of SEQ ID NO:58, and a VL domain comprising the amino acid sequence of SEQ ID NO:59.

[0109] Another embodiment of the invention is a bispecific antibody that binds to human HLA-G and human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3; The first antigen-binding portion comprises A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion It comprises a VH domain comprising the amino acid sequence of SEQ ID NO:66, and a VL domain comprising the amino acid sequence of SEQ ID NO:67.

[0110] Another embodiment of the invention is a bispecific antibody that binds to human HLA-G and human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3; The first antigen-binding portion comprises A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion It comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO: 75.

[0111] In one embodiment, these bispecific antibodies are characterized by one or more of the following properties: a) induction of T cell-mediated cytotoxicity / tumor cell killing in the presence of HLA-G expressing tumor cells, preferably in the presence of JEG3 cells (ATCC number HTB36), and / or b) induction of IFN-gamma secretion by T cells in the presence of HLA-G expressing tumor cells, preferably JEG3 cells (ATCC number HTB36), and / or c) inhibition of tumor growth in vivo (in a mouse xenograft tumor model); d) in vivo antitumor efficacy / tumor regression in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example 13), and / or e) In vivo antitumor efficacy / tumor of HLA-G CD3 T cell bispecifics in humanized NSG mice bearing human breast cancer PDX tumors (BC004) (see Example 14).

[0112] In one embodiment, these bispecific antibodies are further characterized by one or more of the following properties: a) does not cross-react with a modified human HLA-G β2M MHC I complex, in which the HLA-G specific amino acids are replaced by HLA-A consensus amino acids, and the complex comprises SEQ ID NO: 44; and / or b) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 41; and / or c) does not cross-react with rat RT1A β2M MHC I complex containing SEQ ID NO:43.

[0113] In one embodiment, these bispecific antibodies are further characterized by one or more of the following properties: a) inhibiting ILT2 binding to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36); or b) binds to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36) and inhibits ILT2 binding to JEG-3 cells (ATCC No. HTB36); and / or multispecific antibodies

[0114] A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. In a preferred embodiment, the multispecific antibody provided herein is a bispecific antibody. In certain embodiments, one of the binding specificities is for HLA-G and the other specificity is for CD3. In certain embodiments, a bispecific antibody can bind to two (or more) different epitopes of HLA-G. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0115] Techniques for making multispecific antibodies, and particularly bispecific antibodies, include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using general light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605). al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by the preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0116] Also included herein are engineered antibodies having three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual acting FAbs" or "DAFs" that contain antigen-binding sites that bind to HLA-G and another distinct antigen, or to two different epitopes of HLA-G (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820 and WO 2015 / 095539).

[0117] Multispecific antibodies can also be provided in an asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by swapping VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253) or complete Fab arms (see, WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, WO 2016 / 172485.

[0118] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0119] A particular type of multispecific antibody also included herein is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an active, invariant component of the T cell receptor (TCR) complex, e.g., CD3, for retargeting T cells to kill the target cell. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, in which one of the binding specificities is for HLA-G and the other is for CD3.

[0120] Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules, in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("TandAbs"; Kipriyanov et al., J Mol Biol 1999, 2010 ... 293, 41-56 (1999)); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)); and the so-called triomabs, which are whole mouse / rat IgG hybrid molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Certain T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498. A bispecific antibody that binds to HLA-G and CD3

[0121] The present invention also provides bispecific antibodies, ie, antibodies comprising at least two antigen-binding portions capable of specific binding to two different antigenic determinants (a first antigen and a second antigen).

[0122] Based on the developed anti-HLA-G and anti-CD3 antigen-binding moieties, the present inventors developed a bispecific antibody that binds to both HLA-G and CD3.

[0123] As shown in the Examples, these bispecific antibodies have a number of outstanding properties, including good potency and low toxicity.

[0124] Thus, in certain aspects, the invention provides a bispecific antibody comprising (a) a first antigen-binding moiety that binds human HLA-G and (b) a second antigen-binding moiety that specifically binds human CD3, the bispecific antibody having any of the following characteristics: - the bispecific antibody of the invention specifically induces T cell-mediated killing of cells expressing HLA-G. In some embodiments, the bispecific antibody of the invention specifically induces T cell-mediated killing of cells expressing HLA-G. In more specific embodiments, the bispecific antibody specifically induces T cell-mediated killing of cells expressing HLA-G.

[0125] In one embodiment, the induction of T cell-mediated killing by bispecific antibodies is determined using HLA-G expressing cells.

[0126] In one embodiment, activation of T cells by the bispecific antibody is determined by measuring the expression of CD25 and / or CD69 by T cells after incubation with the bispecific antibody in the presence of HLA-G expressing cells, in particular by flow cytometry. In a specific embodiment, the induction of T cell-mediated killing by a bispecific antibody is determined as follows:

[0127] The ability of anti-HLA-G / anti-CD3 TCB to activate T cells in the presence of HLA-G expressing tumor cells is tested on SKOV3 cells transfected with recombinant HLA-G (SKOV3 HLA-G). T cell activation is assessed by FACS analysis of cell surface activation marker CD25 and early activation marker CD69 on T cells. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from human peripheral blood by density gradient centrifugation using Lymphocyte Separating Medium Tubes (PAN#P04-60125). PBMCs and SKOV3 HLA-G cells are seeded in a 96-well U-bottom plate at a ratio of 10:1. The co-cultures are then incubated with different concentrations of HLA-G-TCB as described in Example 12 and incubated for 24 hours at 37°C in an incubator containing 5% Co2. The next day, CD25 and CD69 expression is measured by flow cytometry.

[0128] For flow cytometry analysis, cells are stained with PerCP-Cy5.5 mouse anti-human CD8 (BD Pharmingen#565310), PE mouse anti-human CD25 (eBioscience#9012-0257) and APC mouse anti-human CD69 (BD Pharmingen#555533) at 4°C. Briefly, antibodies are diluted to 2x concentration and 25μl of antibody dilution is added to each well with 25μl of pre-washed co-culture. Cells are stained for 30 minutes at 4°C, washed twice with 200μl / well staining buffer and centrifuged at 300g for 5 minutes. Cell pellets are resuspended in 200μl staining buffer and stained with DAPI for live / dead discrimination at a final concentration of 2μg / ml. Samples are then measured using a BD LSR flow cytometer. Data analysis is performed using FlowJo V.10.1 software.

[0129] The bispecific antibodies of the invention specifically activate T cells in the presence of cells expressing HLA-G. In some embodiments, the bispecific antibodies of the invention specifically activate T cells in the presence of cells expressing HLA-G. In more specific embodiments, the bispecific antibodies specifically activate T cells in the presence of cells expressing HLA-G.

[0130] In one embodiment, the bispecific antibody induces T cell-mediated killing of T cells or activates T cells in the presence of cells expressing HLA-G. In one embodiment, the bispecific antibody induces T cell-mediated killing and / or activates T cells in the presence of cells expressing HLA-G with an EC50 that is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold lower than the EC50 for induction of T cell-mediated killing in the presence of cells expressing HLA-G or activation of T cells in the presence of cells expressing HLA-G.

[0131] According to certain embodiments of the invention, the antigen-binding portion of the bispecific antibody is a Fab molecule (i.e., an antigen-binding domain composed of a heavy and a light chain, each of which comprises a variable domain and a constant domain). In one embodiment, the first and / or second antigen-binding portion is a Fab molecule. In one embodiment, the Fab molecule is human. In a particular embodiment, the Fab molecule is humanized. In yet another embodiment, the Fab molecule comprises human heavy and light chain constant domains.

[0132] Preferably, at least one of the antigen-binding moieties is a crossover Fab molecule. Such a modification reduces mispairing of the heavy and light chains of different Fab molecules, thereby improving the yield and purity of the bispecific antibodies of the invention in recombinant production. In certain crossover Fab molecules useful for the bispecific antibodies of the invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. However, even with this domain exchange, the preparation of bispecific antibodies may contain certain side products due to the so-called Bence-Jones type interactions between the mispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and thus increase the purity and yield of the desired bispecific antibody, charged amino acids with opposite charges can be introduced at specific amino acid positions in the CH1 and CL domains of either the Fab molecule(s) that bind the first antigen (HLA-G) or the Fab molecule that binds the second antigen, an activating T cell antigen, e.g., CD3, as further described herein. The charge modifications are made either in the conventional Fab molecule (e.g., shown in Figures 13A-C, G-J) comprised in the bispecific antibody or in the VH / VL crossover Fab molecule (e.g., shown in Figures 13D-F, K-N) comprised in the bispecific antibody (but not both). In certain embodiments, the charge modifications are made in the conventional Fab molecule comprised in the bispecific antibody (which in certain aspects binds the first antigen, i.e., HLA-G).

[0133] In a particular embodiment according to the invention, the bispecific antibody is capable of simultaneously binding a first antigen (i.e. HLA-G) and a second antigen (e.g. an activating T cell antigen, in particular CD3). In one embodiment, the bispecific antibody is capable of cross-linking T cells with a target cell by simultaneously binding HLA-G and an activating T cell antigen. In even more particular embodiments, such simultaneous binding results in the lysis of a target cell, in particular an HLA-G expressing tumor cell. In one embodiment, such simultaneous binding results in the activation of the T cell. In another embodiment, such simultaneous binding results in a cellular response of T lymphocytes, in particular cytotoxic T lymphocytes, selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. In one embodiment, binding of the bispecific antibody to an activating T cell antigen, in particular CD3, without simultaneous binding to HLA-G does not result in T cell activation.

[0134] In one embodiment, bispecific antibodies are capable of redirecting the cytotoxic activity of T cells to target cells, in certain embodiments, said redirection being independent of MHC-mediated peptide antigen presentation by the target cells and / or the specificity of the T cells.

[0135] In particular, the T cells according to any of the embodiments of the invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells. A first antigen-binding portion that binds to human HLA-G

[0136] The bispecific antibody of the present invention comprises at least one antigen-binding portion, in particular a Fab molecule, that binds to human HLA-G (first antigen). In certain embodiments, the bispecific antibody comprises two antigen-binding portions, in particular Fab molecules, that bind to human HLA-G. In such certain embodiments, each of these antigen-binding portions binds to the same antigenic determinant. In yet further particular embodiments, all of these antigen-binding portions are identical, i.e., they comprise the same amino acid sequence, including the same amino acid substitutions in the CH1 and CL domains (if present) as described herein. In one embodiment, the bispecific antibody comprises no more than two antigen-binding portions, in particular Fab molecules, that bind to human HLA-G.

[0137] In certain embodiments, the antigen-binding moiety that binds to human HLA-G is a conventional Fab molecule. In such embodiments, the antigen-binding moiety that binds to a second antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced by each other.

[0138] In an alternative embodiment, the antigen-binding portion that binds to human HLA-G is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced by one another. In such an embodiment, the antigen-binding portion that binds to the second antigen is a conventional Fab molecule.

[0139] The HLA-G binding moiety can direct the bispecific antibody to a target site, for example, a particular type of tumor cell that expresses human HLA-G.

[0140] The first antigen-binding portion of the bispecific antibody may incorporate any of the features described herein for antibodies that bind to HLA-G, either alone or in combination, unless this is scientifically clearly unreasonable or impossible.

[0141] Thus, in one aspect, the invention provides a bispecific antibody comprising a first antigen-binding portion that binds to a first antigen, wherein the first antigen is human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 39), and the first antigen-binding portion comprises: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, wherein the VH domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the VL domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0142] A term such as "a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, said VH domain comprising an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7" refers to a VH domain having the amino acid sequence of SEQ ID NO: 7, in which the three CDRs are unchanged (i.e. are the same as SEQ ID NO: 7) but for example 0, 1, 2, 3, 4 or 5 amino acid residues in the framework regions of the VH have been changed / substituted with another amino acid without affecting the binding properties of the VH and the antigen binding site. Framework residues that are likely to affect binding properties are well known (see, for example, Foote J. and Winter G., J. Mol. Biol. (1992) 224, 487-499), and framework residues with no or little effect can be selected for substitution. The same applies to the synonymous terms used herein for different VH or VL.

[0143] In one embodiment, the first antigen-binding portion comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:24.

[0144] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, an HLA-G β2M MHC I complex comprising SEQ ID NO: 39) comprises: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, wherein the VH domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the VL domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 26.

[0145] In one embodiment, the first antigen-binding portion comprises a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:26.

[0146] Such anti-HLA-G antibodies exhibit highly valuable properties since they have no N-glycosylation in the antigen binding site (and CDR-L1) (as shown in Example 2), have improved binding properties in terms of maximum binding (Rmax) and / or binding affinity (KD) compared to a (parent) antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8 (as shown in Example 2), do not cross-react with the HLA-A MHC I complex or with mouse or rat MHC I complexes, bind to (HLA-G expressed on) and inhibit ILT2 binding to JEG-3 cells (ATCC No. HTB36) (HLA-G expressed on). A second antigen-binding moiety that binds to human CD3

[0147] The bispecific antibodies of the invention comprise at least one antigen-binding portion, in particular a Fab molecule, that binds to human CD3.

[0148] In certain embodiments, the antigen-binding portion that binds human CD3 is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced by one another). In such embodiments, the antigen-binding portion that binds human HLA-G is preferably a conventional Fab molecule. In embodiments in which the antigen-binding portion that binds human CD3 contained in a bispecific antibody is present, particularly in embodiments in which more than one Fab molecule is present, the antigen-binding portion that binds human CD3 is preferably a crossover Fab molecule and the antigen-binding portion that binds human HLA-G is a conventional Fab molecule.

[0149] In alternative embodiments, the antigen-binding moiety that binds the second antigen is a conventional Fab molecule. In such embodiments, the antigen-binding moiety that binds the first antigen (i.e., HLA-G) is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced. In embodiments in which there are more than one antigen-binding moiety, particularly Fab molecules, that binds the second antigen contained in the bispecific antibody, the antigen-binding moiety that binds human HLA-G is preferably a crossover Fab molecule and the antigen-binding moiety that binds human CD3 is a conventional Fab molecule.

[0150] In some embodiments, the second antigen is an activating T cell antigen (also referred to herein as "activating T cell antigen binding portion or activating T cell antigen binding Fab molecule"). In certain embodiments, a bispecific antibody comprises no more than one antigen binding portion capable of specific binding to an activating T cell antigen. In one embodiment, a bispecific antibody provides monovalent binding to an activating T cell antigen.

[0151] In certain embodiments, the second antigen is CD3, particularly human CD3 (SEQ ID NO: 88) or cynomolgus CD3 (SEQ ID NO: 89), most particularly human CD3. In one embodiment, the second antigen-binding portion is cross-reactive (i.e., specifically binds) to human and cynomolgus CD3. In some embodiments, the second antigen is the epsilon subunit of CD3 (CD3 epsilon).

[0152] In one embodiment, the second antigen-binding portion that binds human CD3 comprises: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54, wherein the amino acid sequence has at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 58. and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the VL domain comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 59.

[0153] In one embodiment, the second antigen-binding portion that binds human CD3 comprises: (a) a VH domain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62; wherein the amino acid sequence has at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 66. and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 64, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 65, wherein the VL domain comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 67.

[0154] In one embodiment, the second antigen-binding portion that binds human CD3 comprises: (a) a VH domain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 68; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, said CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, said CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, said CDR-H2 comprising the amino acid sequence of SEQ ID NO: 74; 3; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73, wherein the VL domain comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment, 98% or 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 75.

[0155] In one embodiment, the second antigen-binding portion that binds human CD3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO:58 and a VL domain comprising the amino acid sequence of SEQ ID NO:59.

[0156] In one embodiment, the second antigen-binding portion that binds human CD3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO:66 and a VL domain comprising the amino acid sequence of SEQ ID NO:67.

[0157] In one embodiment, the second antigen-binding portion that binds human CD3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO:74 and a VL domain comprising the amino acid sequence of SEQ ID NO:75.

[0158] Such CD3 antigen binding portions / sites exhibit highly advantageous properties (e.g., when provided as bispecific antibodies that bind to CD3 and to HLA-G (with the HLA-G antigen binding portion described herein)). They exhibit the following: a) Good thermal stability b) induction of IFN-gamma secretion by T cells in the presence of HLA-G expressing tumor cells, preferably JEG3 cells (ATCC number HTB36) (Example 11), and / or c) induction of T cell-mediated cytotoxicity / tumor cell killing in the presence of HLA-G expressing tumor cells, preferably in the presence of JEG3 cells (ATCC number HTB36) (Example 12), and / or d) inhibition of tumor growth in vivo (in mouse xenograft tumor models); e) in vivo antitumor efficacy / tumor regression in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example 13), and / or f) In vivo antitumor efficacy / tumor of HLA-G CD3 T cell bispecifics in humanized NSG mice bearing human breast cancer PDX tumors (BC004) (see Example 14).

[0159] In some embodiments, the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, in particular the variable domains VL and VH, are replaced by each other (i.e., according to such embodiments, the second antigen-binding moiety is a crossover Fab molecule in which the variable or constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one such embodiment, the first (and, if present, the third) antigen-binding moiety is a conventional Fab molecule.

[0160] In one embodiment, no more than one antigen-binding moiety that binds to a second antigen (e.g., an activating T cell antigen such as CD3) is present in the bispecific antibody (i.e., the bispecific antibody provides monovalent binding to the second antigen). charge modification

[0161] The bispecific antibodies of the invention may contain amino acid substitutions in the Fab molecules contained therein that are particularly efficient in reducing mispairing of light chains with incompatible heavy chains (Bence Jones by-products) that may occur in the production of Fab-based bispecific antibodies / antibodies with a VH / VL exchange in one (or more in the case of molecules containing more than two antigen-binding Fab molecules) binding arms (see also WO 2015 / 150447, which is incorporated herein by reference in its entirety, in particular the Examples therein). The ratio of the desired bispecific antibody compared to undesired by-products, in particular the Bence Jones by-products that arise in bispecific antibodies with a VH / VL domain exchange in one of the binding arms, may be improved by introducing charged amino acids with an opposite charge to certain amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0162] Thus, in some embodiments, the first and second antigen-binding moieties of the bispecific antibody are both Fab molecules, and in one of the antigen-binding moieties (particularly the second antigen-binding moiety), the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other: i) in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index), or ii) in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index).

[0163] Bispecific antibodies do not contain both modifications described under i) and ii): the constant domains CL and CH1 of the antigen-binding moieties in which VH / VL are replaced are not replaced by each other (i.e. remain unexchanged).

[0164] In a more specific embodiment, i) in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (numbering according to Kabat EU index); or ii) in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0165] In one such embodiment, in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0166] In a further embodiment, in the constant domain CL of the first antigen binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the first antigen binding moiety the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0167] In a particular embodiment, in the constant domain CL of the first antigen binding moiety, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CHI of the first antigen binding moiety, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0168] In a more particular embodiment, in the constant domain CL of the first antigen-binding portion the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding portion the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0169] In an even more particular embodiment, in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted by a lysine (K) (Kabat numbering), the amino acid at position 123 is substituted by an arginine (R) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 is substituted by a glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by a glutamic acid (E) (Kabat EU index numbering).

[0170] In a particular embodiment, when the amino acid substitutions according to the above embodiments are made in the constant domain CL and in the constant domain CH1 of the first antigen binding moiety, the constant domain CL of the first antigen binding moiety is of the kappa isotype.

[0171] Alternatively, the amino acid substitutions of the above embodiments may be made in the constant domain CL and constant domain CH1 of the second antigen binding moiety, rather than in the constant domain CL and constant domain CH1 of the first antigen binding moiety. In certain such embodiments, the constant domain CL of the second antigen binding moiety is of the kappa isotype.

[0172] Thus, in one embodiment, in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0173] In a further embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the second antigen binding moiety the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (numbering according to Kabat EU index).

[0174] In yet another embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0175] In one embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CH1 of the second antigen binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0176] In another embodiment, in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by an arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index).

[0177] In certain embodiments, the bispecific antibodies of the invention comprise: I) a first antigen-binding moiety that binds to human HLA-G, said first antigen-binding moiety being A) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 32;24; B) a Fab molecule comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26; a first antigen-binding portion; and II) a second antigen-binding moiety that binds to human CD3, the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, C) a VH domain comprising the amino acid sequence of SEQ ID NO: 58 and a VL domain comprising the amino acid sequence of SEQ ID NO: 59; or D) a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO: 67; or E) a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO: 75; and a second antigen-binding portion, III) in the constant domain CL of the first antigen-binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (in certain embodiments, independently by lysine (K) or arginine (R)) (numbering according to Kabat); and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (in certain embodiments, independently by lysine (K) or arginine (R)) (numbering according to Kabat); and in the constant domain CHI of the first antigen-binding portion, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). Bispecific antibody formats

[0178] The components of the bispecific antibodies of the invention can be fused to each other in a variety of configurations, exemplary configurations are shown in FIG.

[0179] In certain embodiments, the antigen-binding moieties comprised in the bispecific antibody are Fab molecules, in such embodiments, the first, second, third, etc. antigen-binding moieties may be referred to herein as the first, second, third, etc. Fab molecules, respectively.

[0180] In one embodiment, the first and second antigen-binding moieties of the bispecific antibody are fused to each other, optionally via a peptide linker. In a particular embodiment, the first and second antigen-binding moieties are each Fab molecules. In one such embodiment, the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain. In another such embodiment, the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain. In embodiments in which (i) the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain, the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety may further be fused to each other, optionally via a peptide linker.

[0181] Bispecific antibodies having a single antigen-binding moiety (such as a Fab molecule) capable of specific binding to a target cell antigen, such as HLA-G (e.g., as shown in Figures 13A,D,G,H,K,L), are useful when internalization of the target cell antigen is expected, especially after binding of the high affinity antigen-binding moiety. In such cases, the presence of two or more antigen-binding moieties specific for a target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

[0182] However, in other cases, for example, to optimize targeting to a target site or to allow cross-linking of target cell antigens, it may be advantageous to have a bispecific antibody that comprises two or more antigen-binding portions (e.g., Fab molecules) specific for a target cell antigen (see examples shown in Figures 13B, 13C, 13E, 13F, 13I, 13J, 13M, or 13N).

[0183] Thus, in certain embodiments, a bispecific antibody according to the invention comprises a third antigen-binding moiety.

[0184] In one embodiment, the third antigen-binding moiety binds to the first antigen, i.e., HLA-G. In one embodiment, the third antigen-binding moiety is a Fab molecule.

[0185] In certain embodiments, the third antigen-binding portion is identical to the first antigen-binding portion.

[0186] The third antigen-binding portion of the bispecific antibody may incorporate any of the features described herein for the first antigen-binding portion and / or antibodies that bind HLA-G, either alone or in combination, unless this is scientifically clearly unreasonable or impossible.

[0187] In one embodiment, the third antigen binding moiety is a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24; or a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26; or

[0188] In certain embodiments, the third antigen-binding moiety and the first antigen-binding moiety are each a Fab molecule, and the third antigen-binding moiety is identical to the first antigen-binding moiety. Thus, in these embodiments, the first and third antigen-binding moieties comprise the same heavy and light chain amino acid sequences and have the same domain arrangement (i.e., conventional or crossover). Furthermore, in these embodiments, the third antigen-binding moiety, if present, comprises the same amino acid substitutions as the first antigen-binding moiety. For example, the amino acid substitutions described herein as "charge-modified" are made in the constant domains CL and CH1 of the first and third antigen-binding moieties, respectively. Alternatively, the amino acid substitutions may be made in the constant domains CL and CH1 of the second antigen-binding moiety (which in certain embodiments is also a Fab molecule), rather than in the constant domains CL and CH1 of the first and third antigen-binding moieties.

[0189] As with the first antigen-binding moiety, the third antigen-binding moiety is in particular a conventional Fab molecule. However, embodiments in which the first and third antigen-binding moieties are crossover Fab molecules (and the second antigen-binding moiety is a conventional Fab molecule) are also contemplated. Thus, in certain embodiments, the first and third antigen-binding moieties are each conventional Fab molecules, and the second antigen-binding moiety is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced by each other). In other embodiments, the first and third antigen-binding moieties are each crossover Fab molecules, and the second antigen-binding moiety is a conventional Fab molecule.

[0190] When a third antigen-binding moiety is present, in certain embodiments, the first and third antigen-binding moieties bind to human HLA-G and the second antigen-binding moiety binds to a second antigen, human CD3, most specifically CD3 epsilon.

[0191] In certain embodiments, the bispecific antibody comprises an Fc domain composed of a first and a second subunit, the first and second subunits of the Fc domain being capable of stable association.

[0192] Bispecific antibodies according to the invention can have different structures, i.e. the first, second (and optionally third) antigen-binding moieties may be fused to each other in different manners to the Fc domain. The components may be fused to each other directly or, preferably, via one or more suitable peptide linkers. When the fusion of the Fab molecule is to the N-terminus of the Fc domain subunit, it is typically via the immunoglobulin hinge region.

[0193] In some embodiments, the first and second antigen-binding moieties are each Fab molecules, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such embodiments, the first antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety or to the N-terminus of the other subunit of the Fc domain. In certain such embodiments, the first antigen-binding moiety is a conventional Fab molecule and the second antigen-binding moiety is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced by each other). In other such embodiments, the first Fab molecule is a crossover Fab molecule and the second Fab molecule is a conventional Fab molecule.

[0194] In one embodiment, the first and second antigen-binding moieties are each Fab molecules, the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety. In a specific embodiment, the bispecific antibody consists essentially of a first and a second Fab molecule, an Fc domain composed of the first and the second subunits, and optionally one or more peptide linkers, where the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc domain of the first or the second subunit. Such structures are depicted diagrammatically in Figures 13G and 13K (the second antigen-binding domain in these examples is a VH / VL crossover Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0195] In another embodiment, the first and second antigen-binding moieties are each Fab molecules, and the first and second antigen-binding moieties are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain. In a specific embodiment, the bispecific antibody consists essentially of a first and second Fab molecule, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, and the first and second Fab molecules are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain. Such a structure is depicted diagrammatically in Figures 13A and 13D (in these examples, the second antigen-binding moiety is a VH / VL crossover Fab molecule and the first antigen-binding moiety is a conventional Fab molecule). The first and second Fab molecules may be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the first and second Fab molecules are each fused to an Fc domain by an immunoglobulin hinge region, hi a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain.

[0196] In some embodiments, the first and second antigen-binding moieties are each Fab molecules, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such embodiments, the second antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety or to the N-terminus of the other subunit of the Fc domain (as described above). In certain such embodiments, the first antigen-binding moiety is a conventional Fab molecule and the second antigen-binding moiety is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced by each other). In other such embodiments, the first Fab molecule is a crossover Fab molecule and the second Fab molecule is a conventional Fab molecule.

[0197] In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, the first antigen-binding moiety being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding moiety being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety. In a specific embodiment, the bispecific antibody consists essentially of a first and a second Fab molecule, an Fc domain composed of the first and the second subunits, and optionally one or more peptide linkers, the second Fab molecule being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. Such structures are depicted diagrammatically in Figures 13H and 13L (in these examples, the second antigen-binding moiety is a VH / VL crossover Fab molecule and the first antigen-binding moiety is a conventional Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0198] In some embodiments, the third antigen-binding moiety, in particular the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In certain such embodiments, the first and third Fab molecules are each conventional Fab molecules and the second Fab molecule is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced by each other). In other such embodiments, the first and third Fab molecules are each crossover Fab molecules and the second Fab molecule is a conventional Fab molecule.

[0199] In certain such embodiments, the second and third antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific embodiment, the bispecific antibody consists essentially of first, second and third Fab molecules, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are depicted diagrammatically in Figures 13B and 13E (in these examples, the second antigen-binding moiety is a VH / VL crossover Fab molecule and the first and third antigen-binding moieties are conventional Fab molecules) and in Figures 13J and 13N (in these examples, the second antigen-binding moiety is a conventional Fab molecule and the first and third antigen-binding moieties are VH / VL crossover Fab molecules). The second and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the second and third Fab molecules are each fused to the Fc domain by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0200] In another such embodiment, the first and third antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety. In a specific embodiment, the bispecific antibody consists essentially of first, second and third Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are depicted diagrammatically in Figures 13C and 13F (in these examples, the second antigen-binding moiety is a VH / VL crossover Fab molecule and the first and third antigen-binding moieties are conventional Fab molecules) and in Figures 13I and 13M (in these examples, the second antigen-binding moiety is a conventional Fab molecule and the first and third antigen-binding moieties are VH / VL crossover Fab molecules). The first and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the first and third Fab molecules are fused to the Fc domain, respectively, by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0201] In the structure of a bispecific antibody, where the Fab molecule is fused via an immunoglobulin hinge region to the C-terminus of the Fab heavy chain and to the N-terminus of each of the Fc domain subunits, the two Fab molecules, the hinge region and the Fc domain essentially form an immunoglobulin molecule. In a particular embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In a still further particular embodiment, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In a further particular embodiment, the immunoglobulin is a human immunoglobulin. In another embodiment, the immunoglobulin is a chimeric or humanized immunoglobulin. In one embodiment, the immunoglobulin comprises a human constant region, in particular a human Fc region.

[0202] In some of the bispecific antibodies of the invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the structure of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces mispairing of incompatible Fab heavy and light chains and also reduces the number of plasmids required for the expression of some of the bispecific antibodies of the invention.

[0203] The antigen-binding moieties may be fused to the Fc domain or to each other, either directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) nExamples of the linker peptide include a peptide linker. "n" is generally an integer of 1 to 10, typically 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment, a length of 5 to 100 amino acids, and in a further embodiment, a length of 10 to 50 amino acids. In one embodiment, the linker peptide is (GxS) n or (GxS) n G m where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3), or (x=4, n=2, 3, 4 or 5, m=0, 1, 2 or 3), in one embodiment x=4, n=2 or 3, in a further embodiment x=4, n=2. In one embodiment said peptide linker is (G4S)2. A peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-(G4S)2. Another suitable such linker comprises the sequence (G4S)4. In addition, the linker may comprise (part of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, the fusion may be via an immunoglobulin hinge region or part thereof, with or without an additional peptide linker.

[0204] In certain embodiments, a bispecific antibody according to the invention comprises a Fab light chain variable region of a second Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (2) -CH1 (2) and a polypeptide (VH) in which the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (1) -CH1 (1)In some embodiments, the bispecific antibody comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ), and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0205] In certain embodiments, a bispecific antibody of the invention comprises a Fab heavy chain variable region of a second Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn is bound to a polypeptide that shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CL (2) -CH2-CH3(-CH4)), and a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) In some embodiments, the bispecific antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ), and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0206] In some embodiments, a bispecific antibody comprises a polypeptide in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (2) -CH1 (2) -VH (1) -CH1 (1) In other embodiments, the bispecific antibody comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) -VL (2) -CH1 (2) -CH2-CH3(-CH4)).

[0207] In some of these embodiments, the bispecific antibody comprises a crossover Fab light chain polypeptide (VH) of a second Fab molecule in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ), and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1)In some other of these embodiments, the bispecific antibody further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain constant region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of the first Fab molecule. (2) -CL (2) -VL (1) -CL (1) ), or a polypeptide in which the Fab light chain polypeptide of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of a second Fab molecule (VL (1) -CL (1) -VH (2) -CL (2) ).

[0208] Bispecific antibodies according to these embodiments may comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0209] In some embodiments, a bispecific antibody comprises a Fab heavy chain variable region of a second Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CL (2) -VH (1) -CH1 (1) In other embodiments, the bispecific antibody comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) -VH (2) -CL (2) -CH2-CH3(-CH4)).

[0210] In some of these embodiments, the bispecific antibody comprises a Fab light chain variable region of the second Fab molecule that binds a carboxy-terminal peptide bond to a Fab heavy chain constant region (VL (2) -CH1 (2) ) and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1)In other of these embodiments, the bispecific antibody further comprises a crossover Fab light chain polypeptide of a second Fab molecule, which shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of the first Fab molecule (VL (2) -CH1 (2) -VL (1) -CL (1) ), or a polypeptide in which the Fab light chain polypeptide of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of a second Fab molecule (VL (1) -CL (1) -VH (2) -CL (2) ).

[0211] Bispecific antibodies according to these embodiments may comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0212] In all the different structures of the bispecific antibodies according to the invention, the amino acid substitutions described herein may, if present, be in the CH1 and CL domains of the first and (if present) third antigen-binding moiety / Fab molecule, or in the CH1 or CL domains of the second antigen-binding moiety / Fab molecule. Preferably, they are in the CH1 and CL domains of the first and (if present) third antigen-binding moiety / Fab molecule. According to the concept of the invention, when an amino acid substitution described herein is made in the first (and if present) third antigen-binding moiety / Fab molecule, such an amino acid substitution is not made in the second antigen-binding moiety / Fab molecule. Conversely, when an amino acid substitution described herein is made in the second antigen-binding moiety / Fab molecule, such an amino acid substitution is not made in the first (and if present) third antigen-binding moiety / Fab molecule. The amino acid substitutions are in particular made in bispecific antibodies comprising Fab molecules in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain are replaced by each other.

[0213] In certain embodiments of the bispecific antibodies according to the invention, particularly when the amino acid substitutions described herein are made in the first (and third, if present) antigen binding moiety / Fab molecule, the constant domain CL of the first (and third, if present) Fab molecule is of the kappa isotype. In other embodiments of the bispecific antibodies according to the invention, particularly when the amino acid substitutions described herein are made in the second antigen binding moiety / Fab molecule, the constant domain CL of the second antigen binding moiety / Fab molecule is of the kappa isotype. In some embodiments, the constant domain CL of the first (and third, if present) antigen binding moiety / Fab molecule and the constant domain CL of the second antigen binding moiety / Fab molecule are of the kappa isotype.

[0214] In a particular aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a first antigen-binding moiety and a third antigen-binding moiety that bind to a first antigen, the first antigen being HLA-G, and the first antigen-binding moiety and the second antigen-binding moiety each being a (conventional) Fab molecule comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24; b) a second antigen-binding moiety that binds to a second antigen, the second antigen being CD3, the second antigen-binding moiety being a Fab molecule in which the variable domains VL and VH of the Fab light and heavy chains are replaced with each other, the second antigen-binding moiety comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67; or (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75; c) an Fc domain consisting of a first and a second subunit, In the constant domain CL of the first and third antigen binding moieties in a) the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by a lysine (K) or by an arginine (R), most particularly by an arginine (R) (numbering according to Kabat), in the constant domain CHI of the first and third antigen binding moieties in a) the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index), moreover, The first antigen-binding moiety under a) is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety under b) at the C-terminus of the Fab heavy chain, and the second antigen-binding moiety under b) and the third antigen-binding moiety under a) are each fused to the N-terminus of one of the subunits of the Fc domain under c) at the C-terminus of the Fab heavy chain.

[0215] In one embodiment according to these aspects of the invention, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

[0216] In further embodiments according to these aspects of the invention, the first subunit of the Fc domain additionally has the serine residue at position 354 replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine ​​residue), and the second subunit of the Fc domain additionally has the tyrosine residue at position 349 replaced with a cysteine ​​residue (Y349C) (numbering according to Kabat EU index).

[0217] In further embodiments according to these aspects of the invention, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbering according to the Kabat EU index).

[0218] In further embodiments according to these aspects of the invention, the Fc domain is a human IgG1 Fc domain.

[0219] Specific embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, including polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 76, polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 77, polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 78, and polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 79 (wherein amino acid substitutions are made in the VH or VL framework regions or constant regions without affecting the specific binding properties and the properties of the constant regions of such bispecific antibodies).

[0220] Specific embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, comprising a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 76, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 77, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 78, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 79, The bispecific antibody has one or more of the following properties: Bispecific antibodies exhibit a) inhibition of ILT2 and / or ILT4 binding to HLA-G (see Example 10), and / or b) antibody-mediated IFN-gamma secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV3 HLA-G) and / or on JEG3 cells expressing endogenous HLA-G (IFN-gamma secretion was detected) (by Luminex technology) (see Example 11); and / or c) T cell-mediated cytotoxicity / tumor cell killing against SKOV3 cells transfected with recombinant HLA-G (SKOV 3HLA-G) and / or JEG3 cells expressing endogenous HLA-G (cytotoxicity was detected by measuring caspase 8 activation in cells after treatment with the bispecific antibody) (see Example 12), and / or d) in vivo antitumor efficacy / tumor regression in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example 13), and / or e) In vivo antitumor efficacy / tumor of HLA-G CD3 T cell bispecifics in humanized NSG mice bearing human breast cancer PDX tumors (BC004) (see Example 14).

[0221] In more specific embodiments, the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:76, a polypeptide comprising the amino acid sequence of SEQ ID NO:77, a polypeptide comprising the amino acid sequence of SEQ ID NO:78, and a polypeptide comprising the amino acid sequence of SEQ ID NO:79.

[0222] Further specific embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, including polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 80, polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 81, polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 82, and polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 83 (wherein amino acid substitutions are made in the VH or VL framework regions or constant regions without affecting the specific binding properties and the properties of the constant regions of such bispecific antibodies).

[0223] Specific embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, comprising a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 80, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 81, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 82, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 83, The bispecific antibody has one or more of the following properties: Bispecific antibodies exhibit a) inhibition of ILT2 and / or ILT4 binding to HLA-G (see Example 10), and / or b) antibody-mediated IFN-gamma secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV3 HLA-G) and / or on JEG3 cells expressing endogenous HLA-G (IFN-gamma secretion was detected) (by Luminex technology) (see Example 11); and / or c) T cell-mediated cytotoxicity / tumor cell killing against SKOV3 cells transfected with recombinant HLA-G (SKOV 3HLA-G) and / or JEG3 cells expressing endogenous HLA-G (cytotoxicity was detected by measuring caspase 8 activation in cells after treatment with the bispecific antibody) (see Example 12), and / or d) in vivo antitumor efficacy / tumor regression in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example 13), and / or e) In vivo antitumor efficacy / tumor of HLA-G CD3 T cell bispecifics in humanized NSG mice bearing human breast cancer PDX tumors (BC004) (see Example 14).

[0224] In more specific embodiments, the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:80, a polypeptide comprising the amino acid sequence of SEQ ID NO:81, a polypeptide comprising the amino acid sequence of SEQ ID NO:82, and a polypeptide comprising the amino acid sequence of SEQ ID NO:83.

[0225] Further specific embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, including polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 84, polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 85, polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 86, and polypeptides comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 87 (wherein amino acid substitutions are made in the VH or VL framework regions or constant regions without affecting the specific binding properties and the properties of the constant regions of such bispecific antibodies).

[0226] Specific embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, comprising a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 84, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 85, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 86, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 87, The bispecific antibody has one or more of the following properties: Bispecific antibodies are: a) inhibition of ILT2 and / or ILT4 binding to HLA-G (see Example 10), and / or b) antibody-mediated IFN-gamma secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV3 HLA-G) and / or on JEG3 cells expressing endogenous HLA-G (IFN-gamma secretion was detected) (by Luminex technology) (see Example 11); and / or c) T cell-mediated cytotoxicity / tumor cell killing against SKOV3 cells transfected with recombinant HLA-G (SKOV 3HLA-G) and / or JEG3 cells expressing endogenous HLA-G (cytotoxicity was detected by measuring caspase 8 activation in cells after treatment with the bispecific antibody) (see Example 12), and / or d) in vivo antitumor efficacy / tumor regression in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (see Example 13), and / or e) In vivo antitumor efficacy / tumor of HLA-G CD3 T cell bispecifics in humanized NSG mice bearing human breast cancer PDX tumors (BC004) (see Example 14).

[0227] In more specific embodiments, the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:84, a polypeptide comprising the amino acid sequence of SEQ ID NO:85, a polypeptide comprising the amino acid sequence of SEQ ID NO:86, and a polypeptide comprising the amino acid sequence of SEQ ID NO:87. Fc domain

[0228] In a particular embodiment, the bispecific antibody of the invention comprises an Fc domain composed of a first and a second subunit, it being understood that the features of the Fc domain described herein in relation to the bispecific antibody, except for the modifications related to Fc heterodimerization, can be equally applied to the Fc domain comprised in the monospecific anti-HLAG antibody of the invention.

[0229] The Fc domain of a bispecific antibody consists of a pair of polypeptide chains that contain the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each subunit containing the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, a bispecific antibody of the invention does not contain more than one Fc domain.

[0230] In one embodiment, the Fc domain of the bispecific antibody is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU index numbering). This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgG1 Fc domain. Fc domain modifications that promote heterodimerization

[0231] The bispecific antibodies according to the invention comprise different antigen-binding moieties that can be fused to one or the other of the two subunits of the Fc domain, which are thus typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of bispecific antibodies in recombinant production, it is advantageous to introduce modifications in the Fc domain of the bispecific antibody that promote the association of the desired polypeptides.

[0232] Thus, in a particular embodiment, the Fc domain of the bispecific antibody according to the invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The longest site of protein-protein interaction between the two subunits of a human IgG Fc domain is within the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0233] There are several approaches to modifications in the CH3 domain of the Fc domain to enhance heterodimerization, which are fully described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner such that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself but heterodimerize with the other complementarily engineered CH3 domain (the first and second CH3 domains heterodimerize and no homodimers are formed between the two first or two second CH3 domains). These different approaches for improved heavy chain heterodimerization are considered as different alternatives in combination with heavy-light chain modifications in bispecific antibodies that reduce heavy / light chain mispairing and Bence-Jones type by-products (e.g., swapping / replacement of VH and VL in one binding arm and introduction of substitutions of charged amino acids with opposite charges at the CH1 / CL interface).

[0234] In a specific embodiment, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which comprises a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0235] Knobs-into-holes technology is described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Complementary cavities of identical or similar size to the protrusion are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0236] Thus, in certain embodiments, in the CH3 domain of a first subunit of the Fc domain of a bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit which can be repositioned in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be repositioned.

[0237] Preferably, the amino acid residue having a larger side chain mass is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0238] Preferably, the amino acid residue having a smaller side chain mass is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0239] The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example by site-directed mutagenesis or by peptide synthesis.

[0240] In a specific embodiment, in the (CH3 domain of) the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the (CH3 domain of) the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0241] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the replacement of the serine residue at position 354 with a cysteine ​​residue), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine ​​residues leads to the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0242] In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

[0243] In certain embodiments, an antigen binding moiety that binds a second antigen (e.g., an activating T cell antigen) is fused (optionally via a first antigen binding moiety that binds HLA-G and / or a peptide linker) to a first subunit of an Fc domain (comprising a "knob" modification). Without wishing to be bound by theory, the fusion of an antigen binding moiety that binds a second antigen (e.g., an activating T cell antigen) to a knob-containing subunit of an Fc domain (further) minimizes the generation of antibodies that comprise two antigen binding moieties that bind to an activating T cell antigen (steric clash of the two knob-containing polypeptides).

[0244] Other techniques for CH3 modifications to enhance heterodimerization are contemplated as alternatives according to the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1 870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0245] In one embodiment, the heterodimerization approach described in EP 1870459 is used instead. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment of the bispecific antibody of the invention is the amino acid mutations R409D, K370E in one of the two CH3 domains (of the Fc domain) and D399K, E357K in the other CH3 domain of the Fc domain (numbering according to Kabat EU index).

[0246] In another embodiment, a bispecific antibody of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to Kabat EU index).

[0247] In another embodiment, a bispecific antibody of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said bispecific antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain and additionally the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbering according to Kabat EU index).

[0248] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to Kabat EU index). In a further embodiment, the first CH3 domain comprises the further amino acid mutation L351K. In a further embodiment, the second CH3 domain comprises a further amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numbering according to Kabat EU index).

[0249] In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises further amino acid mutations at positions T411, D399, S400, F405, N390 or K392, such as (a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, (b) D399R, D399W, D399Y or D In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutations Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

[0250] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is used instead, e.g., with amino acid modifications at positions selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0251] In one embodiment, the heterodimerization approach described in WO2011 / 090762 is used instead, which also uses the knob-in-hole technique described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to Kabat EU index).

[0252] In one embodiment, the bispecific antibody or its Fc domain is of the IgG2 subclass and the heterodimerization approach described in WO 2010 / 129304 is alternatively used.

[0253] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering effects, e.g., as described in PCT Publication WO 2009 / 089004. Generally, this method involves the replacement of one or more amino acid residues at the interface of the two Fc domain subunits with a charged amino acid residue such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution at K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D) and the second CH3 domain comprises an amino acid substitution at D399, E356, D356 or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution at K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain further comprises, or alternatively, an amino acid substitution at K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (all numbering according to the Kabat EU index).

[0254] In yet further embodiments, the heterodimerization approach described in WO 2007 / 147901 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbering according to the Kabat EU index).

[0255] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 may alternatively be used.

[0256] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to Kabat EU index). Fc domain modifications that reduce Fc receptor binding and / or effector function

[0257] The Fc domain confers favorable pharmacokinetic properties to the bispecific antibody (or antibody), including a long serum half-life that contributes to good accumulation in target tissues, and favorable tissue-blood distribution ratios. However, it may at the same time result in undesirable targeting of the (bispecific) antibody (or antibody) to cells expressing Fc receptors, rather than to the preferred antigen-bearing cells. Furthermore, simultaneous activation of Fc receptor signaling pathways may result in cytokine secretion / release, which, combined with the T cell activation properties (e.g., in bispecific antibody embodiments where the second antigen-binding moiety binds to an activating T cell antigen) and the long half-life of the bispecific antibody, may result in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of immune cells (bearing Fc receptors) other than T cells may even reduce the effectiveness of the bispecific antibody (especially bispecific antibodies where the second antigen-binding moiety binds to an activating T cell antigen), due to potential destruction of T cells, e.g., by NK cells.

[0258] Thus, in a particular embodiment, the Fc domain of the bispecific antibody according to the invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. In one such embodiment, the Fc domain (or a bispecific antibody comprising said Fc domain) exhibits less than 50%, in particular less than 20%, more particularly less than 10%, especially less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain) and / or exhibits less than 50%, in particular less than 20%, more particularly less than 10%, especially less than 5% of the effector function compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or a bispecific antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a bispecific antibody comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, more particularly more than about 90% of the binding affinity to FcRn of a native IgG1 Fc domain (or a bispecific antibody comprising a native IgG1 Fc domain).

[0259] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain of the bispecific antibody comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, a combination of these amino acid mutations may reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, a bispecific antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor compared to a bispecific antibody comprising a non-engineered Fc domain. In a particular embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, and most particularly human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity to the complementary components, particularly to C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to the receptor, is achieved when the Fc domain (or a bispecific antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the non-engineered form of the Fc domain (or a bispecific antibody comprising a non-engineered form of the Fc domain) for FcRn. The Fc domain or a bispecific antibody of the invention comprising the Fc domain may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of the bispecific antibody is engineered to have reduced effector function compared to the non-engineered Fc domain. The reduced effector function may include, but is not limited to, one or more of reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a bispecific antibody comprising a non-engineered Fc domain).

[0260] In one embodiment, the amino acid mutation that reduces the binding affinity and / or effector function of the Fc domain to an Fc receptor is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbering according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to Kabat EU index). In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG").Specifically, in certain embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) in each of the first and second subunits of the Fc domain (Kabat EU index numbering).

[0261] In one such embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (as well as complement) binding of the human IgG1 Fc domain, as described in WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector functions.

[0262] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions compared to IgG1 antibodies. Thus, in some embodiments, the Fc domain of the (bispecific) antibody of the invention is an IgG4 Fc domain, in particular a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, in particular the amino acid substitution S228P (numbering according to Kabat EU index). To further reduce the binding affinity to the Fc receptor and / or its effector functions, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E (numbering according to Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, in particular the amino acid substitution P329G (numbering according to Kabat EU index). In a particular embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically the amino acid substitutions S228P, L235E and P329G (numbering according to Kabat EU index). Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in WO 2012 / 130831, which is incorporated herein by reference in its entirety.

[0263] In certain embodiments, the Fc domain that exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numbering according to Kabat EU index).

[0264] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by screening.

[0265] Binding to an Fc receptor can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare) and Fc receptors that can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain or a (bispecific) antibody comprising an Fc domain for an Fc receptor may be assessed using a cell line known to express a particular Fc receptor, for example human NK cells expressing the FcγIIIa receptor.

[0266] The effector function of an Fc domain or an Fc domain-containing (bispecific) antibody can be measured by methods known in the art. Examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96 (登録商標)Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0267] In some embodiments, binding of the Fc domain to its complement components, particularly to C1q, is reduced. Thus, in some embodiments in which the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. To determine whether an Fc domain or a bispecific antibody comprising an Fc domain is capable of binding C1q and thus has CDC activity, a C1q binding assay may be performed. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0268] Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0269] In a further aspect, an anti-HLA-G antibody according to any of the above embodiments may incorporate any of the features described in paragraphs 1 to 6 below, either alone or in combination. 1. Antibody affinity

[0270] In certain embodiments, the antibodies provided herein have a potency of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant KD of 0.01 M.

[0271] In a preferred embodiment, the KD is determined using a BIACORE™ assay at 25° C. using an immobilized antigen CM5 chip of approximately 10 response units (RU). (登録商標) The kinetics of the Fab is measured using a surface plasmon resonance assay using a 100-μg / ml ELISA kit. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of coupled protein. Following injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of the Fab (0.78 nM to 500 nM) are injected into 0.05% polysorbate 20 (TWEEN-20) and incubated at 4°C for 1 h. (商標) ) in PBS containing surfactant (PBST) at a flow rate of approximately 25 μl / min at 25° C. Association rates (k or ka) and dissociation rates (k or k) were determined by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE (登録商標)The equilibrium dissociation constant, KD, is calculated as the ratio kd / ka (koff / kon). See, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881. If the on-rate exceeds 106 M-1 s-1 by the surface plasmon resonance assay described above, the on-rate is measured using a spectrophotometer equipped with a stop-flow (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirred cuvette. (商標) It can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band pass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen as measured with a spectrometer such as a spectrophotometer (ThermoSpectronic). 2. Antibody fragment

[0272] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315, and WO 93 / 16185, as well as U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments which contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0273] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 0 404 097, WO 1993 / 01161, Hudson, PJ et al., Nat.Med.9 (2003) 129-134, and Holliger, P. et al., Proc.Natl.Acad.Sci.USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat.Med.9 (20039 129-134).

[0274] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0275] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein. 3. Chimeric and humanized antibodies

[0276] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison, SLet al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from those of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0277] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the CDRs, e.g., the CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0278] Humanized antibodies and methods for making them are reviewed, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and in, for example, Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describing SDR (a-CDR) grafting); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing"); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); as well as Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing a "guided selection" approach to FR shuffling).

[0279] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims, MJ et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, LGet al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening of FR libraries (see, e.g., Baca, M. et al., J. Immunol. 151 (1993) 2623-2632). al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (19969 22611-22618). 4. Human antibodies

[0280] In certain embodiments, the antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in the art.Human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr.Opin.Pharmacol.5(2001)368-374 and Lonberg, N., Curr.Opin.Immunol.20(2008)450-459.

[0281] Human antibodies may be prepared by administering an immunogen to an intact human antibody or a transgenic animal that has been modified to produce intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. See also, for example, XENOMOUSE. (商標) See also U.S. Patent Nos. 6,075,181 and 6,150,584, which describe HuMab® technology; 5,770,429, which describes HuMab® technology; 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with a different human constant region.

[0282] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, for example, Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95). Human antibodies produced via human B-cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Further methods include, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, J., Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20 (2005) 927-937 and Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

[0283] Human antibodies can also be made by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below. 5. Library-derived Antibodies

[0284] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom, HR et al., Methods in Molecular Biology 178 (2001) 1-37 and can be found, for example, in McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks, JD et al., J. Mol. Biol. 222 (1992) 581-597; Marks, JD and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, SSet al., J. Mol. Biol. 338 (2004) 299-310; Lee, CV et al. al., J. Mol. Biol. 340 (2004) 1073-1093; Fellowes, FA, Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, CV et al., J. Immunol. Methods 284 (2004) 119-132.

[0285] In one particular phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described by Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455. Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self antigens and also self antigens, as described by Griffiths, AD et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro as described in Hoogenboom, H R and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0286] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments. 6. Antibody Variants

[0287] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics (e.g., antigen binding). a) Substitution, insertion and deletion mutants

[0288] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs (in preferred embodiments, framework residues not involved in the binding properties of the antibody (see, e.g., Foote J. and Winter G., J. Mol. Biol. (1992) 224, 487-499)). Exemplary changes are provided in Table 1 under the heading of "Exemplary Substitutions" and as further described below with reference to amino acid side chain classes. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions". Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. Table 1 JPEG2025037987000003.jpg178170

[0289] Amino acids can be classified according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0290] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0291] One type of substitution variant involves substituting one or more CDRs of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing have modified (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained specific biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage are screened for a specific biological activity (e.g., binding affinity).

[0292] To improve antibody affinity, modifications (e.g., substitutions) may be made, for example, in the CDRs. Such modifications can be made in CDR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196), and / or in the SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction of a secondary library and reselection from the secondary library is described, for example, in Hoogenboom, HR et al., Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. Another method of introducing diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0293] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may be outside the CDR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unaltered or does not contain one or more, two or more, or three or more amino acid substitutions.

[0294] A useful method for identifying antibody residues or regions that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085. In this method, a residue or target group of residues (e.g., charged residues, e.g., arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. Mutants may be screened to determine whether they have the desired properties.

[0295] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. b) Fc region mutants

[0296] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid alteration (e.g., substitution) at one or more amino acid positions.

[0297] Antibodies with reduced effector function include those containing one or more substitutions at residues 238, 265, 269, 270, 297, 327 and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). Certain antibody variants have been described with improved or diminished binding to FcRs. (See, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields, R Let al., J. Biol. Chem. 276 (2001) 6591-6604.)

[0298] In one embodiment of the invention, such an antibody is an IgG1 with the mutations L234A and L235A, or with the mutations L234A, L235A and P329G, or in another embodiment, an IgG4 (numbering according to the EU index in Kabat et al, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) with the mutations S228P and L235E, or S228P, L235E or P329G.

[0299] Antibodies with increased half-life and improved binding to neonatal Fc receptors are responsible for the transfer of mature IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593 and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434) and are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include variants having a substitution at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0300] For other examples of Fc region variants, see also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351. c) Cysteine ​​Engineered Antibody Variants

[0301] In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with a cysteine ​​residue. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other sites, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541. d) Antibody derivative

[0302] In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one polymer is attached, the polymers can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0303] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, N. Wet al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells, but which heat the non-proteinaceous moiety to a temperature that kills cells proximal to the antibody non-proteinaceous moiety. B. Recombinant Methods and Compositions

[0304] The antibodies may be produced, for example, using recombinant methods and compositions described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-HLA-G antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence constituting the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a nucleic acid encoding an amino acid sequence comprising the VH of the antibody, or (2) a vector comprising a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In a preferred embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, a HEK293 cell or a lymphocytic cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-HLA-G antibody or a bispecific antibody is provided, comprising culturing a host cell comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and, optionally, recovering the antibody from the host cell (or host cell culture).

[0305] For recombinant production of anti-HLA-G antibodies, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0306] The host cells most suitable for cloning or expression of vectors encoding the antibodies described herein are eukaryotic cells, preferably mammalian cells.

[0307] Vertebrate cells may also be used as hosts, for example mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney lines (e.g., 293 cells or 293 cells as described in Graham et al., J Gen Virol 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (e.g., Mather et al., Annals NYAcad.Sci.383(1982)44-68, MRC 5 cells and FS4 cells. The most useful mammalian host cell lines include DHFR -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0308] To some extent, prokaryotic cells can also be used, but sometimes higher efforts and more complicated procedures are possible. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237, U.S. Patent No. 5,789,199 and U.S. Patent No. 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibodies of the present invention may be isolated in a soluble fraction from the bacterial cell paste or may be further purified.

[0309] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). (商標) See, for example, US Pat. No. 6,233,363, which is incorporated herein by reference in its entirety C. Assay

[0310] The anti-HLA-G antibodies provided herein may be identified, screened or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. 1. Binding and other assays

[0311] In one embodiment, the antibodies of the invention are tested for their antigen binding activity by known methods, such as, for example, ELISA, Western blot, etc. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris, GE (ed.), Epitope Mapping Protocols, In: Methods in Molecular Biology, Vol. 66, Humana Press, Totowa, NJ (1996). 2. Activity Assay

[0312] In one embodiment, an assay is provided to identify anti-HLA-G antibodies that have biological activity. Biological activity may include, for example, the ability to enhance the activation and / or proliferation of different immune cells, including T cells. For example, they enhance the secretion of immunomodulatory cytokines, such as interferon-gamma (IFN-gamma) and / or tumor necrosis factor alpha (TNFalpha). Other immunomodulatory cytokines that are or can be enhanced are, for example, IL1β, IL6, IL12, granzyme B, etc., that bind to different cell types. Antibodies that have such biological activity in vivo and / or in vitro are also provided.

[0313] In certain embodiments, antibodies of the invention are tested for such biological activities, for example as described in the Examples below. D. Methods and Compositions for Diagnostics and Detection

[0314] In certain embodiments, any of the anti-HLA-G antibodies provided herein are useful for detecting the presence of HLA-G in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as immune cells, or T cell infiltrates, and / or tumor cells.

[0315] In one embodiment, an anti-HLA-G antibody is provided for use in a method of diagnosis or detection. In a further aspect, a method for detecting the presence of HLA-G in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-HLA-G antibody as described herein under conditions that allow binding of the anti-HLA-G antibody to HLA-G, and detecting whether a complex is formed between the anti-HLA-G antibody and HLA-G. Such a method may be an in vitro method or an in vivo method. In one embodiment, for example, where HLA-G is a biomarker for patient selection, the anti-HLA-G antibody is used to select subjects eligible for therapy with an anti-HLA-G antibody.

[0316] In certain embodiments, a labeled anti-HLA-G antibody is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, radioactive labels, etc.) and indirectly detectable moieties (e.g., enzymes or ligands, etc.) via enzymatic reactions or molecular interactions. Exemplary labels include: radioisotopes. 32 P, 14 C. 125 I, 3 H and 131I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like. E. Pharmaceutical Preparations

[0317] Pharmaceutical formulations of the anti-HLA-G antibodies or anti-HLA-G / anti-CD3 bispecific antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies having the desired purity with one or more any pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine. Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein include soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., rHuPH20 (HYLENEX). (登録商標)The sHASEGPs may further include an intervening drug dispersing agent, such as a human soluble PH-20 hyaluronidase glycoprotein, such as PH-20 hyaluronidase glycoprotein (E.G., Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0318] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine acetate buffer.

[0319] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to provide additional. Such active ingredients are suitably present in combination in amounts effective for the purpose intended.

[0320] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

[0321] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0322] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes. F. Treatment Methods and Compositions

[0323] Any of the anti-HLA-G antibodies or anti-HLA-G / anti-CD3 bispecific antibodies provided herein can be used in therapeutic methods.

[0324] In one aspect, an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody is provided for use as a medicament. In a further aspect, an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody is provided for use in the treatment of cancer. In certain embodiments, an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody is provided for use in a method of treatment. In certain embodiments, the invention provides an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of an anti-HLA-G / anti-CD3 bispecific antibody.

[0325] In a further embodiment, the invention provides an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody for use as an immunomodulatory agent, for directly or indirectly inducing the proliferation and / or activation of immune cells (such as T cells, B cells, and myeloid cells including monocytes, macrophages, dendritic cells, plasmacytoid dendritic cells), e.g. by secretion of immunostimulatory cytokines such as TNF alpha (TNFa) and IFN gamma (IFNg), or by recruitment of further immune cells. In certain embodiments, the invention provides an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of immunomodulation / method of directly or indirectly inducing immune cell proliferation, e.g. activation by secretion of immunostimulatory cytokines such as TNFα and IFN-gamma, or recruitment of further immune cells in an individual comprising administering to the individual an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody effective to directly or indirectly induce immune cell proliferation, e.g. activation by secretion of immunostimulatory cytokines such as TNFα and IFN-gamma, or recruitment of further immune cells.

[0326] In further embodiments, the invention provides anti-HLA-G antibodies or anti-HLA-G / anti-CD3 bispecific antibodies for use as immunostimulants and / or for stimulating tumor necrosis factor alpha (TNFalpha) secretion. In certain embodiments, the invention provides anti-HLA-G antibodies or anti-HLA-G / anti-CD3 bispecific antibodies for use in an immunomodulatory method for directly or indirectly inducing immune cell proliferation, e.g. activation by secretion of immunostimulatory cytokines such as TNFa and IFNg, or further recruitment of immune cells in an individual comprising administering to the individual an anti-HLA-G antibody or anti-HLA-G / anti-CD3 bispecific antibody effective to directly or indirectly induce immune cell proliferation, e.g. activation by secretion of immunostimulatory cytokines such as TNFa and IFNg, or further recruitment of immune cells.

[0327] The term "cancer" as used herein includes, for example, lung cancer, non-small cell lung (NSCL) cancer, lung bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, and the like. cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia (including refractory aspects of any of the above cancers), or a combination of one or more of the above cancers.

[0328] The "individual" according to any of the above embodiments is preferably a human. In a further aspect, the invention provides the use of an anti-HLA-G antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer. In a further embodiment, the medicament is for use in a method of treating cancer, the method comprising administering an effective amount of the medicament to an individual having cancer. In a further embodiment, the medicament is for inducing cell-mediated lysis of cancer cells. In a further embodiment, the medicament is for use in a method of inducing cell-mediated lysis of cancer cells in an individual suffering from cancer, the method comprising administering to the individual an amount of the medicament effective to induce apoptosis in the cancer cells / or inhibit cancer cell proliferation. The "individual" according to any of the above embodiments may be a human.

[0329] In a further aspect, the invention provides a method for treating cancer. In one embodiment, the method comprises administering an effective amount of an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody to an individual having cancer. The "individual" according to any of the above embodiments may be a human.

[0330] In a further aspect, the invention provides a method of inducing cell-mediated lysis of cancer cells in an individual suffering from cancer. In one embodiment, the method comprises administering to the individual an effective amount of an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody to induce cell-mediated lysis of cancer cells in the individual suffering from cancer. In one embodiment, the "individual" is a human.

[0331] In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-HLA-G antibodies or anti-HLA-G / anti-CD3 bispecific antibodies provided herein, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-HLA-G antibodies or anti-HLA-G / anti-CD3 bispecific antibodies provided herein and a pharma- ceutically acceptable carrier.

[0332] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, by injection, for example, intravenous or subcutaneous, depending in part on whether administration is temporary or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0333] The antibodies of the invention will be formulated, administered, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. Effective amounts of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages as those described herein, or about 1-99% of the dosages described herein, or at any dosages and by any route of administration that are empirically / clinically determined to be appropriate.

[0334] For the prevention or treatment of disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody of the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg to 10 mg / kg) of the antibody can be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. In repeated administrations over several days or more, depending on the condition, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives about 2 to about 20, or, for example, about 6 doses of the antibody). An initial larger dose may be administered, followed by one or more smaller doses. An exemplary dosing regimen includes administering an initial loading dose of about 4 mg / kg, followed by weekly maintenance doses of the antibody of about 2 mg / kg. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0335] It will be understood that any of the above formulations or methods of treatment may be practiced using an immunoconjugate of the invention in place of, or in addition to, an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody.

[0336] It will be understood that any of the above formulations or methods of treatment may be practiced using an immunoconjugate of the invention in place of, or in addition to, an anti-HLA-G antibody or an anti-HLA-G / anti-CD3 bispecific antibody. II. Manufactured articles

[0337] In another aspect of the invention, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective to treat, prevent, and / or diagnose a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture includes (a) a first container containing a composition, the composition comprising an antibody of the invention, and (b) a second container containing a composition, the composition further comprising a cytopathic or other therapeutic agent. The article of manufacture, in this embodiment of the invention, may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc.

[0338] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Explanation of amino acid sequence Anti-HLA-G antibody / antigen-binding portion (sequence numbers of variable regions and complement determining regions (CDRs)): SEQ ID NO: 1 Heavy chain CDR-H1, HLA-G-0090 SEQ ID NO:2 Heavy chain CDR-H2, HLA-G-0090 SEQ ID NO:3 Heavy chain CDR-H3, HLA-G-0090 SEQ ID NO: 4 Light chain CDR-L1, HLA-G-0090 SEQ ID NO:5 Light chain CDR-L2, HLA-G-0090 SEQ ID NO:6 Light chain CDR-L3, HLA-G-0090 SEQ ID NO: 7 Heavy chain variable domain VH, HLA-G-0090 SEQ ID NO:8 Light chain variable domain VL, HLA-G-0090 SEQ ID NO: 9 Light chain CDR-L1, HLA-G-0090-VL-N31D SEQ ID NO: 10 Light chain variable domain VL, HLA-G-0090-VL-N31D SEQ ID NO: 11 Light chain CDR-L1, HLA-G-0090-VL-N31L SEQ ID NO: 12 Light chain variable domain VL, HLA-G-0090-VL-N31L SEQ ID NO: 13 Light chain CDR-L1, HLA-G-0090-VL-N31Q SEQ ID NO: 14 Light chain variable domain VL, HLA-G-0090-VL-N31Q SEQ ID NO: 15 Light chain CDR-L1, HLA-G-0090-VL-N31S SEQ ID NO: 16 Light chain variable domain VL, HLA-G-0090-VL-N31S SEQ ID NO: 17 Light chain CDR-L1, HLA-G-0090-VL-N31T SEQ ID NO: 18 Light chain variable domain VL, HLA-G-0090-VL-N31T SEQ ID NO: 19 Light chain CDR-L1, HLA-G-0090-VL-N31Y SEQ ID NO: 20 Light chain variable domain VL, HLA-G-0090-VL-N31Y SEQ ID NO: 21 Light chain CDR-L1, HLA-G-0090-VL-N31Y-N38Y SEQ ID NO: 22 Light chain variable domain VL, HLA-G-0090-VL-N31Y-N38Y SEQ ID NO: 23 Light chain CDR-L1, HLA-G-0090-VL-S32P SEQ ID NO: 24 Light chain variable domain VL, HLA-G-0090-VL-S32P SEQ ID NO: 25 Light chain CDR-L1, HLA-G-0090-VL-S33A SEQ ID NO: 26 Light chain variable domain VL, HLA-G-0090-VL-S33A SEQ ID NO: 27 Light chain CDR-L1, HLA-G-0090-VL-S33D SEQ ID NO: 28 Light chain variable domain VL, HLA-G-0090-VL-S33D SEQ ID NO: 29 Light chain CDR-L1, HLA-G-0090-VL-S33P SEQ ID NO: 30 Light chain variable domain VL, HLA-G-0090-VL-S33P Further sequences SEQ ID NO: 31 Exemplary human HLA-G SEQ ID NO: 32 Exemplary human HLA-G extracellular domain (ECD) SEQ ID NO: 33 Exemplary human β2M SEQ ID NO: 34 Modified human HLA-G (HLA-G specific amino acids are replaced by HLA-A consensus amino acids (=degrafted HLA-G also see FIG. 1) ECD) SEQ ID NO: 35 Exemplary human HLA-A2 SEQ ID NO: 36 Exemplary human HLA-A2 ECD SEQ ID NO:37 Exemplary mouse H2Kd ECD SEQ ID NO:38 Exemplary rat RT1A ECD SEQ ID NO: 39 Exemplary human HLA-G β2M MHC class I complex SEQ ID NO: 40 Exemplary modified human HLA-G β2M MHC class I complex (HLA-G specific amino acids are replaced by HLA-A consensus amino acids (=degrafted HLA-G), see also FIG. 2) SEQ ID NO: 41 Exemplary mouse H2Kd β2M MHC class I complex SEQ ID NO: 42 An exemplary human HLA-G / mouse H2Kd β2M MHC class I complex in which positions specific for human HLA-G are grafted onto the mouse H2Kd framework SEQ ID NO: 43 Exemplary rat RT1A β2M MHC class I complex SEQ ID NO: 44 An exemplary human HLA-G / rat RT1A β2M MHC class I complex in which positions specific for human HLA-G are grafted onto the rat RT1A framework SEQ ID NO: 45 Linker and his-Tag SEQ ID NO: 46 Peptide SEQ ID NO: 47 Human kappa light chain constant region SEQ ID NO: 48 Human lambda light chain constant region SEQ ID NO: 49 Human heavy chain constant region from IgG1 SEQ ID NO: 50 Human heavy chain constant region from IgG1 with the mutations L234A, L235A and P329G SEQ ID NO: 51 Human heavy chain constant region from IgG4 Anti-CD3 antibody / antigen-binding portion (variable region and complementarity determining region (CDR)): SEQ ID NO: 52 Heavy chain CDR-H1, P035-093 (abbreviated as P035) SEQ ID NO:53 Heavy chain CDR-H2, P035-093 SEQ ID NO:54 Heavy chain CDR-H3, P035-093 SEQ ID NO: 55 Light chain CDR-L1, P035-093 SEQ ID NO: 56 Light chain CDR-L2, P035-093 SEQ ID NO: 57 Light chain CDR-L3, P035-093 SEQ ID NO: 58 Heavy chain variable domain VH, P035-093 SEQ ID NO: 59 Light chain variable domain VL, P035-093 SEQ ID NO: 60 Heavy chain CDR-H1, clone 22 (abbreviated as Cl22) SEQ ID NO: 61 Heavy chain CDR-H2, clone 22 SEQ ID NO: 62 Heavy chain CDR-H3, clone 22 SEQ ID NO: 63 Light chain CDR-L1, clone 22 SEQ ID NO: 64 Light chain CDR-L2, clone 22 SEQ ID NO: 65 Light chain CDR-L3, clone 22 SEQ ID NO: 66 Heavy chain variable domain VH, clone 22 SEQ ID NO: 67 Light chain variable domain VL, clone 22 SEQ ID NO: 68 Heavy chain CDR-H1, V9 SEQ ID NO: 69 Heavy chain CDR-H2, V9 SEQ ID NO: 70 Heavy chain CDR-H3, V9 SEQ ID NO: 71 Light chain CDR-L1, V9 SEQ ID NO: 72 Light chain CDR-L2, V9 SEQ ID NO: 73 Light chain CDR-L3, V9 SEQ ID NO: 74 Heavy chain variable domain VH, V9 SEQ ID NO: 75 Light chain variable domain VL, V9 Bispecific anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibody: P1AF7977(HLA-G-0090-VL-S32P / CD3 P035-093(P035)): SEQ ID NO: 76 Light Chain 1 P1AF7977 SEQ ID NO: 77 Light chain 2 P1AF7977 SEQ ID NO: 78 Heavy Chain 1 P1AF7977 SEQ ID NO: 79 Heavy Chain 2 P1AF7977 P1AF7978(HLA-G-0090-VL-S32P / CD3 Clone 22(Cl22)): SEQ ID NO: 80 Light chain 1 P1AF7978 SEQ ID NO: 81 Light chain 2 P1AF7978 SEQ ID NO: 82 Heavy Chain 1 P1AF7978 SEQ ID NO: 83 Heavy Chain 2 P1AF7978 P1AF7979(HLA-G-0090-VL-S32P / CD3 V9): SEQ ID NO: 84 Light Chain 1 P1AF7979 SEQ ID NO: 85 Light chain 2 P1AF7979 SEQ ID NO: 86 Heavy Chain 1 P1AF7979 SEQ ID NO: 87 Heavy Chain 2 P1AF7979 Further sequences SEQ ID NO:88 Exemplary human CD3 SEQ ID NO: 89 Exemplary cynomolgus CD3 SEQ ID NO: 90 Human CD3 epsilon stalk-Fc (knob)-Avi SEQ ID NO: 91 Human CD3 delta stalk-Fc(whole)-Avi SEQ ID NO:92 CD3 orig VH SEQ ID NO:93 CD3 orig VL SEQ ID NO:94 CD3 orig IgG HC SEQ ID NO: 95 P035 IgG HC SEQ ID NO:96 CD3 orig / P035 IgG LC Amino acid sequence of HLA-G-0090 antibody (variable region (bold) with underlined complementarity determining regions (CDRs) and unmodified N-glycosylation site of CDR-L1): SEQ ID NO: 7: Heavy chain variable domain VH, HLA-G-0090: QVQLQQSPGGLLKPSQTLSLTCAISGDSVS SNRAAWN WIRQSPSRGLEWLG RTYYRSKWYNDYAVSVQG RITLIPDTSKNQFSLRLNSVTPEDTAVYYCAS VRAVAPFDY WGQGVLVTVSS JPEG2025037987000004.jpg255170 Amino acid sequence of the anti-CD3 binding portion (variable region with underlined complementarity determining regions (CDRs)): SEQ ID NO: 58 Heavy chain variable domain VH, P035-093 (abbreviated as P035) EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMNW VRQAPGKGLEWVS RIRSKYNNYATYYADSVK GRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR ASNFPASYVSYFAY WGQGTLVTVSS SEQ ID NO: 59 Light chain variable domain VL, P035-093 (P035) QAVVTQEPSLTVSPGGTVTLTC GSSTGAVTTSNYAN WVQEKPGQAFRGLIG GTNKRAP GTPARFSGSLLGGKAALTLSGAQPEDEAEYYC ALWYSNLWV FGGGTKLTVL SEQ ID NO: 66 Heavy chain variable domain VH, clone 22 (abbreviated as Cl22) EVQLLESGGGLVQPGGSLRLSCAASGFQFS SYAMN WVRQAPGKGLEWVS RIRSKYNNYATYYADSVKG RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HTTFPSSYVSYYGY WGQGTLVTVSS SEQ ID NO: 67 Light chain variable domain VL, clone 22 (Cl22) QAVVTQEPSLTVSPGGTVTLTC GSSTGAVTTSNYAN WVQEKPGQAFRGLIG GTNKRAP GTPARFSGSLLGGKAALTLSGAQPEDEAEYYC ALWYSNLWV FGGGTKLTVL SEQ ID NO: 74 Heavy chain variable domain VH, V9 EVQLVESGGGLVQPGGSLRLSCAASGYSFT GYTMN WVRQAPGKGLEWVA LINPYKGVSTYNQKFKD RFTISVDKSKNTAYLQMNSLRAEDTAVYYCAR SGYYGDSDWYFDV WGQGTLVTVSS SEQ ID NO: 75 Light chain variable domain VL, V9 DIQMTQSPSSLSASVGDRVTITC RASQDIRNYLN WYQQKPGKAPKLLIY YTSRLES GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPWT FGQGTKVEIK Amino acid sequence of bispecific anti-HLA-G / anti-CD3 T cell bispecific (TCB) antibody: P1AF7977(HLA-G-0090-VL-S32P / CD3 P035-093(P035)): SEQ ID NO: 76 Light Chain 1 P1AF7977 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRASNFPASYVSYFAYWG QGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 77 Light chain 2 P1AF7977 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKV EIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 78 Heavy Chain 1 P1AF7977 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWG QGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPI EKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP SEQ ID NO: 79 Heavy Chain 2 P1AF7977 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGG GTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP P1AF7978(HLA-G-0090-VL-S32P / CD3 Clone 22(Cl22)): SEQ ID NO: 80 Light chain 1 P1AF7978 EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWG QGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 81 Light chain 2 P1AF7978 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKV EIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 82 Heavy Chain 1 P1AF7978 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWG QGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPI EKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP SEQ ID NO: 83 Heavy Chain 2 P1AF7978 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGG GTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP P1AF7979(HLA-G-0090-VL-S32P / CD3 V9): SEQ ID NO: 84 Light Chain 1 P1AF7979 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQ GTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 85 Light chain 2 P1AF7979 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKV EIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 86 Heavy Chain 1 P1AF7979 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWG QGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPI EKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP SEQ ID NO: 87 Heavy Chain 2 P1AF7979 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQG TKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0339] Specific embodiments of the present invention are listed below. 1. An antibody that binds to human HLA-G, A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) An antibody comprising: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0340] 2.Antibody, A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or B) The antibody of embodiment 1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7, and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0341] 3. The antibody of embodiment 1 or 2, wherein the antibody comprises an Fc domain of human origin, in particular of the IgG isotype, more particularly of the IgG1 isotype.

[0342] 4. An antibody according to embodiment 1 or 2, wherein the antibody comprises a constant region of human origin, in particular of the IgG isotype, more particularly of the IgG1 isotype, comprising human CH1, CH2, CH3 and / or CL domains.

[0343] 5.Antibody, a) have improved binding properties in terms of maximum binding (Rmax) and / or binding affinity (KD) compared to a (parent) antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8 (as shown in Example 2), b) does not cross-react with a modified human HLA-G β2M MHC I complex, in which the HLA-G specific amino acids are replaced by HLA-A consensus amino acids, and the complex comprises SEQ ID NO: 40 (as shown in Example 2); and / or c) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 41 (as shown in Example 2); and / or d) An antibody according to any one of embodiments 1 to 4, which does not cross-react with the rat RT1A β2M MHC I complex comprising SEQ ID NO: 43 (as shown in Example 2).

[0344] 6.Antibodies a) inhibiting ILT2 binding to JEG3 cells (ATCC No. HTB36) (as shown in Example 5); or b) An antibody according to any one of embodiments 1 to 3, which binds to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36) and inhibits ILT2 binding to JEG-3 cells (ATCC No. HTB36) (as shown in Example 5).

[0345] 7. The antibody according to any one of embodiments 1 to 4, wherein the antibody is a multispecific antibody (preferably a bispecific antibody).

[0346] 8. The antibody of embodiment 7, wherein the antibody is a bispecific antibody that binds to human HLA-G and human CD3.

[0347] 9. The antibody is a bispecific antibody that binds to human HLA-G and human CD3, and comprises a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3; The first antigen-binding portion that binds to human HLA-G is A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, a second antigen-binding portion that binds to a T cell activation antigen that binds to human CD3, C) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57; or D) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 64, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 65, or E) The antibody of embodiment 7, comprising: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 68, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73.

[0348] 10. The first antigen-binding portion comprises: A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or B) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26; The second antigen-binding portion C) a VH domain comprising the amino acid sequence of SEQ ID NO: 58 and a VL domain comprising the amino acid sequence of SEQ ID NO: 59; or D) a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO: 67; or E) A bispecific antibody according to embodiment 9, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO: 75.

[0349] 11. The first antigen-binding portion comprises: A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion 11. The bispecific antibody of embodiment 10, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 58, and a VL domain comprising the amino acid sequence of SEQ ID NO: 59.

[0350] 12. The first antigen-binding portion comprises: A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion 109. The bispecific antibody of embodiment 108, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 66, and a VL domain comprising the amino acid sequence of SEQ ID NO: 67.

[0351] 13. The first antigen-binding portion comprises: A VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, The second antigen-binding portion 11. The bispecific antibody of embodiment 10, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO: 75.

[0352] 14. Bispecific antibodies exhibit the following: a) inhibition of ILT2 and / or ILT4 binding to HLA-G (as shown in Example 13), and / or b) antibody-mediated IFN-gamma secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV3 HLA-G) and / or on JEG3 cells expressing endogenous HLA-G (IFN-gamma secretion was detected) (by Luminex technology) (as shown in Example 14); and / or c) T cell-mediated cytotoxicity / tumor cell killing against SKOV3 cells transfected with recombinant HLA-G (SKOV 3HLA-G) and / or JEG3 cells expressing endogenous HLA-G (cytotoxicity was detected by measuring caspase 8 activation in cells after treatment with the bispecific antibody) (shown in Example 15); and / or d) in vivo antitumor efficacy / tumor regression in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice (as shown in Example 16); and / or e) A bispecific antibody according to any one of embodiments 8 to 13, showing in vivo antitumor efficacy of HLA-G CD3 T cell bispecific / tumor in humanized NSG mice bearing human breast cancer PDX tumors (BC004) (as shown in Example 17).

[0353] 15. The bispecific antibody of any one of embodiments 9 to 14, wherein the first and second antigen-binding portions are Fab molecules.

[0354] 16. The bispecific antibody according to any one of embodiments 9 to 15, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, in particular the variable domains VL and VH, are replaced by each other.

[0355] 17. The bispecific antibody according to any one of embodiments 9 to 16, wherein the first antigen-binding portion is a Fab molecule, and wherein in the constant domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CH1 the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0356] 18. The bispecific antibody of any one of embodiments 9 to 17, comprising a third antigen-binding portion, which is identical to the first antigen-binding portion.

[0357] 19. The bispecific antibody of any one of embodiments 9 to 18, comprising an Fc domain consisting of the first and second subunits.

[0358] 20. The first antigen-binding moiety, the second antigen-binding moiety, and, if present, the third antigen-binding moiety are each a Fab molecule; (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, 20. The bispecific antibody of embodiment 19, wherein the third antigen-binding portion, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0359] 21. The bispecific antibody according to embodiment 19 or 20, wherein the Fc domain is a human IgG Fc domain, in particular of the IgG1 isotype.

[0360] 22. The bispecific antibody of embodiment 19 or 20, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function.

[0361] 23. The bispecific antibody according to embodiment 22, wherein the antibody is of IgG1 isotype with the mutations L234A, L235A and P329G (numbering according to EU index as per Kabat).

[0362] 24. A bispecific antibody according to any one of embodiments 19 to 23, in which amino acid residues in the CH3 domain of a first subunit of the Fc domain are replaced by amino acid residues having a larger side chain mass, thereby generating a protrusion in the CH3 domain of the first subunit which can be positioned in a cavity in the CH3 domain of the second subunit, and in which amino acid residues in the CH3 domain of a second subunit of the Fc domain are replaced by amino acid residues having a smaller side chain mass, thereby generating a cavity in the CH3 domain of the second subunit, into which the protrusion in the CH3 domain of the first subunit can be positioned.

[0363] 25. The bispecific antibody according to embodiment 24, wherein the antibody is of IgG1 isotype (numbering according to Kabat EU index) with the mutation T366W in the first subunit of the Fc domain and with the mutations Y407V, T366S and L368A in the second subunit of the Fc domain.

[0364] 26. The bispecific antibody according to embodiment 25, wherein the antibody comprises the additional mutation S354C in the first subunit of the Fc domain and the additional mutation Y349C in the second subunit of the Fc domain (numbering according to Kabat EU index).

[0365] 27. The bispecific antibody according to embodiment 25, wherein the antibody comprises an additional mutation Y349C in the first subunit of the Fc domain and an additional mutation S354C in the second subunit of the Fc domain (numbering according to Kabat EU index).

[0366] 28. An isolated nucleic acid encoding an antibody according to any one of embodiments 1 to 4 or a bispecific antibody according to any one of embodiments 9 to 27.

[0367] 29. A host cell, preferably a eukaryotic host cell, comprising a nucleic acid according to embodiment 28.

[0368] 30. A method for producing an antibody according to any one of embodiments 1 to 4, or a bispecific antibody according to any one of embodiments 9 to 227, comprising culturing a host cell according to embodiment 29 to produce the antibody or bispecific antibody.

[0369] 31. The method of embodiment 30, further comprising recovering the antibody or bispecific antibody from the host cell.

[0370] 32. The antibody according to any one of embodiments 1 to 4 or the bispecific antibody according to any one of embodiments 9 to 27, wherein the antibody is produced according to the method according to embodiment 30 or 31, and the host cell is a eukaryotic host cell (in a preferred embodiment, a mammalian host cell, in another preferred embodiment, a CHO cell).

[0371] 33. The antibody according to any one of embodiments 1 to 4 or the bispecific antibody according to any one of embodiments 9 to 27, wherein the antibody is produced in a eukaryotic host cell (in a preferred embodiment, a mammalian host cell, in another preferred embodiment, a CHO cell).

[0372] 34. An antibody according to any one of embodiments 1 to 4 or a bispecific antibody according to any one of embodiments 9 to 27 for use as a medicament.

[0373] 35. An antibody according to any one of embodiments 1 to 4 or a bispecific antibody according to any one of embodiments 9 to 27 for use in the treatment of cancer.

[0374] 36. Use of an antibody according to any one of embodiments 1 to 4 or a bispecific antibody according to any one of embodiments 9 to 27 in the manufacture of a medicament.

[0375] 37. The use according to embodiment 36, wherein the medicament is for the treatment of cancer.

[0376] 38. A method for treating an individual having cancer, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 4 or a bispecific antibody according to any one of embodiments 9 to 27. EXAMPLES

[0377] Recombinant DNA Technology Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. Gene and Oligonucleotide Synthesis

[0378] The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or by PCR. The respective oligonucleotides were prepared by Metabion GmbH (Planeg-Martinsried, Germany). Description of basic / standard mammalian expression plasmids For expression of a desired gene / protein (e.g., a full-length antibody heavy chain, a full-length antibody light chain, or an MHC class I molecule, such as HLA-G, or an MHC class I molecule fused to a peptide and beta2 microglobulin, such as HLA-G binding peptide and / or HLA-G fused to beta2 microglobulin), a transcription unit is used that contains the following functional elements: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) including intron A, - human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - the gene / protein to be expressed (e.g. a full-length antibody heavy chain or an MHC class I molecule), and - bovine growth hormone polyadenylation sequence (BGH pA).

[0379] In addition to the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid is - the origin of replication from the vector pUC18 allowing replication of this plasmid in E. coli, and -Contains the beta-lactamase gene that confers ampicillin resistance to E. coli. Protein determination

[0380] The protein concentration of the purified polypeptide was determined by determining the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence of the purified polypeptide. Example 1 Preparation of HLA-G chimeric molecules

[0381] Due to its high homology with other MHC I molecules (>98%), immunization with HLA-G molecules leads to the production of polyclonal sera composed of a mixture of MHC-I cross-reactive antibodies as well as truly HLA-G specific antibodies.

[0382] So far, no means have been provided to select truly HLA-G specific antibodies without cross-reactivity to other human MHC-I (e.g. HLA-A) and further select those with receptor blocking function.

[0383] The inventors have identified unique HLA-G positions in combination with positions required for structural compatibility and receptor interaction (ILT2 / 4 and KIR2DL4).

[0384] The unique proximal position of human HLA-G was then "grafted" onto MHC class I complex molecules from different rodent species (such as rat RT1A and mouse H2kd) to generate "chimeric" immunogens / screening antigens.

[0385] The antibodies generated were subjected to stringent screening / testing for binding / specificity (no cross-reactivity / no specificity to the counter antigen, respectively).

[0386] Antigens for binding studies: -HLA-G expressed as a human HLA-G β2M MHC complex containing recSEQ ID NO: 43 - HLA-G specific sequences grafted onto rat RT-1 and mouse H2kd (SEQ ID NO: 46: human HLA-G / mouse H2Kd β2M MHC class I complex with positions specific for human HLA-G grafted onto the mouse H2Kd framework and SEQ ID NO: 48: human HLA-G / rat RT1A β2M MHC class I complex with positions specific for human HLA-G grafted onto the rat RT1A framework) - natural HLA-G MHC class I complex expressing cells (e.g. Jeg3 cells) or human HLA-G transfected cell lines SKOV3 HLA-G+ and PA-TU-8902 HLA-G+ Counter antigens for cross-reactivity testing: - Paired antigens (MHC class I complexes) with other HLA-A sequences (HLA-A2 and HLA-G degrafted with HLA-A consensus sequence) in combination with different peptides (see e.g. SEQ ID NO: 35 (HLA-A2) and SEQ ID NO: 40 HLA-A consensus sequence on the HLA-G framework) - Counter antigens (MHC class I complex) from other species such as rat RT-1 and mouse H2kd (SEQ ID NO: 43 and SEQ ID NO: 41) - The non-modified tumor cell lines SKOV3 and PA-TU-8902, characterized by the absence of HLA-G expression. Design of chimeric HLA-G antigens to determine specific binding of anti-HLA-G antibodies (see FIG. 2): Design of a chimeric rat MHC I molecule (RT1-A) carrying the HLA-G unique position (SEQ ID NO: 44) for use in binding assays: HLA-G unique positions were identified by alignment of 2579 HLA-A, 3283 HLA-B, 2133 HLA-C, 15 HLA-E, 22 HLA-F and 50 HLA-G sequences from IMGT (as of Feb. 6, 2014). Residues in HLA-G that are present in less than 1% (mostly about 0%) of the sequences in any of the three sequence sets HLA-A, HLA-B, and the combined set of HLA-C+HLA-E+HLA-F are referred to as HLA-G unique positions.

[0387] Four core HLA-G unique positions (two in alpha-1 and two in alpha-3) show no polymorphism in the set of HLA-G sequences, and none of the other HLA genes contain HLA-G specific residues at these positions (except 1xHLA-A for M100, 1xHLA-B for Q103, and 1xHLA-C for Q103).

[0388] The crystal structure of human HLA-G (Clements, CSet al. PROC. NATL. ACAD. SCI. USA 102:3360-3365 (2005); PDB code: 1YDP) was superimposed on the crystal structure of rat RT1-A (Rudolph, MGet al. J. Mol. Biol. 324:975-990 (2002); PDB code: 1KJM). The overall structure of the alpha chain and the associated beta2-microglobulin are conserved.

[0389] HLA-G unique positions were identified in the RT1-A structure by comparison of sequence and structural alignment. In a first step, unique HLA-G positions that are exposed on the molecular surface of HLA-G and RT1-A and therefore accessible to antibodies were identified. Unique positions that are buried within the protein fold were excluded for engineering. In a second step, structurally proximal residues were identified that also needed to be exchanged to make the corresponding region "HLA-G-like", i.e. to generate real HLA-G epitopes that include unique positions, rather than generating HLA-G / rat RT1-A chimeric epitopes that would be artificial. All positions thus selected for mutation were analyzed for the structural fit of the respective residues from HLA-G to avoid possible local disturbances of the molecular structure upon mutation.

[0390] A chimeric mouse MHC I molecule (H2Kd) was similarly generated containing the HLA-G unique position (SEQ ID NO: 42) for use in binding assays. Design of HLA-A based counter antigens by "degrafting" HLA-G specific positions to the HLA-A consensus sequence for cross-reactivity testing (SEQ ID NO: 40 = modified human HLA-G β2M MHC class I complex (HLA-G specific amino acids are replaced by HLA-A consensus amino acids (=degrafted HLA-G))

[0391] Unique positions derived from a multiple sequence alignment were analyzed in the crystal structure of human HLA-G (PDB code: 1YDP). First, positions that are not exposed on the HLA-G surface and therefore inaccessible to antibodies were excluded for manipulation. Second, surface-exposed residues were analyzed for feasibility of amino acid exchange (i.e., elimination of possible local disturbances of the molecular structure upon mutation of the relevant position). In total, 14 positions were validated for exchange. Amino acids at valid positions were mutated against the HLA-A consensus sequence derived from a multiple sequence alignment of 2579 HLA-A sequences downloaded from IMGT (as of February 6, 2014). Construction of expression plasmids for soluble classical and non-classical MHC class I molecules

[0392] The recombinant MHC class I genes encode an N-terminal extension fusion molecule consisting of the respective MHC class I molecule, beta2 microglobulin, and a peptide known to be bound by the respective MHC class I molecule.

[0393] The expression plasmid for the transient expression of soluble MHC class I molecules contained, in addition to the soluble MHC class I molecule, an origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and a beta-lactamase gene conferring ampicillin resistance in E. coli. The transcription unit for the soluble MHC class I molecule contained the following functional elements: - the immediate early enhancer and promoter from human cytomegalovirus (P-CMV) including intron A, - human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - an N-terminal truncated S. aureus sortase A encoding nucleic acid, and - bovine growth hormone polyadenylation sequence (BGH pA).

[0394] The amino acid sequences of mature soluble MHC class I molecules from various species are as follows: SEQ ID NO: 39: Exemplary human HLA-G β2M MHC class I complex SEQ ID NO: 40: Exemplary modified human HLA-G β2M MHC class I complex (HLA-G specific amino acids have been replaced by HLA consensus amino acids (=degrafted HLA-G, see also FIG. 2) SEQ ID NO: 41: Exemplary mouse H2Kd β2M MHC class I complex SEQ ID NO: 42: An exemplary human HLA-G / mouse H2Kd β2M MHC complex in which positions specific for human HLA-G are grafted onto the mouse H2Kd framework. SEQ ID NO: 43: Exemplary rat RT1A β2M MHC class I complex SEQ ID NO: 44: An exemplary human HLA-G / rat RT1A β2M MHC complex in which positions specific for human HLA-G are grafted onto the rat RT1A framework

[0395] For an exemplary HLA-A2 β2M MHC class I complex, the following components were used and the complex was expressed in E. Coli and purified. MHCI complex HLA-A2 / b2M (SEQ ID NO: 35 and 33) (both with an additional N-terminal methionine) + VLDFAPPGA peptide (SEQ ID NO: 46) + linker and his-Tag (SEQ ID NO: 45) Example 2 Removal of N-glycosylation motifs in CDR-L1

[0396] The CDR-L1 of the anti-HLA-G antibody HLA-G-0090 contains a classical N-glycosylation motif "NSS" that includes positions 31 to 33 of the light chain (LC). It was decided to remove this motif, as it could constitute a liability for potential developability. A homology model of the variable region of HLA-G-0090 showed that LC positions 31-33 are highly solvent accessible. Furthermore, the side chains of N31 and S32 are predicted to point inwards towards the CDR-H3, making them likely to be part of the antibody paratope. Indeed, several published antibody-antigen X-ray complex structures have demonstrated that these residues undergo chemical interactions with the antigen. Therefore, it was considered that there was a high risk that the binding affinity of the antibody would be worsened by introducing mutations in LC positions 31-33. To ameliorate the risk, eleven different variants of the antibody HLA-G-0090 with mutations in LC positions 31, 32 and 33 were designed and produced in HEK293F cells in IgG1 format.

[0397] It should be noted that the mutation variant HLA-G-0090-VL-N31Y-N38Y contains a second mutation (N38Y) apart from the N-glycosylation motif, not associated with its removal, increasing the germline identity.

[0398] Summary of anti-HLA-G antibody sequences (variable region and CDR sequence numbers): JPEG2025037987000005.jpg163170

[0399] The binding and other properties, as well as the biological activity, of the resulting anti-HLA-G specific antibodies were determined as described in the Examples below, and compared to the known reference, HLA-G-0090. Expression and purification

[0400] The expression yields from 0.5 L expression in HEK293F cells after purification by affinity chromatography (MabSelect Sure) and dialysis are shown in the table below. JPEG2025037987000006.jpg162170

[0401] Mutants with mutations involving position LC31 (N31X) showed significantly reduced expression titers and often poor material quality, whereas mutants at positions LC32 and LC33 showed good to acceptable expression titers and good material quality. HLA-G binding wt-HLA-G affinity / kinetics

[0402] The affinity of individual 5 nM anti-HLA-G antibodies to the wt-HLA-G complex (SEQ ID NO: 39) was determined by injecting wt-HLA-G antigen captured with anti-hFc (GE Healthcare BR-1008-39) on a CM3 sensor chip and diluted in HBS-P+ (GE Healthcare) running buffer at concentrations from 11 nM to 300 nM and a flow rate of 60 μl / min with an association time of 120 s and a dissociation time of 600 s. After each cycle, the surface was regenerated by washing with 3 M MgCl2. Kinetic binding curves were evaluated using T200 evaluation software and a 1:1 Langmuir binding model was used to calculate the binding properties.

[0403] RAC (relative activity concentration) of HLAG antibodies (assay scheme shown in Figure 3).

[0404] The RAC of HLA-G binders of individual anti-HLA-G antibodies (10 nM solution in HBS-P+) was determined by capturing with anti-hFc (GE Healthcare BR-1008-39) on a CM3 sensor chip and injecting wt-HLA-G antigen at a concentration of 300 nM diluted in HBS-P+ (GE Healthcare) running buffer and at a flow rate of 10 μl / min with an association time of 60 s and a dissociation time of 600 s. After each cycle, the surface was regenerated by washing with 3 M MgCl2. The final RAC and Rmax values ​​are calculated from the "bound" reporting points and capture levels. Rmax = (MW analyte / MW ligand) x RU captured ligand * stoichiometry of interaction.

[0405] The eleven mutants were evaluated in SPR binding experiments in which the antibody was immobilized via the Fc portion on a CM3 chip and recombinant single chain HLA-G monomer / human HLA-G β2M MHC class I complex (SEQ ID NO: 39) was used as the analyte. Kinetic parameters were determined by single cycle kinetic measurements at 25° C. on a Biacore T200 instrument. JPEG2025037987000007.jpg155170

[0406] Of these, four variants (HLA-G-0090-VL-S32P, HLA-G-0090-VL-S33A, HLA-G-0090-VL-S33D, HLA-G-0090-VL-S33P) with acceptable expression titers were further evaluated with more accurate multi-cycle kinetic measurements using the same SPR instrument and experimental setup. JPEG2025037987000008.jpg70170

[0407] The two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A have improved binding affinity compared to the parent antibody HLA-G-0090, whereas the two variants HLA-G-0090-VL-S33D and HLA-G-0090-VL-S33P lose 5-fold and 2-fold binding affinity, respectively. Surprisingly, for the variants tested, removal of the N-glycosylation motif leads to higher Rmax values ​​in kinetic measurements, indicating a higher rate of successful complex formation than with N-glycosylated antibodies. Cross-reactivity of anti-HLA-G antibodies and mutants to soluble human HLA-G, soluble degrafted human HLA-G with HLA-A consensus-specific sequences, and rat / mouse homologs

[0408] To further investigate the binding properties of the two binding affinity-improved variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A, SPR binding experiments were performed on four counter-screening constructs. These recombinant single-chain peptide-MHC complex constructs were mouse H2-K1 (SEQ ID NO: 41), rat RT1 (SEQ ID NO: 43), and human HLA-G β2M MHC class I complexes in which HLA-G specific amino acids were replaced by HLA-A consensus amino acids (SEQ ID NO: 40). The latter construct constitutes a version of HLA-G in which all HLA-G specific residues have been replaced by their HLA-A consensus counterparts. Again, the antibodies were immobilized on a CM3 chip and the single-chain peptide-MHC constructs were used as analytes on a Biacore T200 instrument at 25°C. JPEG2025037987000009.jpg116170 Stability under stress The parent antibody and the two derived variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A were stressed for 13 days under two different conditions: pH 6.0 20mM His / HisCl, 140mM NaCl; at 40°C (His 40°C) pH 7.4 PBS; at 37°C (PBS 37°C). The material was then analyzed using SEC and SPR to investigate chemical degradation and possible effects on target binding. For reference, the stressed material was compared to material maintained under storage conditions: ·pH6.0 20mM His / HisCl, 140mM NaCl; Freeze at -80℃ (reference)

[0409] The results are listed in the table below (relative % compared to the reference): JPEG2025037987000010.jpg109170

[0410] All three antibodies show very similar stability profiles, however the variant HLAG-0090_VL-S32P retains more HLA-G binding (SPR relative activity concentration (RAC)) after stress in PBS at 37°C than the other two, including the parental antibodies. Thermal Stability Test

[0411] For thermal stability testing of purified proteins, an Uncle device was used (UNCHAINED LABS, Boston, MA, USA). Intrinsic fluorescence in parallel with static light scattering at 266 nm and 473 nm was used to determine the aggregation temperature (Tagg) and melting temperature (Tm) of purified proteins. A temperature gradient was run from 30°C to 90°C with 0.1°C / min steps. A glass cuvette with a volume of 9 μl per sample was used, and the concentration was 1 mg / mL in 20 mM histidine, 140 mM NaCl, pH 6.0 buffer. For analysis, the software UNcle analysis (UNCHAINED LABS) was used. Mass spectrometry and N-glycosylation

[0412] Deconvoluted mass spectra of intact samples document the effect of removal of N-glycosylation sites / NSS motifs on N-glycosylation in HLA-G-0090_VL-S32P and HLA-G-0090_VL-S33A. Samples were prepared with PNGase F to remove all N-linked glycans and obtain only the molecular weight of the antibody. PNGase F is completely specific for cleaving N-linked Fc-glycans, but shows a much lower efficiency when cleaving N-linked Fab-glycans. HLA-G-0090 shows a clear N-glycosylation pattern resulting from incomplete deglycosylation, thus indicating Fab-glycosylation (see Figure 4A). For HLA-G-0090_VL-S32P and HLA-G-0090_VL-S33A, no indication of residual N-glycosylation can be detected (see Figure 4B). Example 3 Inhibition of ILT2 and ILT4 binding by anti-HLA-G antibodies

[0413] The ELISA is set up by coating Fc-tagged ILT2 and ILT4, respectively, on Maxisorp microtiter plates. After incubation and washing steps, the respective antibodies are added at a concentration of 100 nM. Soluble His-tagged monomeric, dimeric or trimeric HLA-G is added to the wells. After incubation and washing steps, detection of bound receptor is performed by anti-His antibody-POD conjugates. The percentage of inhibition is calculated relative to the value obtained from wells with ILT2 / 4+HLA-G (monomer, dimer or trimer) without anti-HLA-G or ILT2 / 4 antibodies (100% binding = 0% inhibition) and is shown in the table. Example 4 Binding of HLA-G antibodies to native or recombinant HLA-G expressed on cells (assessed by FACS analysis)

[0414] For flow cytometry analysis, cells were stained with anti-HLA-G mAb at 4° C. Briefly, each cell suspension was placed in a polypropylene tube (2×10 5 The cells were then transferred to a 100 μl FACS buffer (4° C.) and pre-cooled at 5° C. for 10 min. The cells were then washed with 2 ml of FACS buffer (4° C.) and centrifuged at 300 g for 5 min. The anti-HLA-G antibodies HLAG-0090-VL-S32P, HLAG-0090-VL-S33A, HLAG-0090 were diluted in staining buffer to a starting concentration of 50 μg / ml. Five-fold serial dilutions of the antibodies were performed to obtain the final concentrations (10 μg / ml, 2 μg / ml, 0.4 μg / ml, 0.08 μg / ml, 0.016 μg / ml, 0.0032 μg / ml). The FACS buffer was then aspirated from the tubes and the cell pellet was resuspended in 100 μl of antibody solution and incubated at 5° C. for 1 h. The cells were then washed once with 2 ml of staining buffer and centrifuged at 300 g for 5 min.

[0415] For detection, fluorescently labeled anti-species antibody (Goat anti-human IgG (H+L) conjugated to Alexa 488, Life technologies #A11013) was diluted to 10 μg / ml in staining buffer and cell pellets were resuspended in 100 μl of detection antibody. After 1 h incubation at 5° C., cells were washed once again with 2 ml of staining buffer, resuspended in 500 μl of staining buffer and measured on a FACS CELESTA.

[0416] Exemplary FACS staining for anti-HLA-G antibodies HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S32P (10 μg / ml) is shown in the FACS overlay in Figure 5: HLA-G 0090, HLA-G-0090-VL-S32P and deglycosylated variants of both HLA-G-0090-VL-S32P show good binding to HLA-G expressing SKOV3 and JEG cells but not to parental SKOV3 cells. The MFI values ​​of each HLA-G antibody are shown in the histograms. Example 5 Anti-HLA-G antibodies inhibit / modulate ILT2 binding to HLA-G expressed on JEG3 cells

[0417] For analysis, JEG3 cells (ATCC HTB36) were stained with ILT2-c-Myc-Fc fusion protein (control = no inhibition) with or without preincubation with different anti-HLA-G antibodies.

[0418] Binding / binding inhibition was determined as follows: recombinant ILT2-c-Myc-Fc protein was added to either JEG3 cells preincubated with anti-HLA-G mAb as indicated or to untreated JEG3 cells as a reference. For preincubation with anti-HLA-G antibody, 2 × 10 5 The cells were transferred to polypropylene tubes. Anti-HLA-G antibodies HLAG-0090-VL-S32P, HLAG-0090-VL-S33A and HLA-G-0090 were diluted in staining buffer to a concentration of 80 μg / ml, and 25 μl of the antibody solution was added to the prepared cells and incubated at 5° C. for 1 hour. ILT2-c-Myc-Fc or (control human IgG (Jackson-Immuno-Research#009-000-003)) was diluted in staining buffer to a 2x concentration of 20 μg / ml, and added to the prepared cells at a final concentration of 10 μg / ml and incubated at 5° C. for 2 hours. The cells were washed twice with 200 μl of staining buffer. Human ILT2-c-Myc-Fc protein was detected with fluorescently labeled anti-Myc tag (9E10) Alexa Fluor 647 (abcam; #ab223895) at a dilution of 10 μg / ml in staining buffer. Cells were resuspended in 50 μl of detection antibody dilution and incubated for 1 hour at 5°C. Cells were then washed once with 2 ml of staining buffer and resuspended in 500 μl of staining buffer before measurement on a FACS CELESTA.

[0419] As a control, anti-HLA-G antibodies bound to JEG-3 pre-incubated cells were detected by using an anti-species antibody (Goat anti-human IgG (H+L) conjugated to Alexa 488, Life technologies#A11013), diluted to 10 μg / ml in staining buffer, and the cell pellet was resuspended in 100 μl / well of detection antibody. After 1 h incubation at 5° C., the cells were washed once again with staining buffer, resuspended in 500 μl of staining buffer, and measured on a FACS CELESTA.

[0420] The histograms in Figure 6 show the respective capacity of HLA-G antibodies to alter / inhibit the interaction and binding of recombinant ILT2 to naturally expressed HLA-G on JEG3 tumor cells.

[0421] The table below summarizes the results of the experiment. Binding of anti-HLA-G antibodies to JEG3 cells is shown as +=weak binding -+++=strong binding. Ability of anti-HLA-G antibodies to inhibit / block or increase binding of ILT2 to HLA-G expressing JEG3 cells. The last column shows / quantifies binding of recombinant ILT2 to the cells or its inhibition / blocking (staining of ILT2-c-Myc-Fc in the absence of anti-HLA-G antibodies was set to 100% binding = 0% inhibition): JPEG2025037987000011.jpg57170Example 6 Generation of optimized CD3 binders

[0422] The VH and VL sequences of SEQ ID NOs: 92 and 93 are included in the "CD3 orig Starting from a previously described CD3 binder called " (for details see, for example, WO 2014 / 131712, which is incorporated herein by reference), the inventors aimed to optimize the properties of this binder by removal of the two asparagine deamidation sequence motifs at positions 97 and 100 according to the Kabat method of the heavy chain CDR3.

[0423] To this end, we generated an antibody library of heavy chains with deleted asparagines at both Kabat positions 97 and 100, suitable for phage display, and further generate...

Claims

1. An antibody that binds to human HLA-G, A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) An antibody comprising: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

2. The antibody, A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24; or B) The antibody of claim 1, comprising a VH domain having the amino acid sequence of SEQ ID NO:7, and a VL domain having the amino acid sequence of SEQ ID NO:

26.

3. The antibody according to claim 1 or 2, wherein the antibody comprises an Fc domain of human origin, in particular of the IgG isotype, more particularly of the IgG1 isotype.

4. The antibody according to any one of claims 1 to 3, wherein the antibody comprises a constant region of human origin, in particular of the IgG isotype, more particularly of the IgG1 isotype, comprising human CH1, CH2, CH3 and / or CL domains.

5. The antibody, a) does not cross-react with a modified human HLA-Gβ2M MHC I complex, in which HLA-G specific amino acids are replaced by HLA-A consensus amino acids, said complex comprising SEQ ID NO: 40; and / or b) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 41; and / or c) An antibody according to any one of claims 1 to 4 which does not cross-react with the rat RT1A β2M MHC I complex comprising SEQ ID NO:

43.

6. The antibody, a) inhibiting ILT2 binding to (HLA-G expressed in) JEG3 cells (ATCC No. HTB36); or b) an antibody according to any one of claims 1 to 5, which binds to (HLA-G expressed on) JEG3 cells (ATCC No. HTB36) and inhibits ILT2 binding to (HLA-G expressed on) JEG-3 cells (ATCC No. HTB36).

7. The antibody according to any one of claims 1 to 4, wherein the antibody is a multispecific antibody.

8. The antibody according to any one of claims 1 to 4, which is a bispecific antibody that binds to human HLA-G and human CD3.

9. the antibody is a bispecific antibody that binds to human HLA-G and human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3; a first antigen-binding portion that binds to human HLA-G; A) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, or B) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; a second antigen-binding portion that binds to a T cell activation antigen that binds to human CD3; C) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57, or D) (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62, and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 64, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 65, or E) The antibody of claim 5, comprising: (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 68, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

73.

10. The first antigen-binding portion comprises: A) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24; or B) a VH domain comprising the amino acid sequence of SEQ ID NO: 7, and a VL domain comprising the amino acid sequence of SEQ ID NO: 26; The second antigen-binding moiety comprises: C) a VH domain comprising the amino acid sequence of SEQ ID NO: 58 and a VL domain comprising the amino acid sequence of SEQ ID NO: 59; or D) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO: 67; or E) A bispecific antibody according to claim 9, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 74, and a VL domain comprising the amino acid sequence of SEQ ID NO:

75.

11. The first antigen-binding portion comprises: a VH domain comprising the amino acid sequence of SEQ ID NO:7, and a VL domain comprising the amino acid sequence of SEQ ID NO:24; The second antigen-binding moiety comprises:

11. The bispecific antibody of claim 10, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 58 and a VL domain comprising the amino acid sequence of SEQ ID NO:

59.

12. The first antigen-binding portion comprises: a VH domain comprising the amino acid sequence of SEQ ID NO:7, and a VL domain comprising the amino acid sequence of SEQ ID NO:24; The second antigen-binding moiety comprises:

11. The bispecific antibody of claim 10, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and a VL domain comprising the amino acid sequence of SEQ ID NO:

67.

13. The first antigen-binding portion comprises: a VH domain comprising the amino acid sequence of SEQ ID NO:7, and a VL domain comprising the amino acid sequence of SEQ ID NO:24; The second antigen-binding moiety comprises:

11. The bispecific antibody of claim 10, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 74 and a VL domain comprising the amino acid sequence of SEQ ID NO:

75.

14. The bispecific antibody comprises: a) inhibition of binding of ILT2 and / or ILT4 to HLA-G, and / or b) antibody-mediated IFN-gamma secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV3 HLA-G) and / or on JEG3 cells expressing endogenous HLA-G (IFN-gamma secretion was detected); and / or c) T cell-mediated cytotoxicity / tumor cell killing against SKOV3 cells transfected with recombinant HLA-G (SKOV 3HLA-G) and / or JEG3 cells expressing endogenous HLA-G (said cytotoxicity was detected by measuring caspase 8 activation in cells after treatment with the bispecific antibody), and / or d) in vivo anti-tumor efficacy / tumor regression in humanized NSG mice bearing SKOV3 human ovarian carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G) humanized NSG mice, and / or e) In vivo antitumor efficacy / tumor of the HLA-G CD3 T cell bispecific in humanized NSG mice bearing human breast cancer PDX tumors (BC004).

15. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 4 or a bispecific antibody according to any one of claims 7 to 13.

16. A host cell, preferably a eukaryotic host cell, comprising a nucleic acid according to claim 15.

17. A method for producing an antibody according to any one of claims 1 to 4, or a bispecific antibody according to any one of claims 7 to 13, comprising culturing a host cell according to claim 16 to produce the antibody or bispecific antibody.

18. 31. The method of claim 30, further comprising recovering the antibody or bispecific antibody from the host cell.

19. The antibody of any one of claims 1 to 4 or the bispecific antibody of any one of claims 7 to 13, wherein the antibody is produced in a eukaryotic host cell.

20. A pharmaceutical formulation comprising an antibody according to any one of claims 1 to 4 or a bispecific antibody according to any one of claims 7 to 13, and a pharma- ceutically acceptable carrier.

21. An antibody according to any one of claims 1 to 4 or a bispecific antibody according to any one of claims 7 to 13 for use as a medicament.

22. 14. The antibody of any one of claims 1 to 4 or the bispecific antibody of any one of claims 7 to 13 for use in the treatment of cancer.

23. Use of an antibody according to any one of claims 1 to 4 or a bispecific antibody according to any one of claims 7 to 13 in the manufacture of a medicament.

24. 24. The use according to claim 23, wherein the medicament is for the treatment of cancer.

25. 14. A method of treating an individual having cancer, comprising administering to said individual an effective amount of an antibody according to any one of claims 1 to 4, or a bispecific antibody according to any one of claims 7 to 13.

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