Multispecific antibodies and use thereof

Abstract

To provide multispecific antibodies that bind to HLA-G ant a T cell activating antigen, to provide preparation thereof, to provide formulations thereof, and to provide methods of using the same.SOLUTION: Disclosed is a multispecific antibody that binds to human HLA-G and a T cell activation antigen (particularly human CD3), comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to a T cell activation antigen (particularly human CD3). The multispecific antibody does not cross-react with a modified human HLA-Gβ2M MHCI complex containing a specific sequence (in which HLA-G-specific amino acids are replaced with HLA-A consensus amino acids).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to multispecific antibodies that bind to HLA-G and T cell activation antigens, their preparation, formulations, and methods of using them. [Background technology]

[0002] Background of the Invention 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 non-classical class I heavy chain paralogs of HLA. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is membrane-anchored but can be shed / secreted. The heavy chain 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 MHC I proteins. The second chain is beta-2 microglobulin, which binds to the heavy chain like other MHC I proteins.

[0003] In the case of HLA-G, seven isoforms exist, three of which are secreted and four of which are membrane-bound (schematically shown in Figure 1).

[0004] 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 are also 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).

[0005] HLA-G is predominantly expressed in placental cytotrophoblasts. Multiple 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). This 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 cancer prognosis. Tumor cells evade host immune surveillance by inducing immune tolerance / suppression through HLA-G expression. TIFF2025118635000001.tif63170

[0006] HLA-G shares high homology (>98%) with other MHC I molecules, and therefore it is difficult to generate truly HLA-G-specific antibodies that do not have cross-reactivity with other MHC I molecules.

[0007] Specific antibodies that interact differently with HLA-G have been previously described: Tissue Antigens, 55 (2000) 510-518, describes monoclonal antibodies such as 87G and MEM-G / 9; Neoplasma 50 (2003) 331-338, describes specific monoclonal antibodies that recognize both intact HLA-G oligomeric complexes (e.g., 87G and MEM-G9) and heavy chains without HLA-G (e.g., 4H84, MEM-G / 1, and MEM-G / 2); Hum Immunol. 64 (2003) 315-326, describes several antibodies tested on HLA-G-expressing JEG3 tumor cells (e.g., MEM-G / 09 and -G / 13, which react only with native HLA-G1 molecules). MEM-G / 01 (like 4H84 mAb) recognizes denatured HLA-G heavy chains of all isoforms, whereas MEM-G / 04 selectively recognizes denatured HLA-G1, -G2, and -G5 isoforms; Wiendl et al Brain 2003 176-85 relate to different monoclonal HLA-G antibodies, e.g., 87G, 4H84, MEM-G / 9.

[0008] The above papers report antibodies that bind to human HLA-G or the human HLA-G / β2M MHC complex. However, due to the high polymorphism and homology of the HLA family, many antibodies lack any truly specific HLA-G binding properties, and moreover often bind or cross-react with other HLA family members (either in MHC complex with β2M or in its β2M-free form), or simply do not inhibit the binding of the HLA-G β2M MHC complex to its receptors ILT2 and / or ILT4 (and are considered non-antagonist antibodies).

[0009] Bispecific antibodies (also referred to herein as T cell bispecific antibodies or "TCBs") that bind to both a surface antigen on a target cell and a T cell activation antigen, such as CD3 on a T cell, hold great potential for the treatment of various cancers. Simultaneous binding of such antibodies to both targets forces a transient interaction between the target cell and the T cell, leading to crosslinking of the T cell receptor and subsequent activation of cytotoxic T cells and subsequent lysis of the target cell. Given their potency in killing target cells, target selection and target specificity are paramount for T cell bispecific antibodies to avoid on-target and off-target toxicity. Intracellular proteins such as WT1 represent attractive targets but are only accessible to T cell receptor (TCR)-like antibodies that bind to major histocompatibility complex (MHC) antigens that present peptide antigens derived from intracellular proteins on the cell surface. An inherent problem with TCR-like antibodies is potential cross-reactivity with the MHC molecule itself or with MHC molecules presenting peptides other than the desired peptide, which can compromise organ or tissue selectivity. Summary of the Invention

[0010] The present invention provides multispecific antibodies that bind to human HLA-G and T cell activation antigens (particularly human CD3), comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to a T cell activation antigen (particularly human CD3).

[0011] In one aspect, a multispecific 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, does not cross-react with a modified human HLA-G β2M MHC I complex comprising SEQ ID NO: 44 (in which HLA-G specific amino acids are replaced by HLA-A consensus amino acids).

[0012] In one embodiment of the invention, the multispecific antibody is bispecific; and The first antigen-binding portion antibody that binds to human HLA-G is A) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; wherein the second antigen-binding portion that binds to a T cell activation antigen binds to human CD3; and E) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. Includes:

[0013] In one embodiment of the invention, the first antigen-binding moiety comprises: A) i) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8; ii) or humanized variants of VH and VL of the antibody of i). contains; or i) comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; or B) comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16; or C) comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24; or D) comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32; and the second antigen-binding portion is E) It comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63.

[0014] In one embodiment of the present invention, the first antigen-binding portion i) comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32; or ii) comprises the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; and the second antigen-binding portion is It comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63.

[0015] In one embodiment of the invention, the multispecific antibody comprises: a) does not cross-react with modified human HLA-G β2M MHC I complexes comprising SEQ ID NO: 44; and / or b) does not cross-react with the human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 39 and SEQ ID NO: 37; and / or c) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 45; and / or d) does not cross-react with the rat RT1A β2M MHC I complex containing SEQ ID NO: 47; and / or e) inhibits binding of ILT2 to a monomeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43); and / or f) inhibits binding of ILT2 to the trimeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 60%) (when compared to binding without the antibody) (see Example 4b); and / or g) inhibits binding of ILT2 to monomeric and / or dimeric and / or trimeric HLA-G β2M MHC I complexes (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 80%) (when compared to binding without antibody) (see Example 4b); and / or h) inhibits (by more than 50%, and in one embodiment by more than 80%) the binding of ILT2 to JEG3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or i) binds to JEG3 cells (ATCC No. HTB36) (HLA-G above) (see Example 5) and inhibits (by more than 50%, in one embodiment by more than 80%) the binding of ILT2 to JEG-3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or j) inhibits binding of CD8a to HLAG by more than 80% (compared to binding without antibody) (see e.g., Example 4c); and / or k) restore HLA-G-specific suppressive immune responses (e.g., suppressed tumor necrosis factor (TNF) alpha release) by monocytes co-cultured with JEG-3 cells (ATCC HTB36); and / or l) Induce T cell-mediated cytotoxicity in the presence of HLAG-expressing tumor cells (e.g., JEG-3 cells (ATCC HTB36)) (see Example 12).

[0016] In one embodiment of the invention, the first and second antigen-binding moieties are Fab molecules (each is a Fab molecule).

[0017] In one embodiment of the invention, 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 substituted for each other.

[0018] In one embodiment of the invention, the first antigen-binding portion is a Fab molecule in which 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).

[0019] In one embodiment of the invention, the first antigen-binding moiety and the second antigen-binding moiety are fused to each other, optionally via a peptide linker.

[0020] In one embodiment of the invention, the first and second antigen-binding moieties are each Fab molecules, wherein (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, 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.

[0021] In one embodiment of the invention, the multispecific antibody comprises a third antigen-binding moiety.

[0022] In one embodiment of the invention, such a third antigen-binding moiety is identical to the first antigen-binding moiety.

[0023] In one embodiment of the invention, the multispecific antibody comprises an Fc domain composed of a first and a second subunit.

[0024] In one embodiment of the invention, the first, second, and, if present, third antigen-binding moieties are each Fab molecules; wherein (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 attached at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; and wherein the third antigen-binding moiety, 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.

[0025] The present invention provides an isolated nucleic acid encoding an antibody according to any one of the preceding claims.

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

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

[0028] The present invention provides such methods of producing antibodies, further comprising recovering the antibodies from the host cells.

[0029] The invention provides pharmaceutical formulations comprising an antibody described herein and a pharmaceutically acceptable carrier.

[0030] The present invention provides an antibody as described herein for use as a pharmaceutical.

[0031] The present invention provides the antibodies described herein for use in the treatment of cancer.

[0032] The invention provides the use of an antibody as described herein in the manufacture of a medicament, hi one embodiment, the medicament is for the treatment of cancer.

[0033] The present invention provides a method of treating an individual with cancer, comprising administering to the individual an effective amount of an antibody described herein.

[0034] The screening method described herein allowed the selection of novel anti-HLA-G antibodies that exhibit highly useful properties, such as strong inhibition of ILT2 binding to HLA-G expressed on JEG3 cells or inhibition of ILT2 binding to monomeric and / or dimeric and / or trimeric HLA-G β2M MHC I complexes.

[0035] Furthermore, the antibodies according to the invention are able to restore HLA-G specific suppressive immune responses, ie restore LPS-induced TNFα production by monocytes in co-culture with HLA-G expressing cells.

[0036] In addition, the antibody is highly specific and shows no cross-reactivity with HLA-A MHC I complexes or MHC I complexes of mouse or rat origin. [Brief explanation of the drawings]

[0037] [Figure 1] Different isoforms of HLA-G [Figure 2A] Schematic representation of HLA-G containing molecules associated with β2M [Figure 2B] Structures of HLA-G molecules associated with specific receptors: HLA-G structures in complex with given receptors, ILT4 and KIR2DL1. ILT4 structure (PDB code: 2DYP). The KIR2DL1 structure was obtained from PDB code 1IM9 (KIR2DL1:HLA-Cw4 complex structure) and placed on top of HLA-G by superimposing the HLA-Cw4 and HLA-G structures. The receptor is represented by ribbons, and HLA-G is represented on the molecular surface. HLA-G residues unique to or conserved in other HLA paralogs are shown in white and gray, respectively. Unique surface residues were replaced by HLA consensus sequences in the chimeric counter-antigen. [Figure 3A] HLA-G antibodies that inhibit (or stimulate) HLA-G interaction / binding with ILT2 and ILT4 and CD8: ILT2 inhibition [Figure 3B] HLA-G antibodies that inhibit (or stimulate) HLA-G interaction / binding with ILT2 and ILT4 and CD8: ILT4 inhibition [Figure 3C] HLA-G antibodies that inhibit (or stimulate) HLA-G interaction / binding with ILT2 and ILT4 and CD8: CD8 inhibition [Figure 4A] Flow cytometry analysis of cell surface expression of HLA-G in JEG3 (cells naturally expressing HLA-G), SKOV-3 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)), and PA-TU-8902 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)) using HLA-G antibody: HLA-G-0031 (#0031). [Figure 4B]Flow cytometry analysis of cell surface expression of HLA-G in JEG3 (cells naturally expressing HLA-G), SKOV-3 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)), and PA-TU-8902 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)) using HLA-G antibody: HLA-G-0039 (#0039). [Figure 4C] Flow cytometry analysis of cell surface expression of HLA-G in JEG3 (cells naturally expressing HLA-G), SKOV-3 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)), and PA-TU-8902 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)) using HLA-G antibody: HLA-G-0041 (#0041). [Figure 4D] Flow cytometry analysis of cell surface expression of HLA-G in JEG3 (cells naturally expressing HLA-G), SKOV-3 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)), and PA-TU-8902 cells (wild-type (wt) vs. HLAG-transfected cells (HLAG+)) using HLA-G antibody: HLA-G-0090 (#0090). [Figure 5A]Anti-HLA-G antibodies (0031, 0039, 0041, and 0090) block / modulate the interaction of HLA-G expressed on JEG3 cells with human ILT2 Fc chimeras. Staining of cell surface HLA-G by novel anti-HLA-G antibodies was assessed using an anti-rat IgG secondary antibody conjugated to Alexa488 (top panel). FACS histograms show cells stained with the secondary antibody alone (gray dotted line) and with the anti-HLA-G antibody (black solid line). The bottom panel shows human ILT2-Fc bound to HLA-G on JEG3 cells (black dotted line) compared to cells stained with the secondary antibody alone (gray dotted line). The effect of preincubating JEG3 cells with HLA-G antibodies on ILT2Fc chimera binding can be seen (solid black line): HLA-G-0031 and HLA-G-0090 showed almost complete inhibition of ILT2-Fc chimera binding to JEG3 cells. Interestingly, the two antibodies 0039 and 0041 even increased ILT2:fc binding to the cells. [Figure 5B] Effect of commercial / reference anti-HLA-G antibodies on ILT2 Fc chimera binding to HLA-G on JEG3 cells: Staining of cell surface HLA-G by commercial / reference anti-HLA-G antibodies was assessed using species-specific secondary antibodies conjugated to Alexa488 (top panel). FACS histograms show cells stained with secondary antibody alone (gray dotted line) and anti-HLA-G antibody (black solid line). The bottom panel shows human ILT2 Fc chimera binding to HLA-G on JEG3 cells (black dotted line) compared to cells stained with secondary antibody alone (gray dotted line). The effect of preincubating JEG3 cells with the reference antibody on ILT2 Fc chimera binding can be seen (black solid line). None of the tested reference antibodies were able to block the interaction of ILT2 Fc chimera with cell surface HLA-G on JEG3 cells. [Figure 6A]Effect of HLA-G blockade with inhibitory anti-HLA-G antibodies on the restoration of TNFα production evaluated in different donors. Anti-HLA-G antibodies HLA-G-0031 (#0031), HLA-G-0039 (#0039), and HLA-G-0041 (#0041) evaluated in representative monocyte donors. [Figure 6B] Effect of HLA-G blockade by inhibitory anti-HLA-G antibodies on the recovery of TNFα production evaluated in a different donor. Anti-HLAG antibody HLA-G-0090 (#0090) evaluated in another monocyte donor. [Figure 6C] Effect of HLA-G blockade with inhibitory anti-HLA-G antibodies on the restoration of TNFα production assessed in different donors. Western blot analysis of HLAG expression in wild-type JEG-3 cells and knockdown mutants. [Figure 7] Binding of HLA-G TCB antibodies to native or recombinant HLA-G expressed on cells by anti-HLA-G / anti-CD3 bispecific antibodies (P1AA1185 and P1AD9924) (assessed by FACS analysis) [Figure 8] T cell activation via HLA-G TCB (anti-HLA-G / anti-CD3 bispecific TCB antibodies (P1AA1185 and P1AD9924)) [Figure 9] IFN-gamma secretion by T cells via HLA-G TCB (anti-HLA-G / anti-CD3 bispecific TCB antibodies P1AA1185 and P1AD9924) [Figure 10] Induction of T cell-mediated cytotoxicity / tumor cell killing by anti-HLA-G / anti-CD3 bispecific TCB antibodies (P1AA1185 and P1AD9924) [Figures 11A-F]Exemplary configurations of bispecific antigen-binding molecules of the invention. (A, D) Diagram of a "1+1 CrossMab" molecule. (B, E) Diagram of a "2+1 IgG Crossfab" molecule with the Crossfab and Fab components in an alternative order ("flipped"). (C, F) Diagram of a "2+1 IgG Crossfab" molecule. ++, --: amino acids of opposite charge optionally introduced into the CH1 and CL domains. Although the Crossfab molecules are shown as comprising swapped VH and VL regions, in embodiments where no charge modifications have been introduced into the CH1 and CL domains, they may alternatively comprise swapped CH1 and CL domains. [Figure 11G-N] Exemplary configurations of bispecific antigen-binding molecules of the invention. (G, K) Diagrams of a "1+1 IgG Crossfab" molecule with the Crossfab and Fab components in an alternative order ("flipped"). (H, L) Diagrams of a "1+1 IgG Crossfab" molecule. (I, M) Diagrams of a "2+1 IgG Crossfab" molecule with two CrossFabs. (J, N) Diagrams of a "2+1 IgG Crossfab" molecule with two CrossFabs and with the Crossfab and Fab components in an alternative order ("flipped"). ++, --: amino acids of opposite charge optionally introduced into the CH1 and CL domains. Although the Crossfab molecules are shown as comprising swapped VH and VL regions, in embodiments where no charge modifications have been introduced into the CH1 and CL domains, they may alternatively comprise swapped CH1 and CL domains. [Figure 11O-V] Exemplary configurations of bispecific antigen-binding molecules of the invention. (O, S) Diagram of a "Fab-Crossfab" molecule. (P, T) Diagram of a "Crossfab-Fab" molecule. (Q, U) Diagram of a "(Fab)2-Crossfab" molecule. (R, V) Diagram of a "Crossfab-(Fab)2" molecule. ++, --: amino acids of opposite charge optionally introduced into the CH1 and CL domains. Although Crossfab molecules are shown as comprising swapped VH and VL regions, in embodiments where no charge modifications have been introduced into the CH1 and CL domains, they may alternatively comprise swapped CH1 and CL domains. [Figure 11W-Z]Exemplary configurations of bispecific antigen-binding molecules of the invention. (W, Y) Diagram of a "Fab-(Crossfab)2" molecule. (X, Z) Diagram of a "(Crossfab)2-Fab" molecule. Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: amino acids of opposite charge optionally introduced into the CH1 and CL domains. Although Crossfab molecules are shown as comprising swapped VH and VL regions, in embodiments where no charge modifications have been introduced into the CH1 and CL domains, they may alternatively comprise swapped CH1 and CL domains. [Figure 12] In vivo antitumor effects of anti-HLA-G / anti-CD3 bispecific TCB antibodies (P1AA1185 and P1AD9924) DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description of the Invention As used herein, the term "HLA-G," "human HLA-G," refers 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.

[0039] As used herein, an antibody that "binds to human HLA-G," "specifically binds to human HLA-G," "binds to human HLA-G," or "anti-HLA-G antibody" refers to an antibody that binds to the human HLA-G antigen or its extracellular domain (ECD) with a specific binding affinity of 5.0x10 -8 K in mol / l or less D value, in one embodiment, 1.0x10 -9 K in mol / l or less D value, in one embodiment, 5.0x10 -8 mol / l to 1.0x10 -13 K in mol / l DIn one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO:43.

[0040] Binding affinity is determined by standard binding assays, such as surface plasmon resonance (BIAcore®, GE-Healthcare Uppsala, Sweden), using a construct comprising the HLA-G extracellular domain (e.g., in its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined by standard binding assays using an exemplary soluble HLA-G comprising an MHC class I complex comprising SEQ ID NO:43.

[0041] HLA-G has a regular 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 MHC I proteins. Chain 2 is beta 2 microglobulin, which is shared with various other MHC I proteins.

[0042] 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 forms of HLA-G5 can form disulfide-linked dimers with an intermolecular Cys42-Cys42 disulfide bond. Furthermore, membrane-bound forms of 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 have also been found on the cell surface of trophoblast cells (Apps, R., Tissue Antigens, 68:359 (2006)).

[0043] HLA-G is predominantly expressed in placental cytotrophoblasts. Multiple 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). This 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 cancer prognosis. Tumor cells evade host immune surveillance by inducing immune tolerance / suppression through HLA-G expression.

[0044] In the case of HLA-G, seven isoforms exist, three of which are secreted and four of which are membrane-bound (schematically shown in Figure 1). The most important functional isoforms of HLA-G include the b2-microglobulin-binding HLA-G1 and HLA-G5. However, the immunological effects of these isoforms on tolerogenicity differ and depend on the form of the ligand (monomer, dimer) and the affinity of the ligand-receptor interaction.

[0045] HLA-G proteins can be produced using standard molecular biology techniques. The nucleic acid sequences of HLA-G isoforms are known in the art. See, for example, GENBANK Accession No. AY359818.

[0046] HLA-G allotypes promote signaling through ILTs, particularly ILT2, ILT4, or a combination thereof.

[0047] ILTs: ILTs represent Ig-type activating and inhibitory receptors that are involved in regulating 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 ones (ILT2, ILT3, ILT4, ILT5, and LIR8) that contain a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and transmit inhibitory signals; (ii) activating ones (ILT1, ILT7, ILT8, and LIR6alpha) that contain a short cytoplasmic tail and charged amino acid residues in the transmembrane domain and deliver activation signals through the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of the binding common gamma chain of Fc receptors; and (iii) the soluble molecule ILT6, which lacks a transmembrane domain. Many recent studies have emphasized the immunoregulatory role of ILTs on the surface of antigen-presenting cells (APCs). The best-characterized immunosuppressive receptors, ILT2, ILT3, and ILT4 receptors, are predominantly expressed on myeloid and plasmacytoma DCs. ILT3 and ILT4 are upregulated by exposure of immature DCs to known immunosuppressive factors, including IL-10, vitamin D3, or suppressor CD8 T cells (Chang, CC, et al., Nat Immunol, 3:237-243 (2002)). ILT expression on DCs is tightly regulated by inflammatory stimuli, cytokines, and growth factors and is downregulated following DC activation (Ju, XS, et al., Gene, 331:159-164 (2004)). Expression of the ILT2 and ILT4 receptors is highly regulated by histone acetylation, which contributes to tightly controlled gene expression that is restricted to the myeloid lineage of cells (Nakajima, H., J Immunol, 171:6611-6620 (2003)).

[0048] Engagement of the inhibitory receptors ILT2 and ILT4 can alter the cytokine and chemokine secretion / release profile 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 described in detail by Suciu-Foca's group (Suciu-Foca, N., Int Immunopharmacol, 5:7-11 (2005)). Although the ligand for ILT3 is unknown, 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 for multiple inhibitory ILT receptors is HLA-G. HLA-G potentially plays a role in maternal-fetal tolerance and as a mechanism 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 very 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) and have the ability 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, interaction of HLA-G with DCs, which highly express ILT2 and ILT4 receptors, resulted in the downregulation of multiple genes involved in the MHC class II presentation pathway. IFN-gamma-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, as in vivo T cell responses to selected 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 interfere with the assembly and transport of MHC class II molecules to the cell surface, which may reduce the efficiency of presentation or expression of structurally abnormal MHC class II molecules. HLA-G was determined to significantly reduce the transcription of invariant chain (CD74), HLA-DMA, and HLA-DMB genes on human monocyte-derived DCs, which highly express ILT inhibitory receptors (Ristich, V., et al; Eur J Immunol 35:1133-1142 (2005)).

[0049] Another receptor for HLA-G is KIR2DL4, as KIR2DL4 binds to cells expressing HLA-G (U.S. Patent Application Publication No. 2003232051; Cantoni, C. et al. Eur J Immunol 28 (1998) 1980; Rajagopalan, S. and EO Long. [Errata 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 named 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 a positively charged amino acid in the transmembrane region, a characteristic 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 EO Long. [Erratum in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093).

[0050] HLA-G has also been shown to interact with CD8 on cytotoxic T cells (Sanders et al., J. Exp. Med., 1991) and induce CD95-mediated apoptosis in activated CD8+ cytotoxic T cells (Fournel et al., J. Immun., 2000). This mechanism of cytotoxic T cell elimination has been reported to be one of the mechanisms underlying immune escape and tolerance induction in pregnancy, inflammatory diseases, and cancer (Amodio G. et al., Tissue Antigens, 2014).

[0051] As used herein, an anti-HLA-G antibody that "does not cross-react with" or "does not specifically bind to" a modified human HLA-G β2M MHC I complex comprising SEQ ID NO:44; a mouse H2Kd β2M MHC I complex comprising SEQ ID NO:45; a rat RT1A β2M MHC I complex comprising SEQ ID NO:47; a human HLA-A2 β2M MHC I complex comprising SEQ ID NO:39 and SEQ ID NO:37 refers to an anti-HLA-G antibody that does not substantially bind to any of these counter-antigens. In one embodiment, an anti-HLA-G antibody that "does not cross-react with" or "does not specifically bind to" a modified human HLA-G β2M MHC I complex comprising SEQ ID NO:44; a mouse H2Kd β2M MHC I complex comprising SEQ ID NO:45; a rat RT1A β2M MHC I complex comprising SEQ ID NO:47; and / or a human HLA-A2 β2M MHC I complex comprising SEQ ID NO:39 and SEQ ID NO:37 is ... -6 K mol / l or more (until no further binding affinity is detectable) D " refers to an anti-HLA-G antibody that exhibits only nonspecific binding with a binding affinity of the value indicated. Binding affinity is determined using standard binding assays such as surface plasmon resonance (BIAcore®, GE-Healthcare Uppsala, Sweden) for the respective antigens, i.e., modified human HLA-G β2M MHC I complex comprising SEQ ID NO: 44; mouse H2Kd β2M MHC I complex comprising SEQ ID NO: 45; rat RT1A β2M MHC I complex comprising SEQ ID NO: 47; and / or human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 39 and SEQ ID NO: 37. The assay setup and antigen construction / preparation are described in the Examples.

[0052] The term "inhibiting binding of ILT2 to HLAG on JEG-3 cells (ATCC HTB36)" refers to the inhibition of the binding interaction of recombinant ILT2 in an assay such as that described in Example 6.

[0053] The terms "restoration of an HLA-G-specific suppressive immune response" or "restoring an HLA-G-specific suppressive immune response" refer to the restoration of lipopolysaccharide (LPS)-induced TNF-alpha production by monocytes in coculture with HLA-G-expressing cells, particularly JEG-3 cells. Thus, the antibodies of the present invention restore the HLA-G-specific release of TNF-alpha in LPS-stimulated cocultures of HLA-G-expressing JEG-3 cells (ATCC HTB36) and monocytes compared to untreated cocultured JEG-3 cells (using an untreated coculture as a 0% negative reference and a monocyte-only culture as a 100% positive reference, where the TNF-alpha section is not suppressed by any HLA-G / IL-T2-specific effect (see Example 7)). In this context, "HLA-G-specific suppressive immune response" refers to the immunosuppression of monocytes due to HLA-G expression on JEG-3 cells. In contrast, the anti-HLA-G antibodies of the present invention are unable to restore immune responses by monocytes co-cultured with JEG3 cells with HLA-G knockout.Other commercially available anti-HLA-G antibodies can induce TNF-alpha by monocytes co-cultured with JEG3 cells with HLA-G knockout, suggesting non-HLA-G-specific TNF-alpha release by these antibodies.

[0054] As used herein, "T cell activation antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, which can induce T cell activation upon interaction with an antibody. Specifically, interaction of an antibody with a T cell activation antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In certain embodiments, the T cell activation antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 189), NCBI RefSeq no. NP_000724, SEQ ID NO: 76 for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 77 for the cynomolgus monkey [Macaca fascicularis] sequence).

[0055] Unless otherwise specified, "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). The term encompasses "full-length," unprocessed CD3 and any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is set forth in UniProt (www.uniprot.org) under accession number P07766 (version 189) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724. See also SEQ ID NO: 76. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 77.

[0056] As used herein, an antibody that "binds to human CD3," "specifically binds to human CD3," "binds to human v," or "anti-HLA-G antibody" refers to an antibody that binds to the human CD3 antigen or its extracellular domain (ECD) with a specific binding affinity of 5.0x10 -8 K in mol / l or less D value, in one embodiment, 1.0x10 -9 K in mol / l or less D value, in one embodiment, 5.0x10 -8 mol / l to 1.0x10 -13 K in mol / l D In one embodiment, the antibody binds to CD3 comprising SEQ ID NO: 76.

[0057] Binding affinity is determined by standard binding assays, such as surface plasmon resonance (BIAcore®, GE-Healthcare Uppsala, Sweden), using a construct comprising the HLA-G extracellular domain (e.g., in its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined by standard binding assays using an exemplary CD3 comprising SEQ ID NO:76.

[0058] 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, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

[0059] For purposes herein, an "acceptor human framework" is a framework comprising 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 it may contain 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. A preferred VH acceptor human framework for the resulting humanized variant of antibody HLAG-0031 is HUMAN_IGHV1-3. The preferred VL acceptor human framework for the resulting humanized variant of antibody HLAG-0031 is HUMAN_IGKV1-17 (V domain, one additional backmutation at position R46F, Kabat numbering).

[0060] The term "antibody" as used herein is used 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.

[0061] "Antibody fragment" refers to a molecule other than an intact antibody that binds to an antigen bound by the intact antibody and contains a portion of the intact antibody. 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.

[0062] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

[0063] The term "bispecific" means that an antibody can specifically bind to at least two different antigenic determinants. Typically, a bispecific antibody contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two separate cells.

[0064] As used herein, the term "valency" refers to the presence of a specific number of antigen-binding sites in an antibody. Thus, the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-binding site that is specific for the antigen in the antibody.

[0065] "Antigen-binding site" refers to the site of an antibody, i.e., one or more amino acid residues, that provides interaction with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues from the complementarity-determining regions (CDRs). A native immunoglobulin molecule generally has two antigen-binding sites, while a Fab molecule generally has a single antigen-binding site.

[0066] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can target the entity to which it is bound (e.g., a second antigen-binding moiety) to a target site, e.g., a specific type of tumor cell bearing the antigenic determinant. In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, e.g., a T cell receptor complex antigen. Antigen-binding moieties include antibodies and fragments thereof, as further defined below. Particular antigen-binding moieties comprise the antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding moiety 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.

[0067] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-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).

[0068] 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.

[0069] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IGA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0070] An "effective amount" of an agent, eg, a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0071] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This 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 Fc region of a human IgG heavy chain is usually defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a specific nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or a truncated variant of the full-length heavy chain (also referred to herein as a "truncated variant heavy chain"). This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, 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 an Fc domain subunit as specified herein, contained in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to EU index of Kabat). In one embodiment of the invention, a heavy chain comprising an Fc domain subunit as specified herein, contained in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbered according to EU index of Kabat). Compositions of the invention, such as the pharmaceutical compositions described herein, comprise populations 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.A population of antibodies or bispecific antibodies can consist of a mixture of molecules with full-length heavy chains and molecules with truncated mutant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or bispecific antibodies have truncated mutant heavy chains. In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises antibodies or bispecific antibodies comprising heavy chains that comprise subunits 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). In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises antibodies or bispecific antibodies comprising heavy chains that comprise subunits of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention, such compositions comprise a population of antibodies or bispecific antibodies comprised of molecules comprising a heavy chain comprising an Fc domain subunit as specified herein; molecules comprising a heavy chain comprising an Fc domain subunit as specified herein with an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat); and molecules comprising a heavy chain comprising an Fc domain subunit as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the 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 above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association.For example, the subunits of the IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0072] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0073] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0074] By "fused" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0075] 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 a light chain (a "Fab light chain").

[0076] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been exchanged (i.e., replaced with each other), i.e., the crossover Fab molecule comprises a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, N-terminal to C-terminal), and a peptide chain consisting of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, N-terminal to C-terminal). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain have been 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 have been exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0077] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., comprising a heavy chain composed of a heavy chain variable and constant domain (VH-CH1, N-terminal to C-terminal), and a light chain composed of a light chain variable and constant domain (VL-CL, N-terminal to C-terminal). The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0078] A "human" antibody is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or a human cell, or of an antibody derived from a non-human source that utilizes the human antibody repertoire, or to sequences encoding other human antibodies. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0079] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup of Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0080] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs 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 HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of 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 undergone humanization.

[0081] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains residues that contact the antigen ("antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein are: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) a combination of (a), (b), and / or (c), comprising HVR amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35 (H1), 50-63 (H2), and 95-102 (H3). Includes:

[0082] Unless otherwise specified, HVR residues and other residues within the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0083] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0084] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0085] An "isolated" antibody is one that has been separated from a component of its natural environment. 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, e.g., Flatman, S., et al., J. Chromatogr. B 848 (2007) 79-87.

[0086] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0087] An "isolated nucleic acid encoding an anti-HLA-G antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0088] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for variant antibodies that may contain, for example, naturally occurring mutations or that arise during the production of the monoclonal antibody preparation, and which may generally be present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an 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; these and other exemplary methods for producing monoclonal antibodies are described herein.

[0089] A "modification that promotes association between a first subunit and a second subunit of an Fc domain" refers to manipulation of the peptide backbone or post-translational modification of an Fc domain subunit that reduces or prevents the formation of homodimers by association of a polypeptide comprising the Fc domain subunit with an identical polypeptide. As used herein, a modification that promotes association specifically includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) whose association is desired, where the modifications are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can alter the structure or charge of one or both of the Fc domain subunits to sterically or electrostatically favor their association, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may be non-identical in the sense that the additional 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 a particular embodiment, the association-promoting modifications comprise distinct amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0090] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 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 domain (CL). Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ).

[0091] The term "package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of such therapeutic product.

[0092] "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, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods 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 aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and its source code, along 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, CA) or can be compiled from its source code. The ALIGN-2 program must 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.

[0093] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with (or relative to) a given amino acid sequence B (alternatively, given amino acid sequence A can be said to have or contain a particular % amino acid sequence identity with (or relative to) 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 that program's 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 differs from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values used herein are obtained as described in the previous paragraph using the ALIGN-2 computer program.

[0094] The term "pharmaceutical formulation" refers to a preparation 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 are unacceptably toxic to the subject to which the formulation is administered.

[0095] A "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, additives, stabilizers, or preservatives.

[0096] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and achieving remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0097] The term "variable region" or "variable domain" refers to the domain of an antibody 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 hypervariable regions (HVR). (See, e.g., 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 specific antigen can be isolated using a VH or VL domain derived from an antibody that binds to that antigen, and a library of complementary VL or VH domains, respectively, can be screened. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0098] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are 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."

[0099] I. Compositions and Methods In one aspect, the present invention is based, in part, on the discovery that the multispecific antibodies (e.g., bispecific antibodies) described herein use selected anti-HLA-G antibodies as the first antigen-binding site / moiety. These anti-HLA-G antibodies bind with high specificity to specific epitopes of HLA-G (without cross-reactivity with other species or the human HLA-A consensus sequence) and have the ability to specifically inhibit ILT2 or ILT4 binding to HLA-G. They specifically reverse HLA-G-mediated immunosuppression of monocytes, for example, by inhibiting ILT2 binding to HLA-G and increasing the secretion of immunomodulatory cytokines such as TNF-alpha upon appropriate stimuli (e.g., lipopolysaccharide (LPS)), and show no effect on HLA-G knockout cells.

[0100] At the same time, the multispecific antibodies (e.g., bispecific antibodies) described herein bind to a T cell activation antigen (particularly CD3, especially CD3 epsilon) with a second antigen-binding site (moiety).

[0101] A. Exemplary Multispecific Anti-HLA-G / Anti-CD3 Antibodies In one embodiment of the invention, the multispecific antibody is a bispecific antibody that binds to human HLA-G and human CD3, which comprises a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3.

[0102] In one embodiment, the first antigen-binding portion antibody that binds to human HLA-G is A) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; wherein the second antigen-binding portion that binds to a T cell activation antigen binds to human CD3; and C) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. Includes:

[0103] In one embodiment of the invention, the first antigen-binding moiety comprises: A) comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; or B) comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32; and the second antigen-binding portion is C) It comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63.

[0104] In one embodiment of the present invention, the first antigen-binding portion comprises the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; and the second antigen-binding portion is It comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63.

[0105] In one embodiment of the present invention, the first antigen-binding portion comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32; and the second antigen-binding portion is It comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63.

[0106] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: A) (a) a VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 33; and (b) a VL domain comprising: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:34; or B) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:9; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:11; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:15. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16; or C) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 23. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 14; or D) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 31. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; wherein the VL domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 32. Includes:

[0107] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. and wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (as determined by surface plasmon resonance assay) as an antibody comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34 (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody).

[0108] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the VH domain comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 33; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; wherein the VL domain comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 34. Includes; wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody) as an antibody comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34 (as determined by surface plasmon resonance assay); and / or The antibodies are independently characterized by the following properties: Anti-HLA-G antibodies are a) does not cross-react with modified human HLA-G β2M MHC I complexes comprising SEQ ID NO: 44; and / or b) does not cross-react with the human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 39 and SEQ ID NO: 37; and / or c) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 45; and / or d) does not cross-react with the rat RT1A β2M MHC I complex containing SEQ ID NO: 47; and / or e) inhibits binding of ILT2 to a monomeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43); and / or f) inhibits binding of ILT2 to the trimeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 60%) (when compared to binding without the antibody) (see Example 4b); and / or g) inhibits binding of ILT2 to monomeric and / or dimeric and / or trimeric HLA-G β2M MHC I complexes (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 80%) (when compared to binding without antibody) (see Example 4b); and / or h) inhibits (by more than 50%, and in one embodiment by more than 80%) the binding of ILT2 to JEG3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or i) binds to JEG3 cells (ATCC No. HTB36) (HLA-G above) (see Example 5) and inhibits (by more than 50%, in one embodiment by more than 80%) the binding of ILT2 to JEG-3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or j) inhibits binding of CD8a to HLAG by more than 80% (compared to binding without antibody) (see e.g., Example 4c); and / or k) Restoring HLA-G-specific suppressive immune responses (e.g., suppressed tumor necrosis factor (TNF) alpha release) by monocytes co-cultured with JEG-3 cells (ATCC HTB36).

[0109] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) binds to the same epitope as an antibody comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34.

[0110] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; and wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (as determined by surface plasmon resonance assay) as an antibody comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16 (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody).

[0111] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 15; and and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16. Includes; wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody) as an antibody comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16 (as determined by surface plasmon resonance assay); and / or The antibodies are independently characterized by the following properties: Anti-HLA-G antibodies are a) does not cross-react with modified human HLA-G β2M MHC I complexes comprising SEQ ID NO: 44; and / or b) does not cross-react with the human HLA-A2β2M MHC I complex comprising SEQ ID NO: 39 and SEQ ID NO: 37; and / or c) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 45; and / or d) does not cross-react with the rat RT1A β2M MHC I complex containing SEQ ID NO: 47; and / or e) inhibits binding of ILT2 to a monomeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43); and / or f) inhibits binding of ILT2 to the trimeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 60%) (when compared to binding without the antibody) (see Example 4b); and / or g) inhibits binding of ILT2 to monomeric and / or dimeric and / or trimeric HLA-G β2M MHC I complexes (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 80%) (when compared to binding without antibody) (see Example 4b); and / or h) inhibits (by more than 50%, and in one embodiment by more than 80%) the binding of ILT2 to JEG3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or i) binds to JEG3 cells (ATCC No. HTB36) (HLA-G above) (see Example 5) and inhibits (by more than 50%, in one embodiment by more than 80%) the binding of ILT2 to JEG-3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or j) inhibits binding of CD8a to HLAG by more than 80% (compared to binding without antibody) (see e.g., Example 4c); and / or k) Restoring HLA-G-specific suppressive immune responses (e.g., suppressed tumor necrosis factor (TNF) alpha release) by monocytes co-cultured with JEG-3 cells (ATCC HTB36).

[0112] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) binds to the same epitope as an antibody comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16.

[0113] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22. and wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (as determined by surface plasmon resonance assay) as an antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24 (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody).

[0114] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; wherein the VH domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 23; and (b) a VL domain comprising: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 24. Includes; wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody) as an antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24 (as determined by surface plasmon resonance assay); and / or The antibodies are independently characterized by the following properties: Anti-HLA-G antibodies are a) does not cross-react with modified human HLA-G β2M MHC I complexes comprising SEQ ID NO: 44; and / or b) does not cross-react with the human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 39 and SEQ ID NO: 37; and / or c) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 45; and / or d) does not cross-react with the rat RT1A β2M MHC I complex containing SEQ ID NO: 47; and / or e) inhibits binding of ILT2 to a monomeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43); and / or f) inhibits binding of ILT2 to the trimeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 60%) (when compared to binding without the antibody) (see Example 4b); and / or g) inhibits binding of ILT2 to monomeric and / or dimeric and / or trimeric HLA-G β2M MHC I complexes (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 80%) (when compared to binding without antibody) (see Example 4b); and / or h) inhibits (by more than 50%, and in one embodiment by more than 80%) the binding of ILT2 to JEG3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or i) binds to JEG3 cells (ATCC No. HTB36) (HLA-G above) (see Example 5) and inhibits (by more than 50%, in one embodiment by more than 80%) the binding of ILT2 to JEG-3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or j) inhibits binding of CD8a to HLAG by more than 80% (compared to binding without antibody) (see e.g., Example 4c); and / or k) Restoring HLA-G-specific suppressive immune responses (e.g., suppressed tumor necrosis factor (TNF) alpha release) by monocytes co-cultured with JEG-3 cells (ATCC HTB36).

[0115] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) binds to the same epitope as an antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24.

[0116] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. and wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (as determined by surface plasmon resonance assay) as an antibody comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32 (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody).

[0117] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; wherein the VH domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 31; and (b) a VL domain comprising: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 32. Includes; wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (in one embodiment, the KD value of the binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of the binding affinity is reduced by up to 5-fold compared to said antibody) as an antibody comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32 (as determined by surface plasmon resonance assay); and / or The antibodies are independently characterized by the following properties: Anti-HLA-G antibodies are a) does not cross-react with modified human HLA-G β2M MHC I complexes comprising SEQ ID NO: 44; and / or b) does not cross-react with the human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 39 and SEQ ID NO: 37; and / or c) does not cross-react with the mouse H2Kd β2M MHC I complex containing SEQ ID NO: 45; and / or d) does not cross-react with the rat RT1A β2M MHC I complex containing SEQ ID NO: 47; and / or e) inhibits binding of ILT2 to a monomeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43); and / or f) inhibits binding of ILT2 to the trimeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 60%) (when compared to binding without the antibody) (see Example 4b); and / or g) inhibits binding of ILT2 to monomeric and / or dimeric and / or trimeric HLA-G β2M MHC I complexes (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 80%) (when compared to binding without antibody) (see Example 4b); and / or h) inhibits (by more than 50%, and in one embodiment by more than 80%) the binding of ILT2 to JEG3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or i) binds to JEG3 cells (ATCC No. HTB36) (HLA-G above) (see Example 5) and inhibits (by more than 50%, in one embodiment by more than 80%) the binding of ILT2 to JEG-3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody) (see Example 6); and / or j) inhibits binding of CD8a to HLAG by more than 80% (compared to binding without antibody) (see e.g., Example 4c); and / or k) Restoring HLA-G-specific suppressive immune responses (e.g., suppressed tumor necrosis factor (TNF) alpha release) by monocytes co-cultured with JEG-3 cells (ATCC HTB36).

[0118] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) binds to the same epitope as an antibody comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32.

[0119] In one embodiment, the second binding moiety that binds to human CD3 (in one embodiment, CD3 comprising SEQ ID NO: 76) is (a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58; and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. Includes:

[0120] In one embodiment, the second binding moiety that binds to human CD3 (in one embodiment, CD3 comprising SEQ ID NO: 76) is It comprises the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63.

[0121] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) A VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 62. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 63. Includes:

[0122] In one embodiment, the first binding moiety that binds to human HLA-G (in one embodiment, to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43) comprises: a) A VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 62. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 63. Includes; wherein the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43 with substantially the same binding affinity (in one embodiment, the KD value of binding affinity is reduced by up to 10-fold compared to said antibody, and in one embodiment, the KD value of binding affinity is reduced by up to 5-fold compared to said antibody) as an antibody comprising the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63 (as determined by surface plasmon resonance assay);

[0123] multispecific antibodies In a preferred embodiment, the multispecific antibodies provided herein are bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have 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, multispecific antibodies have three or more binding specificities. In certain embodiments, one of the binding specificities is for HLA-G and the other (two or more) specificities are for CD3. In certain embodiments, bispecific antibodies can bind to two (or more) different epitopes of HLA-G. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0124] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-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)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using common light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431); using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448). (1993)); by using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by preparing triabodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0125] Also included herein are engineered antibodies having three or more antigen-binding sites, such as "octopus antibodies" or DVD-Igs (see, e.g., 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 different antigen, or two different epitopes of HLA-G (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0126] Multispecific antibodies can also be provided in asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging 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, e.g., WO 2009 / 080251, WO 2016 / 016299, and 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.

[0127] 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).

[0128] A particular type of multispecific antibody also included herein is a bispecific antibody designed to simultaneously bind to an invariant activating component of the T cell receptor (TCR) complex, e.g., CD3, and to a surface antigen on a target cell, e.g., a tumor cell, to retarget the T cell for killing the target cell. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, in which one binding specificity is for HLA-G and the other is for CD3.

[0129] 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 their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 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 all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific 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.

[0130] A bispecific antibody that binds to HLA-G and CD3 The present invention also provides bispecific antibodies, ie, antibodies comprising at least two antigen-binding portions capable of specifically binding to two different antigenic determinants (first and second antigens).

[0131] Based on the HLA-G antibody they developed, the inventors have developed a bispecific antibody that binds to HLA-G and a further antigen, in particular a T cell activation antigen such as CD3.

[0132] As shown in the Examples, these bispecific antibodies have many remarkable properties, including good potency and low toxicity.

[0133] Accordingly, in certain aspects, the present invention provides bispecific antibodies comprising (a) a first antigen-binding portion that binds to a first antigen, where the first antigen is HLA-G, and (b) a second antigen-binding portion that specifically binds to a second antigen, wherein the bispecific antibody has any of the following characteristics:

[0134] Bispecific antibodies of the invention specifically induce T cell-mediated killing of cells expressing HLA-G. In some embodiments, bispecific antibodies of the invention specifically induce T cell-mediated killing of cells expressing HLA-G. In more specific embodiments, bispecific antibodies specifically induce T cell-mediated killing of cells expressing HLA-G.

[0135] In one embodiment, the induction of T cell-mediated killing by the bispecific antibody is determined using HLA-G expressing cells.

[0136] In one embodiment, activation of T cells by the bispecific antibody is determined, in particular by flow cytometry, by measuring the expression of CD25 and / or CD69 by T cells, in particular peptide-pulsed T2 cells, after incubation with the bispecific antibody, in particular in the presence of HLA-G expressing cells.

[0137] In a particular embodiment, induction of T cell-mediated killing by a bispecific antibody is determined as follows.

[0138] The ability of anti-HLA-G / anti-CD3TCB to activate T cells in the presence of HLAG-expressing tumor cells was tested using SKOV3 cells transfected with recombinant HLAG (SKOV3HLAG). T cell activation was assessed by FACS analysis of the cell surface activation marker CD25 and the early activation marker CD69 on T cells. Briefly, PBMCs were isolated from human peripheral blood by density gradient centrifugation using lymphocyte separation medium tubes (PAN#P04-60125). PBMCs and SKOV3HLAG cells were seeded at a 10:1 ratio in 96-well U-bottom plates. The co-cultures were then incubated with different concentrations of HLAG-TCB as described in Example 10 and incubated at 37°C in a 5% CO2 incubator for 24 hours. The following day, CD25 and CD69 expression was measured by flow cytometry.

[0139] For flow cytometry analysis, cells were 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 were diluted to half concentration, and 25 μl of antibody dilution was added to each well along with 25 μl of pre-washed co-culture medium. Cells were stained for 30 minutes at 4°C, washed twice with 200 μl / well of staining buffer, and centrifuged at 300 g for 5 minutes. The cell pellet was resuspended in 200 μl of staining buffer and stained with DAPI for live / dead differentiation at a final concentration of 2 μg / ml. Samples were then measured using a BD LSR flow cytometer. Data analysis was performed using FlowJo V.10.1 software. The geometric means of the mean fluorescence intensities are exported and the ratio of the isotype and antibody geometric means is calculated.

[0140] Bispecific antibodies of the invention specifically activate T cells in the presence of cells expressing HLA-G. In some embodiments, bispecific antibodies of the invention specifically activate T cells in the presence of cells expressing HLA-G. In more specific embodiments, bispecific antibodies specifically activate T cells in the presence of cells expressing HLA-G.

[0141] In one embodiment, the bispecific antigen binding does not significantly induce T cell-mediated killing or activate 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 inducing T cell-mediated killing or activating T cells in the presence of cells expressing HLA-G.

[0142] According to certain embodiments of the invention, the antigen-binding moieties comprised in the bispecific antibody are Fab molecules (i.e., antigen-binding domains composed of a heavy chain and a light chain, each comprising a variable and a constant domain). In one embodiment, the first and / or second antigen-binding moieties are Fab molecules. In one embodiment, the Fab molecules are human. In certain embodiments, the Fab molecules are humanized. In yet another embodiment, the Fab molecules comprise human heavy and light chain constant domains.

[0143] Preferably, at least one of the antigen-binding portions is a crossover Fab molecule. Such a modification reduces mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the bispecific antibodies of the invention during 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 by-products due to so-called Bence Jones interactions between 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 improve the purity and yield of the desired bispecific antibody, charged amino acids with opposite charges can be introduced into specific amino acid positions in the CH1 and CL domains of the Fab molecule(s) that bind to the first antigen (HLA-G) or the Fab molecule that binds to the second antigen (a T cell activation antigen such as CD3). This is described below. The charge modifications are made in either the conventional Fab molecule(s) comprised in the bispecific antibody (e.g., as shown in Figures 11A-C, GJ) or in the VH / VL crossover Fab molecule(s) comprised in the bispecific antibody (e.g., as shown in Figures 11D-F, KN), but not both. In certain embodiments, the charge modifications are made in the conventional Fab molecule(s) comprised in the bispecific antibody (in certain embodiments, those that bind to the first antigen, i.e., HLA-G).

[0144] In particular embodiments according to the invention, a bispecific antibody is capable of simultaneously binding a first antigen (i.e., HLA-G) and a second antigen (e.g., a T cell activation antigen, particularly CD3). In one embodiment, the bispecific antibody is capable of crosslinking a T cell to a target cell by simultaneously binding HLA-G and a T cell activation antigen. In a more particular embodiment, such simultaneous binding results in lysis of the target cell, particularly an HLA-G-expressing tumor cell. In one embodiment, such simultaneous binding causes activation of the T cell. In another embodiment, such simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, binding of a bispecific antibody to a T cell activation antigen, particularly CD3, without simultaneous binding to HLA-G does not result in T cell activation.

[0145] In one embodiment, the bispecific antibody is capable of redirecting the cytotoxic activity of a T cell to a target cell, which in certain embodiments is independent of MHC-mediated presentation of peptide antigens by the target cell and / or the specificity of the T cell.

[0146] In particular, the T cells according to any of the embodiments of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells.

[0147] First antigen-binding moiety Bispecific antibodies of the present invention comprise at least one antigen-binding portion, particularly a Fab molecule, that binds to HLA-G (first antigen). In certain embodiments, bispecific antibodies comprise two antigen-binding portions, particularly Fab molecules, that bind to HLA-G. In certain such embodiments, each of these antigen-binding portions binds to the same antigenic determinant. In even more particular embodiments, these antigen-binding portions are all identical, i.e., they comprise the same amino acid sequence, including the same amino acid substitutions in the CH1 and CL domains (if any), as described herein. In one embodiment, a bispecific antibody comprises no more than two antigen-binding portions, particularly Fab molecules, that bind to HLA-G.

[0148] In certain embodiments, the antigen-binding moiety(ies) that bind to HLA-G are conventional Fab molecules. In such embodiments, the antigen-binding moiety(ies) that bind to a second antigen are crossover Fab molecules as described herein, i.e., Fab molecules 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 / substituted for each other.

[0149] In an alternative embodiment, the antigen-binding moiety(s) that bind to HLA-G are crossover Fab molecules as described herein, i.e., Fab molecules 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 / substituted for each other. In such an embodiment, the antigen-binding moiety(s) that bind to the second antigen are conventional Fab molecules.

[0150] The HLA-G binding moiety can direct the bispecific antibody to a target site, for example, to a particular type of tumor cell that expresses HLA-G.

[0151] 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 it is scientifically clearly unreasonable or impossible.

[0152] Thus, in one aspect, the present invention provides a bispecific antibody comprising: (a) a first antigen-binding portion that binds to a first antigen, wherein the first antigen is HLA-G.

[0153] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is A) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. Includes:

[0154] One embodiment of the invention is an isolated antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is A) i) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8; ii) or humanized variants of VH and VL of the antibody of i). contains; or iii) comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; or B) comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16; or C) i) comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24; or D) i) comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32.

[0155] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. Includes:

[0156] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. Includes:

[0157] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22. Includes:

[0158] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. Includes:

[0159] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is i) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8; ii) or humanized variants of VH and VL of the antibody of i). Includes:

[0160] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is i) comprises the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34.

[0161] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is It comprises the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16.

[0162] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is It comprises the VH sequence of SEQ ID NO:23 and the VL sequence of SEQ ID NO:24.

[0163] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is It comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32.

[0164] One embodiment of the invention is an (isolated) antibody that binds to human HLA-G (in one embodiment, the antibody binds to an HLA-G β2M MHC I complex comprising SEQ ID NO: 43), wherein the antibody is A) (a) a VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 33; and (b) a VL domain comprising: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:34; or B) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:9; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:11; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:15. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16; or C) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 23. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 14; or D) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 31. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; wherein the VL domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 32. Includes:

[0165] In one embodiment, the first antigen-binding moiety comprises a human constant region. In one embodiment, the first antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs: 51 and 52 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 53 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the first antigen-binding moiety comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 52, particularly an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51. In particular, the light chain constant region may comprise the amino acid mutations described in the "Charge Modification" section herein and / or, in crossover Fab molecules, deletion or substitution of one or more (particularly two) N-terminal amino acids. In some embodiments, the first antigen-binding portion comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 53. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise the amino acid mutations described in the "Charge Modifications" section herein.

[0166] A second antigen-binding moiety that binds to T cell activation antigens, particularly CD3 The bispecific antibodies of the invention comprise at least one antigen-binding portion, particularly a Fab molecule, that binds to a T cell activation antigen, particularly CD3.

[0167] In certain embodiments, the antigen-binding portion that binds a T cell activation antigen, particularly 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 / substituted for one another. In such embodiments, the antigen-binding portion(s) that bind HLA-G are preferably conventional Fab molecules. In embodiments in which there are two or more antigen-binding portions, particularly Fab molecules, that bind a T cell activation antigen, particularly CD3, contained in a bispecific antibody, the antigen-binding portion that binds a T cell activation antigen, particularly CD3, is preferably a crossover Fab molecule, and the antigen-binding portion that binds HLA-G is a conventional Fab molecule.

[0168] 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 / substituted for one another. 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 HLA-G is preferably a crossover Fab molecule, and the antigen-binding moiety that binds CD3 is a conventional Fab molecule.

[0169] In some embodiments, the second antigen is a T cell activation antigen (also referred to herein as a "T cell activation antigen-binding portion, or a T cell activation antigen-binding Fab molecule"). In certain embodiments, a bispecific antibody comprises no more than one antigen-binding portion capable of specifically binding to a T cell activation antigen. In one embodiment, a bispecific antibody provides monovalent binding to a T cell activation antigen.

[0170] In certain embodiments, the second antigen is CD3, particularly human CD3 (SEQ ID NO: 76) or cynomolgus CD3 (SEQ ID NO: 77), most particularly human CD3. In one embodiment, the second antigen-binding moiety is cross-reactive with (i.e., specifically binds to) human and cynomolgus CD3. In some embodiments, the second antigen is the epsilon subunit of CD3 (CD3 epsilon).

[0171] In one embodiment, the second antigen-binding portion that binds to human CD3 comprises a VH domain comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 58, and (b) a VL domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61.

[0172] In one embodiment, the second antigen-binding portion that binds to human CD3 comprises a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 58; wherein the VH domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 62. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61; wherein the VL domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 63.

[0173] In some embodiments, the second antigen-binding moiety is (derived from) a humanized antibody. In one embodiment, the VH is a humanized VH and / or the VL is a humanized VL. In one embodiment, the second antigen-binding moiety comprises the CDRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0174] In one embodiment, the second antigen-binding portion that binds human CD3 comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62. In one embodiment, the second antigen-binding portion comprises a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63.

[0175] In one embodiment, the second antigen-binding portion that binds to human CD3 comprises a VH comprising the amino acid sequence of SEQ ID NO:62, and a VL comprising the amino acid sequence of SEQ ID NO:63.

[0176] In one embodiment, the second antigen-binding moiety that binds to human CD3 comprises a human constant region. In one embodiment, the second antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs: 51 and 52 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 53 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the second antigen-binding moiety comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 52, particularly an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51. In particular, the light chain constant region may comprise the amino acid mutations described in the "Charge Modification" section herein and / or, in crossover Fab molecules, deletion or substitution of one or more (particularly two) N-terminal amino acids. In some embodiments, the second antigen-binding portion comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 53. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise the amino acid mutations described in the "Charge Modifications" section herein.

[0177] 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 substituted for one another (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 third, if present) antigen-binding moiety is a conventional Fab molecule. In one embodiment, no more than one antigen-binding moiety that binds to a second antigen (e.g., a T cell activation antigen such as CD3) is present in the bispecific antibody (i.e., the bispecific antibody provides monovalent binding to the second antigen).

[0178] Charge modification Bispecific antibodies of the invention may comprise amino acid substitutions in the Fab molecules contained therein that are effective in reducing mispairing of light chains with incompatible heavy chains (Bence Jones by-products), particularly in the generation of Fab-based bispecific antibodies having a VH / VL exchange in one of their binding arms (or two or more, in the case of molecules comprising three or more antigen-binding Fab molecules) (see also PCT Publication No. WO2015 / 1504447, especially the examples therein, which is incorporated herein by reference in its entirety). The ratio of desired bispecific antibodies compared to undesired by-products, particularly Bence Jones by-products that arise in bispecific antibodies having a VH / VL domain exchange in one of their binding arms, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modifications").

[0179] Thus, in some embodiments where 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 Fab heavy chain are replaced by each other: i) in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (Kabat numbering) and in the constant domain CH1 of said 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 (Kabat EU index numbering); 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 (Kabat numbering), 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 (Kabat EU index numbering).

[0180] Bispecific antibodies do not contain both of the modifications mentioned in i) and ii): the constant domains CL and CH1 of the antigen-binding site with VH / VL exchange are not replaced by each other (i.e., remain unexchanged).

[0181] In a more specific embodiment, i) in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CH1 of said first antigen-binding moiety the amino acid at position 147 or 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering); or ii) in the constant domain CL of said second antigen-binding moiety, the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (Kabat numbering); and in the constant domain CH1 of said second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0182] In one such embodiment, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (Kabat numbering), 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 independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0183] In a further embodiment, in the constant domain CL of said first antigen-binding moiety, the amino acid at position 124 is substituted independently by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering), and in the constant domain CH1 of said first antigen-binding moiety, the amino acid at position 147 is substituted independently by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

[0184] 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) (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).

[0185] In a more particular embodiment, in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat); and in the constant domain CHI of said first 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).

[0186] In a more particular embodiment, in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat); and in the constant domain CHI of said first 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).

[0187] 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.

[0188] Alternatively, the amino acid substitutions according to the above embodiments may be made in the constant domains CL and CH1 of the second antigen-binding moiety instead of the constant domains CL and 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.

[0189] Thus, in one embodiment, in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (Kabat numbering), 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 independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0190] In a further embodiment, in the constant domain CL of said second antigen-binding moiety, the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CH1 of said second antigen-binding moiety, the amino acid at position 147 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0191] and in the constant domain CHI of said second 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), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0192] In one embodiment in the constant domain CL of said 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 CHI of said 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).

[0193] In another embodiment, in the constant domain CL of said second antigen-binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat); and in the constant domain CHI of said 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).

[0194] In a particular embodiment, the bispecific antibody of the invention comprises: I) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is A) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. a first antigen-binding portion which is a Fab molecule comprising: and II) a second antigen-binding moiety that binds to human CD3, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced by each other; E) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. and and wherein in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (in particular embodiments, independently by lysine (K) or arginine (R)) (Kabat numbering), and 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 said first antigen-binding moiety the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (in particular embodiments, independently by lysine (K) or arginine (R)) (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). The second antigen-binding moiety is included.

[0195] In a particular embodiment, the bispecific antibody of the invention comprises: I) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) i) a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8; ii) or humanized variants of VH and VL of the antibody of i). contains; or iii) comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; or B) comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16; or C) comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24; or D) Comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32 a first antigen-binding portion that is a Fab molecule; and II) a second antigen-binding moiety that binds to human CD3, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced by each other; E) comprising the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63; and and wherein in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (in particular embodiments, independently by lysine (K) or arginine (R)) (Kabat numbering), and 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 said first antigen-binding moiety the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (in particular embodiments, independently by lysine (K) or arginine (R)) (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). The second antigen-binding moiety is included.

[0196] Bispecific antibody format The components of the bispecific antibodies according to the invention can be fused to each other in a variety of configurations, exemplary configurations are shown in Figure 11.

[0197] In certain embodiments, the antigen-binding portions comprised in the bispecific antibody are Fab molecules. In such embodiments, the first, second, third, etc. antigen-binding portions may be referred to herein as first, second, third, etc. Fab molecules, respectively.

[0198] 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 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 another such embodiment, 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 embodiments in which (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, 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, 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.

[0199] Bispecific antibodies (e.g., as shown in Figures 11A, D, G, H, K, L) having a single antigen-binding moiety (such as a Fab molecule) capable of specifically binding to a target cell antigen, such as HLA-G, are particularly useful when internalization of the target cell antigen is expected following 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 can enhance internalization of the target cell antigen, thereby reducing its availability.

[0200] However, in other cases, it may be advantageous to have a bispecific antibody that comprises two or more antigen-binding portions (such as Fab molecules) specific for target cell antigens, e.g., to optimize targeting to the target site or to allow cross-linking of target cell antigens (see examples shown in Figures 11B, 11C, 11E, 11F, 11I, 11J, 11M, or 11N).

[0201] Thus, in a particular embodiment, the bispecific antibody according to the invention comprises a third antigen-binding moiety.

[0202] 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.

[0203] In certain embodiments, the third antigen-binding portion is identical to the first antigen-binding portion.

[0204] 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 to HLA-G, either alone or in combination, unless it is scientifically clearly unreasonable or impossible.

[0205] In one embodiment, the third antigen binding moiety that binds to HLA-G is A) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. Includes:

[0206] In one embodiment, the third antigen binding moiety that binds to HLA-G is A) (a) a VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 33; and (b) a VL domain comprising: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:34; or B) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:9; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:11; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:15. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16; or C) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 23. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; wherein the VL domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 14; or D) (a) A VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; wherein the VH domain comprises an amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 31. and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; wherein the VL domain comprises an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 32. Includes:

[0207] In one embodiment, the third antigen-binding moiety is A) iv) the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8; v) or humanized variants of VH and VL of the antibody of i) contains; or vi) comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; or B) comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16; or C) comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24; or D) It comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32.

[0208] In some embodiments, the third antigen-binding moiety is (derived from) a human antibody. In one embodiment, the VH is a human VH and / or the VL is a human VL. In one embodiment, the third antigen-binding moiety comprises the CDRs as in any of the above embodiments and further comprises a human framework, e.g., a human immunoglobulin framework.

[0209] In one embodiment, the third antigen-binding portion comprises (i) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8; (ii) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15, and a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; (iii) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24; (iv) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31, and a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32; (v) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33, and a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 34. Includes:

[0210] In one embodiment, the third antigen-binding moiety is (i) a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 23 and a VL comprising the amino acid sequence of SEQ ID NO: 24; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 32; (iv) VH comprising the amino acid sequence of SEQ ID NO: 33 and VL comprising the amino acid sequence of SEQ ID NO: 34 Includes:

[0211] In one embodiment, the third antigen-binding moiety is It comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8.

[0212] In one embodiment, the third antigen-binding moiety is It comprises a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0213] In one embodiment, the third antigen-binding moiety is It comprises a VH comprising the amino acid sequence of SEQ ID NO:23 and a VL comprising the amino acid sequence of SEQ ID NO:24.

[0214] In one embodiment, the third antigen-binding moiety is It comprises a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VL comprising the amino acid sequence of SEQ ID NO: 32.

[0215] In one embodiment, the third antigen-binding moiety is It comprises a VH comprising the amino acid sequence of SEQ ID NO: 33, and a VL comprising the amino acid sequence of SEQ ID NO: 34.

[0216] In one embodiment, the third antigen-binding moiety comprises a human constant region. In one embodiment, the third antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs: 51 and 522 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 53 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the third antigen-binding moiety comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 52, particularly an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 51. In particular, the light chain constant region may comprise the amino acid mutations described in the "Charge Modification" section herein and / or, in crossover Fab molecules, deletion or substitution of one or more (particularly two) N-terminal amino acids. In some embodiments, the second antigen-binding portion comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 51. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise the amino acid mutations described in the "Charge Modifications" section herein.

[0217] In certain embodiments, the third and first antigen-binding moieties are each Fab molecules, 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 contains the same amino acid substitutions, if present, as the first antigen-binding moiety. For example, amino acid substitutions described herein as "charge-modifying" may be made in the constant domains CL and CHI of the first and third antigen-binding moieties, respectively. Alternatively, the amino acid substitutions may be made in the constant domains CL and CHI of the second antigen-binding moiety (which in certain embodiments is also a Fab molecule), but not in the constant domains CL and CHI of the first and third antigen-binding moieties.

[0218] Like the first antigen-binding portion, the third antigen-binding portion is particularly a conventional Fab molecule. However, embodiments in which the first and third antigen-binding portions are crossover Fab molecules (and the second antigen-binding portion is a conventional Fab molecule) are also contemplated. Thus, in certain embodiments, the first and third antigen-binding portions are each conventional Fab molecules, and the second antigen-binding portion 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 / substituted for each other. In other embodiments, the first and third antigen-binding portions are each crossover Fab molecules, and the second antigen-binding portion is a conventional Fab molecule.

[0219] When a third antigen-binding moiety is present, in certain embodiments, the first and third antigen-binding moieties bind to HLA-G and the second antigen-binding moiety binds to a second antigen, particularly a T cell activation antigen, more particularly CD3, most particularly CD3 epsilon.

[0220] In certain embodiments, the bispecific antibody comprises an Fc domain composed of a first subunit and a second subunit, wherein the first and second subunits of the Fc domain are capable of stable association.

[0221] Bispecific antibodies according to the invention can have different configurations, i.e. the first, second (and optionally third) antigen-binding moieties can be fused to each other and to the Fc domain in different ways. The components can be fused to each other directly or, preferably, via one or more suitable peptide linkers. When the fusion of a Fab molecule is to the N-terminus of a subunit of the Fc domain, it is typically via the immunoglobulin hinge region.

[0222] 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 Fc domain subunit. In such embodiments, the first antigen-binding moiety can 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 another one of the Fc domain subunits. 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 have been swapped / substituted for 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.

[0223] In one embodiment, the first and second antigen-binding moieties are each Fab molecules, and the second antigen-binding moiety is fused at the C-terminus of its 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 its Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety. In certain embodiments, the bispecific antibody consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, where the first Fab molecule is fused at the C-terminus of its 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 its Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such configurations are shown schematically in Figures 11G and 11K (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 can further be fused to each other.

[0224] 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 certain embodiments, the bispecific antibody consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, where 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 an arrangement is shown schematically in Figures 11A and 11D (in these examples, the second antigen-binding domain is a VH / VL crossover Fab molecule and the first antigen-binding moiety is a conventional Fab molecule). The first and second Fab molecules can 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 the Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when the Fc domain is an IgG1 Fc domain.

[0225] 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 Fc domain subunit. In such embodiments, the second antigen-binding moiety can 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 another one of the Fc domain subunits (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 have been swapped / substituted for 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.

[0226] In one embodiment, the first and second antigen-binding moieties are each Fab molecules, with the first antigen-binding moiety 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 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 certain embodiments, the bispecific antibody consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, where 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, and the first 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. Such configurations are shown schematically in Figures 11H and 11L (in these examples, the second antigen-binding domain 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 can further be fused to each other.

[0227] In some embodiments, a third antigen-binding moiety, particularly a 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 have been swapped / substituted for 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.

[0228] 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 certain embodiments, 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 first Fab molecule is fused at the C-terminus of its 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 its 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 its Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such configurations are shown schematically in Figures 11B and 11E (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 Figures 11J and 11N (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 can 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 via an immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when 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 can be further fused to each other.

[0229] 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 particular 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 its Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of its 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 its Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such configurations are shown schematically in Figures 11C and 11F (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 Figures 11I and 11M (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 can be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the first and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when 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 can be further fused to each other.

[0230] In a bispecific antibody configuration, in which a Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each subunit of the Fc domain via an immunoglobulin hinge region, the two Fab molecules, the hinge region, and the Fc domain essentially form an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In a more specific embodiment, the immunoglobulin is an immunoglobulin of the IgG1 subclass. In another embodiment, the immunoglobulin is an immunoglobulin of the IgG4 subclass. In a more specific embodiment, the immunoglobulin is a human immunoglobulin. In another embodiment, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the immunoglobulin comprises a human constant region, particularly a human Fc region.

[0231] In some bispecific antibodies of the invention, the Fab light chain of a first Fab molecule and the Fab light chain of a second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of a first Fab molecule can be fused at its C-terminus to the N-terminus of the Fab light chain of a second Fab molecule, or the Fab light chain of a second Fab molecule can be fused at its C-terminus to the N-terminus of the Fab light chain of a first Fab molecule. Fusing 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 to express some bispecific antibodies of the invention.

[0232] The antigen-binding portions can be fused to the Fc domain, directly, or to each other via a peptide linker comprising one or more amino acids (usually about 2-20 amino acids). Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or 4 (SG4) nPeptide linkers are included. "n" is usually an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker is at least 5 amino acids in length, in one embodiment from 5 to 100 amino acids in length, and in a further embodiment from 10 to 50 amino acids in length. In one embodiment, the peptide linker is (GxS) n or (GxS) n G m wherein G=glycine, S=serine, (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, and in a further embodiment x=4, n=2. In one embodiment, the peptide linker is (G4S)2. A particularly suitable peptide linker 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 linking the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-(G4S)2 (SEQ ID NOs: 110 and 111). Another suitable such linker comprises the sequence (G4S)4. Furthermore, 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, it may be fused via the immunoglobulin hinge region or part thereof, with or without an additional peptide linker.

[0233] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VL) 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 an Fc domain subunit. (2) -CH1 (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 further 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 disulfide bonds.

[0234] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain variable region of a second Fab molecule 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). (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 further 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 disulfide bonds.

[0235] 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 the 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 the 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 (VH (1) -CH1 (1) -VL (2) -CH1 (2) -CH2-CH3(-CH4)).

[0236] In some of these embodiments, the bispecific antibody further 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 other of these embodiments, the bispecific antibody optionally comprises a polypeptide (VH) 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, which in turn shares a carboxy-terminal peptide bond with the 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 the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (VL (1) -CL (1) -VH (2) -CL (2) ) further includes.

[0237] Bispecific antibodies according to these embodiments may further comprise (i) a subunit polypeptide of the Fc domain (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the 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 disulfide bonds.

[0238] In some embodiments, a bispecific antibody comprises a polypeptide in which the Fab heavy chain variable region of a second Fab molecule 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 the 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 the 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)).

[0239] In some of these embodiments, the bispecific antibody further comprises a crossover Fab light chain polypeptide (VL) of a second Fab molecule, 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. (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 optionally comprises a polypeptide (VL) 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, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule. (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 the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (VL (1) -CL (1) -VH (2) -CL (2) ) further includes.

[0240] Bispecific antibodies according to these embodiments may further comprise (i) a subunit polypeptide of the Fc domain (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the 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 disulfide bonds.

[0241] In certain embodiments, the bispecific antibody does not comprise an Fc domain. In certain such embodiments, the first Fab molecule, and, if present, the third Fab molecule, 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 have been swapped / substituted for one another. In other such embodiments, the first Fab molecule, and, if present, the third Fab molecule, are each crossover Fab molecules, and the second Fab molecule is a conventional Fab molecule.

[0242] In one such embodiment, the bispecific antibody consists essentially of first and second antigen-binding moieties, and optionally one or more peptide linkers, where the first and second antigen-binding moieties are both 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 Fab heavy chain of the second antigen-binding moiety. Such an arrangement is shown schematically in Figures 11O and 11S (in these examples, the second antigen-binding domain is a VH / VL crossover Fab molecule and the first antigen-binding moiety is a conventional Fab molecule).

[0243] In another such embodiment, the bispecific antibody consists essentially of first and second antigen-binding moieties, and optionally one or more peptide linkers, where the first and second antigen-binding moieties are both 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 Fab heavy chain of the first antigen-binding moiety. Such an arrangement is shown schematically in Figures 11P and 11T (in these examples, the second antigen-binding domain is a VH / VL crossover Fab molecule and the first antigen-binding moiety is a conventional Fab molecule).

[0244] In some embodiments, the first Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule, and the bispecific antibody further comprises a third antigen-binding moiety, particularly a third Fab molecule, wherein said third Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the bispecific antibody consists essentially of first, second, and third Fab molecules and optionally one or more peptide linkers, wherein the first Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule. Such configurations are shown schematically in Figures 11Q and 11U (in these examples, the second antigen-binding domain is a VH / VL crossover Fab molecule and the first and third antigen-binding moieties are each conventional Fab molecules), or in Figures 11X and 11Z (in these examples, the second antigen-binding domain is a conventional Fab molecule and the first and third antigen-binding moieties are each VH / VL crossover Fab molecules).

[0245] In some embodiments, the second Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule, and the bispecific antibody further comprises a third antigen-binding moiety, particularly a third Fab molecule, wherein said third Fab molecule is fused at its N-terminus to the C-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the bispecific antibody consists essentially of first, second, and third Fab molecules and optionally one or more peptide linkers, wherein the second Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule, and the third Fab molecule is fused at its N-terminus to the C-terminus of the Fab heavy chain of the first Fab molecule. Such configurations are shown schematically in Figures 11R and 11V (in these examples, the second antigen-binding domain is a VH / VL crossover Fab molecule and the first and third antigen-binding moieties are each conventional Fab molecules), or in Figures 11W and 11Y (in these examples, the second antigen-binding domain is a conventional Fab molecule and the first and third antigen-binding moieties are each VH / VL crossover Fab molecules).

[0246] In a particular embodiment, a bispecific antibody according to the invention 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 by a light chain variable region) (VH (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the bispecific antibody further 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) ) is included.

[0247] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VL) sharing 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 constant region of a first Fab molecule. (2) -CH1 (2) -VH (1) -CH1 (1) In some embodiments, the bispecific antibody further 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) ) is included.

[0248] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VH) sharing 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 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. (2) -CL (2) -VH (1) -CH1 (1) In some embodiments, the bispecific antibody further 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) ) is included.

[0249] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VL) sharing 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 constant region of a first Fab molecule. (2) -CH1 (2) -VH (1) -CH1 (1) In some embodiments, the bispecific antibody further 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) ) is included.

[0250] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the 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) (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the bispecific antibody further 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 some embodiments, the bispecific antibody comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0251] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the 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) (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VH (2) -CL (2) In some embodiments, the bispecific antibody further 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 some embodiments, the bispecific antibody comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0252] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VL) in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the 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 the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (2)-CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some embodiments, the bispecific antibody further 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 some embodiments, the bispecific antibody comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0253] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the 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 the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some embodiments, the bispecific antibody further 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 some embodiments, the bispecific antibody comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0254] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain of a second Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the first Fab molecule (i.e., the first 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 light chain variable region of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region). (2) -CH1 (2) -VL (1) -CH1 (1) -VL (3) -CH1 (3) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH (1) -CL (1) ), and the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH (3) -CL (3) ) is included.

[0255] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first 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 the Fab heavy chain variable region of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region). (2) -CH1 (2) -VH (1) -CL (1) -VH (3) -CL (3) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL (1) -CH1 (1) ), and the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL (3) -CH1 (3) ) is included.

[0256] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VL) in which the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (i.e., the third 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 the Fab light chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first 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 the Fab heavy chain of a second Fab molecule. (3) -CH1 (3) -VL (1) -CH1 (1) -VH (2) -CH1 (2) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH (1) -CL (1) ), and the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH (3) -CL (3) ) is included.

[0257] In a particular embodiment, a bispecific antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (i.e., the third 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 the Fab heavy chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first 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 the Fab heavy chain of a second Fab molecule. (3) -CL (3) -VH (1) -CL (1) -VH (2) -CH1 (2) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL (1) -CH1 (1) ), and the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In some embodiments, the bispecific antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL (3) -CH1 (3) ) is included.

[0258] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. a first antigen-binding portion which is a Fab molecule comprising: and b) a second antigen-binding moiety that binds to human CD3, 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 and heavy chains are replaced by one another; E) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. a second antigen-binding moiety comprising: c) an Fc domain composed of the first and second subunits A bispecific antibody comprising: where (i) a first antigen-binding moiety according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second antigen-binding moiety according to b) and the second antigen-binding moiety according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c); or (ii) a bispecific antibody in which the second antigen-binding moiety according to b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety according to a) at the C-terminus of the Fab heavy chain, and the first antigen-binding moiety according to a) is fused to the N-terminus of one of the subunits of the Fc domain according to c) at the C-terminus of the Fab heavy chain.

[0259] In certain embodiments, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. a first antigen-binding portion which is a Fab molecule comprising: and b) a second antigen-binding moiety that binds to human CD3, 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 and heavy chains are replaced by one another; E) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. a second antigen-binding moiety comprising: c) a third antigen-binding portion that binds to the first antigen and is identical to the first antigen-binding portion; and d) Fc domain composed of the first and second subunits A bispecific antibody comprising: where (i) the first antigen-binding moiety according to a) 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 according to b), and the second antigen-binding moiety according to b) and the third antigen-binding moiety according to c) 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 according to d); or (ii) a bispecific antibody wherein the second antigen-binding moiety according to b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety according to a) at the C-terminus of the Fab heavy chain, and the first antigen-binding moiety according to a) and the third antigen-binding moiety according to c) are each fused to the N-terminus of one of the subunits of the Fc domain according to d) at the C-terminus of the Fab heavy chain.

[0260] In another embodiment, the present invention provides a method for producing a composition comprising: a) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. a first antigen-binding portion which is a Fab molecule comprising: and b) a second antigen-binding moiety that binds to human CD3, 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 and heavy chains are replaced by one another; E) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. a second antigen-binding moiety comprising: c) an Fc domain composed of the first and second subunits A bispecific antibody comprising: where A bispecific antibody is provided, wherein the first antigen-binding moiety according to a) and the second antigen-binding moiety according to b) 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 according to c).

[0261] In all of the different configurations of bispecific antibodies according to the invention, the amino acid substitutions described herein, if present, can be present either in the CH1 and CL domains of the first and (if present) third antigen-binding moiety / Fab molecule or in the CH1 and CL domains of the second antigen-binding moiety / Fab molecule. Preferably, such substitutions are present in the CH1 and CL domains of the first and (if present) third antigen-binding moiety / Fab molecule. In accordance with the concept of the present invention, when an amino acid substitution described herein is made in the first (and, if present, third) antigen-binding moiety / Fab molecule, none of such amino acid substitutions is 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, none of such amino acid substitutions is made in the first (and, if present, third) antigen-binding moiety / Fab molecule. Amino acid substitutions are particularly made in bispecific antibodies comprising Fab molecules in which the variable domains VL and VH1 of the Fab light and heavy chains are replaced by each other.

[0262] In particular embodiments of bispecific antibodies according to the invention, particularly where the amino acid substitutions described herein are made in the first (and, if present, third) antigen-binding moiety / Fab molecule, the constant domain CL of the first (and, if present, third) Fab molecule is of the kappa isotype. In other embodiments of bispecific antibodies according to the invention, particularly where 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, if present, third) antigen-binding moiety / Fab molecule and the constant domain CL of the second antigen-binding moiety / Fab molecule are of the kappa isotype.

[0263] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) a first antigen-binding portion which is a Fab molecule comprising a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; and b) a second antigen-binding moiety that binds to human CD3, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced by each other; E) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. a second antigen-binding moiety comprising: c) an Fc domain composed of the first and second subunits A bispecific antibody comprising: in which in the constant domain CL of the first antigen-binding moiety according to a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most particularly by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the first antigen-binding moiety according to a) 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); where (i) a first antigen-binding moiety according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second antigen-binding moiety according to b) and the second antigen-binding moiety according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c); or (ii) a bispecific antibody in which the second antigen-binding moiety according to b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety according to a) at the C-terminus of the Fab heavy chain, and the first antigen-binding moiety according to a) is fused to the N-terminus of one of the subunits of the Fc domain according to c) at the C-terminus of the Fab heavy chain.

[0264] In certain embodiments, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. a first antigen-binding portion which is a Fab molecule comprising: and b) a second antigen-binding moiety that binds to human CD3, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced by each other; E) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. a second antigen-binding moiety comprising: c) a third antigen-binding portion that binds to the first antigen and is identical to the first antigen-binding portion; and d) Fc domain composed of the first and second subunits A bispecific antibody comprising: wherein in the constant domain CL of the first antigen-binding moiety according to a) and the third antigen-binding moiety according to c) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most particularly by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the first antigen-binding moiety according to a) 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); where (i) the first antigen-binding moiety according to a) 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 according to b), and the second antigen-binding moiety according to b) and the third antigen-binding moiety according to c) 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 according to d); or (ii) a bispecific antibody wherein the second antigen-binding moiety according to b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety according to a) at the C-terminus of the Fab heavy chain, and the first antigen-binding moiety according to a) and the third antigen-binding moiety according to c) are each fused to the N-terminus of one of the subunits of the Fc domain according to d) at the C-terminus of the Fab heavy chain.

[0265] In another embodiment, the present invention provides a method for producing a composition comprising: a) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. a first antigen-binding portion which is a Fab molecule comprising: and b) a second antigen-binding moiety that binds to human CD3, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced by each other; E) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. a second antigen-binding moiety comprising: c) an Fc domain composed of the first and second subunits A bispecific antibody comprising: in which in the constant domain CL of the first antigen-binding moiety according to a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most particularly by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the first antigen-binding moiety according to a) 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); (i) A bispecific antibody is provided, in which a first antigen-binding moiety according to a) and a second antigen-binding moiety according to b) 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 according to c).

[0266] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first antigen-binding moiety that binds to HLAG, wherein the first antigen-binding moiety is: A) a VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30. a first antigen-binding portion which is a Fab molecule comprising: and b) a second antigen-binding moiety that binds to human CD3, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced by each other; E) A VH domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58, and a VL domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. a second antigen-binding moiety comprising: c) an Fc domain composed of the first and second subunits A bispecific antibody comprising: in which in the constant domain CL of the first antigen-binding moiety according to a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most particularly by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the first antigen-binding moiety according to a) 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); where (i) a first antigen-binding moiety according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second antigen-binding moiety according to b) and the second antigen-binding moiety according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c); or (ii) a bispecific antibody in which the second antigen-binding moiety according to b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety according to a) at the C-terminus of the Fab heavy chain, and the first antigen-binding moiety according to a) is fused to the N-terminus of one of the subunits of the Fc domain according to c) at the C-terminus of the Fab heavy chain.

[0267] In certain embodiments, the present invention provides a bispecific antibody comprising:

[0268] In a particular aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) first and third antigen-binding moieties that bind to a first antigen; wherein the first antigen is HLA-G, and the first and second antigen-binding moieties are (conventional) Fab molecules each comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32, or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34; b) a second antigen-binding portion that binds to a second antigen; wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 63; c) an Fc domain composed of the first and second subunits; 1. A bispecific antibody comprising: where in the constant domain CL of the first and third antigen-binding moieties according to a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most particularly by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the first and third antigen-binding moieties according to a) 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); moreover, The first antigen-binding moiety according to a) 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 according to b), and the second antigen-binding moiety according to b) and the third antigen-binding moiety according to a) 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 according to c). Bispecific antibodies are provided.

[0269] 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 substituted with a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V), optionally the threonine residue at position 366 is substituted with a serine residue (T366S), and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0270] In further embodiments according to these aspects of the invention, the first subunit of the Fc domain further comprises a substitution of the serine residue at position 354 with a cysteine residue (S354C) or the glutamic acid residue at position 356 with a cysteine residue (E356C), particularly a substitution of the serine residue at position 354 with a cysteine residue, and the second subunit of the Fc domain further comprises a substitution of the tyrosine residue at position 349 with a cysteine residue (Y349C) (numbering according to the Kabat EU index).

[0271] In yet another embodiment 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 substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbering according to the Kabat EU index).

[0272] In yet another embodiment according to these aspects of the invention, the Fc domain is a human IgG1 Fc domain.

[0273] Certain embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, wherein the antibodies comprise a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 64, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 65, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 66, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 67.

[0274] In further specific embodiments, the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 64, a polypeptide comprising the amino acid sequence of SEQ ID NO: 65, a polypeptide comprising the amino acid sequence of SEQ ID NO: 66, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 67.

[0275] Certain embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, wherein the antibodies comprise a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 68, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 69, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 70, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 71.

[0276] In further specific embodiments, the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 68, a polypeptide comprising the amino acid sequence of SEQ ID NO: 69, a polypeptide comprising the amino acid sequence of SEQ ID NO: 70, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 71.

[0277] Certain embodiments of the invention are bispecific antibodies that bind to human HLA-G and human CD3, wherein the antibodies comprise a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 72, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 73, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 74, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 75.

[0278] In further specific embodiments, the bispecific antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 72, a polypeptide comprising the amino acid sequence of SEQ ID NO: 73, a polypeptide comprising the amino acid sequence of SEQ ID NO: 74, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 75.

[0279] Fc domain In a particular embodiment, a bispecific antibody of the invention comprises an Fc domain composed of a first and a second subunit, and the characteristics of the Fc domain described herein in relation to bispecific antibodies apply equally to the Fc domain comprised in an antibody of the invention.

[0280] The Fc domain of a bispecific antibody consists of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, a bispecific antibody of the invention comprises no more than one Fc domain.

[0281] 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 (Kabat EU index numbering), in particular the amino acid substitution S228P. 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.

[0282] Fc domain modifications that promote heterodimerization Bispecific antibodies according to the present invention contain different antigen-binding moieties, which can be fused to one or the other of the two subunits of the Fc domain, which are therefore typically contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization results in multiple possible combinations of the two polypeptides. Therefore, to improve the yield and purity of bispecific antibodies in recombinant production, it would be advantageous to introduce modifications to the Fc domain of the bispecific antibody that promote the association of the desired polypeptides.

[0283] Thus, in a particular embodiment, the Fc domain of a bispecific antibody according to the invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of the Fc domain of human IgG is in the CH3 domain of the Fc domain. Thus, in one embodiment, said modification is in the CH3 domain of the Fc domain.

[0284] There are several approaches for modifications in the CH3 domain of an Fc domain to effect heterodimerization, which are described in detail in, for example, WO 96 / 27011, WO 98 / 050431, EP1870459, 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 Fc domain subunit and the CH3 domain of the second Fc domain subunit are both engineered to be complementary to each other, such that each CH3 domain (or its constituent heavy chain) is unable to homodimerize with itself but is forced to heterodimerize with the other complementary engineered CH3 domain (thus heterodimerizing the first and second CH3 domains and preventing homodimerization between the two first CH3 domains or the two second CH3 domains). These different approaches to improving heavy chain heterodimerization are considered as different alternatives in combination with heavy-light chain modifications in bispecific antibodies to reduce heavy / light chain mispairing and Bence Jones by-products (e.g., swapping / substituting VH and VL in one binding arm and introducing oppositely charged amino acid substitutions at the CH1 / CL interface).

[0285] In certain embodiments, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, comprising 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.

[0286] Knob-into-hole technology has been described, for example, in U.S. Patent Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protuberance can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger one (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine).

[0287] 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 substituted with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that can be positioned within a cavity within the CH3 domain of a second subunit, and in the CH3 domain of a second subunit of the Fc domain, an amino acid residue is substituted with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit that can be positioned within the protuberance within the CH3 domain of the first subunit.

[0288] Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0289] Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0290] The protuberances and cavities can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0291] In a particular embodiment, in the CH3 domain of the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is substituted 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 substituted with a valine residue (Y407V). In one embodiment, the second subunit of the Fc domain further comprises a substitution of the threonine residue at position 366 with a serine residue (T366S) and a substitution of the leucine residue at position 368 with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0292] In yet another embodiment, the first subunit of the Fc domain further comprises a substitution of a cysteine residue for the serine residue at position 354 (S354C) or a cysteine residue for the glutamic acid residue at position 356 (E356C) (particularly a substitution of a cysteine residue for the serine residue at position 354), and a cysteine residue for the tyrosine residue at position 349 (Y349C) (numbering according to the Kabat EU index) in the second subunit of the Fc domain. Introduction of these two cysteine residues results in 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)).

[0293] In a particular 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 EU index of Kabat).

[0294] In certain embodiments, an antigen-binding moiety that binds a second antigen (e.g., a T cell activation 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, fusing an antigen-binding moiety that binds a second antigen, such as a T cell activation antigen, to a knob-containing subunit of an Fc domain would minimize the generation of antibodies comprising two antigen-binding moieties that (further) bind to the T cell activation antigen (steric clash of a polypeptide comprising two knobs).

[0295] Other techniques of CH3 modification to effect heterodimerization are contemplated as alternatives according to the present invention and are 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 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0296] In one embodiment, the heterodimerization technique described in EP 1 870 459 is used instead. This technique is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment of the bispecific antibody of the invention comprises the amino acid mutants 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 the Kabat EU index).

[0297] In another embodiment, a bispecific antibody of the invention comprises the amino acid mutations 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 further comprises 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).

[0298]

[0044] 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 further 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 numbered according to Kabat EU index).

[0299] In one embodiment, the heterodimerization technique 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 the Kabat EU index). In a further embodiment, the first CH3 domain comprises the additional amino acid mutation L351K. In a further embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E, preferably L368E (numbering according to the Kabat EU index).

[0300] 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 and Y407A, and the second CH3 domain comprises the amino acid mutations T366A and K409F. In a further embodiment, the second CH3 domain comprises, at positions T411, D399, S400, F405, N390, or K392, e.g., a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W; b) D399R, D399W, D399Y, or D399K; c) S400E, S400F, or S400G; and / or F400D, S400R, or S400K; d) F405I, F405M, F405T, F405S, F405V, or F405W; e) N390R, N390K, or N390D; or f) K392V, K392M, K392R, K392L, K392F, or K392E (numbering according to the Kabat EU index). 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 mutation 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).

[0301] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is alternatively used, e.g., using amino acid modifications at positions selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0302] In one embodiment, the heterodimerization approach described in WO 2011 / 090762 is alternatively used, which also employs the knobs-into-holes 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 the Kabat EU index).

[0303] 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.

[0304] In another embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, e.g., as described in WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, such that homodimer formation is electrostatically unfavorable, while heterodimerization is electrostatically favored. 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 also or alternatively comprises 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 numbered according to the Kabat EU index).

[0305] In yet another embodiment, the heterodimerization approach described in WO 2007 / 147901 is alternatively used. 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).

[0306] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 can alternatively be used.

[0307] 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 the Kabat EU index).

[0308] Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain confers favorable pharmacokinetic properties to the bispecific antibody (or antibody), including a long serum half-life and favorable tissue-to-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, at the same time, the Fc domain can result in undesirable targeting of the bispecific antibody (or antibody) to cells expressing Fc receptors rather than to preferred antigen-bearing cells. Furthermore, simultaneous activation of the Fc receptor signaling pathway can lead to cytokine release, which, combined with the T cell activating properties (e.g., in bispecific antibody embodiments in which the second antigen-binding moiety binds to a T cell-activating antigen) and the long half-life of the bispecific antibody, can result in excessive activation of cytokine receptors, causing severe side effects when administered systemically. Activation of immune cells other than T cells (Fc receptor-bearing) can even reduce the effectiveness of the bispecific antibody (particularly bispecific antibodies in which the second antigen-binding moiety binds to a T cell-activating antigen) due to potential destruction of T cells, for example, by NK cells.

[0309] Thus, in certain embodiments, the Fc domain of a 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%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or a bispecific antibody comprising a native IgG1 Fc domain), and / or exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a bispecific 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 certain 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 specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a specific 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%, and more particularly more than about 90% of the binding affinity of a native IgG1 Fc domain (or a bispecific antibody comprising a native IgG1 Fc domain).

[0310] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to an unengineered Fc domain. In certain embodiments, the Fc domain of a bispecific antibody comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an 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 an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor are present, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or at least 50-fold. In one embodiment, a bispecific antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and 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 specific 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, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly C1q, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain for said receptor, is achieved when the Fc domain (or a bispecific antibody comprising said Fc domain) exhibits a binding affinity for FcRn that is greater than about 70% of that of an unengineered form of the Fc domain (or a bispecific antibody comprising said unengineered form of the Fc domain). An Fc domain, or a bispecific antibody of the invention comprising said Fc domain, may exhibit more than about 80%, or even more than about 90%, of such affinity. In certain embodiments, the Fc domain of the bispecific antibody has been engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function can include, but is not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent T 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 that induces apoptosis, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group consisting 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).

[0311] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or the effector function 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 the 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 the Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly 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, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the 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 the 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., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (Kabat EU index numbering).

[0312] In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly 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 function.

[0313] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions compared to IgG1 antibodies. Accordingly, in some embodiments, the Fc domain of a bispecific antibody of the invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce binding affinity to Fc receptors and / or its effector functions, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering according to the Kabat EU index). In a particular embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, in particular the amino acid substitutions S228P, L235E and P329G (numbering according to the Kabat EU index). Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in PCT Publication No. WO2012 / 130831, which is incorporated herein by reference in its entirety.

[0314] In particular 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 the Kabat EU index).

[0315] In certain embodiments, N-glycosylation of the Fc domain is removed. In one such embodiment, the Fc domain comprises an amino acid substitution at position N297, particularly replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the Kabat EU index).

[0316] In addition to the Fc domains described above and in PCT Publication No. WO2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056 and P329G (Kabat EU index numbering)). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

[0317] Variant 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 can include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be verified, for example, by sequencing.

[0318] Binding to Fc receptors can be readily measured, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), and such Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain, or a bispecific antibody comprising an Fc domain, to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, for example, human NK cells expressing the FcγIIIa receptor.

[0319] The effector function of an Fc domain, or a bispecific antibody comprising an Fc domain, can be measured by methods known in the art. Exemplary 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 assays may be used (e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assays (see CellTechnology, Inc. Mountain View, CA; and CytoTox 96® Non-radioactive Cytotoxicity Assay (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 can be assessed in vivo, e.g., in an animal model, such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0320] In some embodiments, binding of the Fc domain to complement components, particularly 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. A C1q binding assay can be performed to determine whether the Fc domain, or a bispecific antibody comprising the Fc domain, is capable of binding C1q and therefore has CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. CDC assays can also be performed to assess complement activation (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)).

[0321] 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).

[0322] In further aspects, an anti-HLA-G antibody according to any of the above embodiments may incorporate any of the features, either alone or in combination, as described in Sections 1-6 below.

[0323] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a cytotoxicity 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 to 10 -13 M, e.g. 10 -9 M to 10 -13It has a dissociation constant KD of 0.05 M.

[0324] In one preferred embodiment, KD is measured using a surface plasmon resonance assay with a BIACORE® at 25°C using an antigen CM5 chip immobilized at approximately 10 response units (RU). 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 (~0.2 μM) in 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were incubated in 0.05% polysorbate 20 (TWEEN-20) at 25°C and a flow rate of approximately 25 μl / min. TM ) in PBS containing surfactant (PBST). The association rate (k or ka) and dissociation rate (k or k) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore® Evaluation Software version 3.2). The equilibrium dissociation constant (KD) is calculated as the ratio kd / ka (k / k). See, for example, Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881. If the association rate by the surface plasmon resonance assay described above is 10 6 M -1 s -1 If the association rate exceeds 100 kJ / s, the association rate can be measured using a spectrophotometer, e.g., a spectrophotometer equipped with a flow stop (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirred cuvette. TMMeasurements can be made using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass = 16 nm) 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 by a spectrophotometer (ThermoSpectronic).

[0325] 2. Antibody fragment 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 specific antibody fragments, see Hudson et al., Nat Med 9, 1299 (2003). For a review of scFv fragments, see, e.g., Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-lives in vivo.

[0326] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 0404097; 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 (2003) 129-134.

[0327] Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 (B1)).

[0328] 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 phage), as described herein.

[0329] 3. Chimeric and humanized antibodies 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, SL et 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 mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0330] 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. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody will also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0331] Humanized antibodies and methods for their production are reviewed, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and further described, for example, in 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, SV 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"); and 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).

[0332] Human framework regions that can 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 a particular subgroup 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, LG et 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 obtained from screening of FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (19969 22611-22618)).

[0333] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are reviewed 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.

[0334] Human antibodies can be prepared by administering an immunogen to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. For example, the XENO mouse. TM See also U.S. Patent Nos. 6,075,181 and 6,150,584, describing HuMab® technology, U.S. Patent No. 5,770,429, describing HuMab® technology, U.S. Patent No. 7,041,870, describing KM Mouse® technology, and U.S. Patent Application Publication No. 2007 / 0061900, describing VelociMouse® technology. The human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0335] 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, e.g., Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, B.R. 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 by 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 those described, for example, in 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, H.P. and Brandlein, S., Histology and Histopathology 20 (2005) 927-937 and Vollmers, H.P. and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

[0336] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0337] 5. Library-derived antibodies Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). 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 further described, 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, SS et al., J. Mol. Biol. 338 (2004) 299-310; Lee, CV et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, FA, Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, CV et al., J. Immunol. Methods 284 (2004) 119-132.

[0338] In a specific phage display method, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR), randomly recombined in phage libraries, and then screened for antigen-binding phage, as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455. Phages typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without immunization, 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, using PCR primers containing random sequences to encode the highly variable CDR3 regions, and achieving 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.

[0339] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0340] 6. Antibody Variants 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 an antibody can 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 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, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0341] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Exemplary changes are shown in Table 1 under the heading of "Exemplary Substitutions" and are further described below with reference to classes of amino acid side chains. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." Amino acid substitutions can be introduced into an antibody of interest, and the products can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0342] TIFF2025118635000002.tif168170

[0343] Amino acids can be grouped 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 that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0344] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0345] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have modified (e.g., improved) certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0346] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in "hot spots" of HVRs, i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196), and / or in SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting a secondary library is described, for example, in Hoogenboom, HR et al. in 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 any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0347] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs 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 can be made in HVRs. Such modifications may be outside of HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR contains unchanged or only one, two, or three amino acid substitutions.

[0348] A useful method for identifying antibody residues or regions that can 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, target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antigen and the antibody is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact and neighboring residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0349] 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 includes an antibody with an N-terminal methionine 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.

[0350] b) Fc region mutants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0351] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant, which has substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0352] 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, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0353] In one embodiment of the invention, such an antibody is an IgG1 comprising the mutations L234A and L235A, or the mutations L234A, L235A and P329G. In another embodiment, such an antibody is an IgG4 comprising the mutations S228P and L235E, or S228P, L235E and / or P329G (numbering according to the EU index of Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0354] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those 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, for example, a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0355] See also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Patent Nos. 5,648,260; 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0356] c) Cysteine Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In certain embodiments, the substituted residues are located at accessible sites of the antibody. By substituting 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 moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be substituted 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.

[0357] d) Antibody derivative In certain embodiments, the antibodies provided herein can be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Moieties suitable 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 (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used in therapy under defined conditions.

[0358] In another embodiment, a conjugate of an antibody and a nonprotein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonprotein moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, including, but not limited to, wavelengths that heat the nonprotein moiety to a temperature that is not harmful to normal cells but that kills cells proximal to the antibody-nonprotein moiety.

[0359] B. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-HLA-G antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising 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 nucleic acids are provided. In a further ...

Claims

1. A multispecific 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 multispecific antibody that does not cross-react with a modified human HLA-G β2M MHC I complex comprising SEQ ID NO: 44, in which HLA-G specific amino acids are replaced by HLA-A consensus amino acids.

2. the antibody is bispecific; wherein the first antigen-binding portion of the antibody that binds to human HLA-G is A) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 3; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or B) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 11; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or C) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 19; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or D) (a) a VH domain comprising (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 27; and (b) a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 30; the second antigen-binding portion that binds to a T cell activation antigen binds to human CD3, and E) (a) a VH domain comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 56; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 57; and (iii) an HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 58; and (b) a VL domain comprising: (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and (iii) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:

61.

2. The multispecific antibody of claim 1 , comprising:

3. the first antigen-binding moiety A) vii) the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8; viii) or humanized variants of VH and VL of the antibody of i). contains; or ix) comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; or B) comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16; or C) comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24; or D) comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32; and the second antigen-binding moiety is E) comprising the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63; The bispecific antibody of claim 2.

4. the first antigen-binding portion i) comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32; or ii) comprises the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 34; and the second antigen-binding moiety is comprising the VH sequence of SEQ ID NO: 62 and the VL sequence of SEQ ID NO: 63; The bispecific antibody of claim 3.

5. The antibody, a) does not cross-react with the human HLA-A2 β2M MHC I complex comprising SEQ ID NO: 39 and SEQ ID NO: 37; and / or b) does not cross-react with the mouse H2Kd β2M MHC I complex comprising SEQ ID NO: 45; and / or c) does not cross-react with the rat RT1A β2M MHC I complex containing SEQ ID NO: 47; and / or d) inhibits binding of ILT2 to a monomeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43); and / or e) inhibits binding of ILT2 to the trimeric HLA-G β2M MHC I complex (comprising SEQ ID NO: 43) by more than 50% (in one embodiment by more than 60%) (when compared to binding without the antibody); and / or f) inhibits binding of ILT2 to monomeric and / or dimeric and / or trimeric HLA-G β2M MHC I complexes (comprising SEQ ID NO: 43) by more than 50% (in one embodiment, more than 80%) (when compared to binding without the antibody); and / or g) inhibits (by more than 50%, and in one embodiment by more than 80%) the binding of ILT2 to JEG3 cells (ATCC No. HTB36) (HLA-G above) when compared to binding without antibody; and / or h) binds to JEG-3 cells (ATCC No. HTB36) (HLA-G above) (see Example 5) and inhibits (by more than 50%, in one embodiment more than 80%) the binding of ILT2 to JEG-3 cells (ATCC No. HTB36) (HLA-G above) (when compared to binding without antibody); and / or i) inhibits binding of CD8a to HLAG by more than 80% (compared to binding without the antibody); and / or j) restores HLA-G-specific suppressive immune responses by monocytes co-cultured with JEG-3 cells (ATCC HTB36); and / or k) Inducing T cell-mediated cytotoxicity in the presence of HLAG-expressing tumor cells (e.g., JEG-3 cells (ATCC HTB36)). A multispecific antibody according to any one of claims 1 to 4.

6. 6. The multispecific antibody of claim 1 , wherein the first and second antigen-binding moieties are Fab molecules.

7. 7. A multispecific antibody according to any one of claims 1 to 6, wherein the second antigen-binding moiety is a Fab molecule, in which the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are substituted for each other.

8. 8. The multispecific antibody according to any one of claims 1 to 7, wherein the first antigen-binding moiety is a Fab molecule in which in the constant domains 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 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).

9. 9. The multispecific antibody of claim 1 , wherein the first and second antigen-binding moieties are fused to each other, optionally via a peptide linker.

10. 10. The multispecific antibody of claim 1 , wherein the first and second antigen-binding moieties are each Fab molecules, and wherein (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, 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.

11. 11. The multispecific antibody of claim 1 , comprising a third antigen-binding moiety.

12. 12. The multispecific antibody of claim 11, wherein the third antigen-binding portion is identical to the first antigen-binding portion.

13. 13. An isolated nucleic acid encoding a multispecific antibody according to any one of claims 1 to 12.

14. 13. A pharmaceutical formulation comprising the multispecific antibody of any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

15. 13. A multispecific antibody according to any one of claims 1 to 12 for use in the treatment of cancer.

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