Treatment of cancer with anti-HLA-G / anti-CD3 bispecific antibodies and 4-1BB (CD137) agonists

Abstract

The present invention relates to the treatment of cancer, in particular to the treatment of cancer using anti-HLA-G / anti-CD3 bispecific antibodies and 4-1BB (CD137) agonists.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the treatment of cancer, particularly HLA-G-expressing cancers, using anti-HLA-G / anti-CD3 bispecific antibodies and 4-1BB (CD137) agonists. [Background technology]

[0002] background T cell-activating bispecific antibodies are promising cancer therapies designed to target cytotoxic T cells against tumor cells. The simultaneous binding of such antibodies to CD3 on T cells and to antigens expressed on tumor cells forces a transient interaction between the tumor cell and the T cell, leading to T cell activation and subsequent tumor cell lysis.

[0003] Human major histocompatibility complex class I, 6, also known as human leukocyte antigen G (HLA-G), is a protein encoded by the HLA-G gene in humans. HLA-G belongs to the HLA nonclassical class I heavy chain paralogs. This class I molecule is a heterodimer (beta-2 microglobulin) consisting of a heavy chain and a light chain. The heavy chain is membrane-anchored but can also be excreted / secreted. HLA-G is primarily expressed on placental cytotrophoblasts. Several tumors (e.g., pancreatic, breast, skin, colorectal, gastric, and ovarian) express HLA-G (Lin, A. et al., Mol Med. 21 (2015) 782-791 (Non-Patent Document 1); Amiot, L., et al., Cell Mol Life Sci. 68 (2011) 417-431 (Non-Patent Document 2)). Its expression has also been reported to be associated with pathologies such as inflammatory diseases, GvHD, and cancer. HLA-G expression has been reported to be associated with poor cancer prognosis, and tumor cells evade host immune surveillance by inducing immune tolerance / suppression through HLA-G expression.

[0004] Because HLA-G shares high homology (>98%) with other MHC I molecules, it is difficult to generate truly HLA-G-specific antibodies that lack cross-reactivity to other MHC I molecules. Due to the high polymorphism and homology of the HLA family, the majority of antibodies lack truly specific HLA-G binding properties and often bind or cross-react with other HLA family members (either as an 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 (considered non-antagonistic antibodies). Antibodies that specifically bind to HLA-G are described in International Publication Nos. WO 2019 / 202040 (Patent Document 1), WO 2019 / 202041 (Patent Document 2), WO 2022 / 24024 (Patent Document 3), and WO 2022 / 129120 (Patent Document 4). These documents also describe T cell bispecific antibodies (TCBs) that contain a binding moiety that specifically binds to HLA-G.

[0005] The antitumor activity of HLA-G TCB has been demonstrated in vitro using different HLA-G-positive tumor cell lines and in vivo using mouse models. The T cell activation, IFNγ secretion, and cytotoxicity induced by HLA-G TCB were shown to be dose-dependent and correlated with the density of HLA-G on the cell surface and the percentage of HLA-G-positive cells. Consistent with the TCB's mode of action, tumor growth inhibition was accompanied by increased cytokine secretion (including IFNγ), tumor T cell infiltration, and activation, as reflected by increased expression of T cell activation markers such as CD69, CD25, and granzyme B. However, T cell activation and cytokine secretion were transient and returned to baseline after treatment. Furthermore, activated T cells can become exhausted and undergo activation-induced cell death. Therefore, additional therapeutic agents that maintain sustained T cell activation are needed to achieve complete tumor cell elimination.

[0006] Costimulation of T cells through the 4-1BB receptor (e.g., by 4-1BBL binding) leads to activation of multiple signaling cascades in T cells, potently enhancing T cell activation. When combined with T cell receptor activators, agonistic 4-1BB antibodies enhance T cell proliferation, stimulate cytokine secretion, and reduce T cell susceptibility to activation-induced cell death.

[0007] Therefore, it would be desirable to enhance the efficacy of HLA-G-targeted T cell-activating antibodies to maximize their therapeutic benefit, particularly in solid tumors. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2019 / 202040 [Patent Document 2] International Publication No. 2019 / 202041 [Patent Document 3] International Publication No. 2022 / 24024 [Patent Document 3] International Publication No. 2022 / 129120 [Non-patent literature]

[0009] [Non-Patent Document 1] Lin,A.et al.,Mol Med.21(2015)782-791 [Non-patent document 2] Amiot, L., et al., Cell Mol Life Sci.68(2011)417-431 Summary of the Invention

[0010] Brief description of the invention The present invention enhances T cell responses (e.g., in solid tumor cancers) by combining an HLA-G-targeting T cell-activating bispecific antibody with a 4-1BB (CD137) agonist that provides a positive costimulatory signal (4-1BBBL) to T cells, particularly a 4-1BB (CD137) agonist that targets a stromal antigen such as fibroblast activation protein (FAP). Fibroblast activation protein (FAP) is highly expressed by cancer-associated fibroblasts (CAFs) and has the ability to regulate the tumor microenvironment (TME) by remodeling the extracellular matrix (ECM). As described, FAP is upregulated on cancer-associated fibroblasts in tumors, and thus the FAP specificity of the 4-1BB (CD137) agonist provides T cell costimulation strictly restricted to the tumor.

[0011] The inventors have discovered that the combination of an HLA-G-targeting T cell-activating bispecific antibody with a 4-1BB (CD137) agonist results in enhanced activity in HLA-G-expressing cancers compared to the HLA-G-targeting T cell-activating bispecific antibody alone.

[0012] Using in vivo data from patient-derived tumor models, the inventors surprisingly found that tumor cell lysis induced by an anti-HLA-G / anti-CD3 bispecific antibody in the presence of FAP-expressing fibroblasts was enhanced by the addition of the 4-1BB agonist FAP4-1BBL, even in patient-derived tumor models and / or at doses where the anti-HLA-G / anti-CD3 bispecific antibody alone showed little or no activity.

[0013] Thus, in a first aspect, the present invention provides an anti-HLA-G / anti-CD3 bispecific antibody for use in the treatment of cancer in an individual, the treatment comprising administration of the anti-HLA-G / anti-CD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

[0014] In a further aspect, the present invention provides a 4-1BB (CD137) agonist for use in the treatment of cancer in an individual, the treatment comprising administration of a 4-1BB (CD137) agonist in combination with an anti-HLA-G / anti-CD3 bispecific antibody.

[0015] In one aspect, the invention provides the use of an anti-HLA-G / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of cancer in an individual, the treatment comprising administration of the anti-HLA-G / anti-CD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

[0016] In a further aspect, the present invention provides the use of a 4-1BB (CD137) agonist in the manufacture of a medicament for the treatment of cancer in an individual, the treatment comprising administration of a 4-1BB (CD137) agonist in combination with an anti-HLA-G / anti-CD3 bispecific antibody.

[0017] In a still further aspect, the present invention provides a method for treating cancer in an individual comprising administering to the individual an anti-HLA-G / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist.

[0018] In one aspect, the present invention also provides a kit comprising a first medicament comprising an anti-HLA-G / anti-CD3 bispecific antibody and a second medicament comprising a 4-1BB (CD137) agonist, and optionally further comprising a package insert containing instructions for administering the first medicament and the second medicament in combination to treat cancer in an individual.

[0019] The anti-HLA-G / anti-CD3 bispecific antibodies, 4-1BB (CD137) agonists, methods, uses or kits described above and herein may incorporate any of the features described below, either alone or in combination (unless the context dictates otherwise).

[0020] The anti-HLA-G / anti-CD3 bispecific antibodies herein are bispecific antibodies that specifically bind to CD3 and HLA-G, particularly HLA-G, on JEG3 cells (ATCC No. HTB36). In particular, useful anti-HLA-G / anti-CD3 bispecific antibodies for use in the present invention inhibit ILT2 binding to JEG3 cells (ATCC No. HTB36) (on which HLA-G is expressed) and / or bind to and inhibit ILT2 binding to JEG3 cells (ATCC No. HTB36) (on which HLA-G is expressed). Particularly useful anti-HLA-G / anti-CD3 bispecific antibodies for use in the present invention are described, for example, in WO 2022 / 129120 (incorporated herein by reference in its entirety). DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Invention definition The term "bispecific" means that an antibody can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antibody contains two antigen-binding sites, each specific for a different antigenic determinant. In some embodiments, a bispecific antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

[0022] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a stretch of contiguous amino acids or a configuration consisting of distinct stretches of non-contiguous amino acids) 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 tumor cell surfaces, virus-infected cell surfaces, other diseased cell surfaces, immune cell surfaces, serum educts, and / or in the extracellular matrix (ECM).

[0023] As used herein, the terms "antigen-binding moiety" and "antigen-binding domain" are used interchangeably and refer to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can target a moiety to which it binds (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 herein. Particular antigen-binding moieties include the antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, antigen-binding moieties can include antibody constant regions known in the art, as further defined below. 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.

[0024] As used herein, the term "specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or nonspecific interactions. The binding ability of an antigen-binding moiety to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to the antigen, as measured, for example, by SPR. In certain aspects, an antigen-binding portion, or an antibody comprising an antigen-binding portion, that binds to an antigen has a denaturing activity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13Dissociation constant (K D )

[0025] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y is usually measured by the dissociation constant (K D ), and the dissociation rate constant and association rate constant (k off and k on ) is the ratio of the rate constants. Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, such as those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0026] Unless otherwise indicated, "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 as well as 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 (accession version 217) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO: 44. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 45.

[0027] As used herein, the terms "HLA-G," "human HLA-G," or "HLAG" refer to HLA-G human major histocompatibility complex, class I, G, also known as human leukocyte antigen G (HLA-G) (exemplary SEQ ID NO: 41). Typically, HLA-G forms an MHC class I complex with β2 microglobulin (B2M or β2m). In one embodiment, HLA-G refers to the MHC class I complex of HLA-G and β2 microglobulin. In a preferred embodiment, HLA-G refers to the cell surface-bound MHC class I complex of HLA-G and β2 microglobulin, also known as HLA-G1 (see, e.g., Figure 1 in WO 2022 / 129120 and Blaschitz et al., Molecular Human Reproduction, 11 (2005) 699-710, especially Figure 1).

[0028] As used herein, a "binds to human HLA-G," "specifically binds to human HLA-G," "binds to human HLA-G," or "anti-HLA-G" antibody (whether monospecific, multispecific, or bispecific) or antigen-binding portion is defined as 5.0 x 10 -8 KD value of 1.0×10 mol / l or less, in one embodiment 1.0×10 -9 KD value of 5.0×10 mol / l or less, in one embodiment 5.0×10 -8 mol / l ~ 1.0 × 10 -13 It refers to an antibody / antigen-binding portion that specifically binds to the human HLA-G antigen or its extracellular domain (ECD) with a binding affinity of K in mol / l. In one embodiment, the antibody binds to the HLA-G β2M MHC I complex comprising SEQ ID NO: 42.

[0029] Binding affinity is determined in a standard binding assay, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare, Uppsala, Sweden), using a construct comprising the HLA-G extracellular domain (e.g., its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined in a standard binding assay using an exemplary soluble HLA-G comprising an MHC class I complex comprising SEQ ID NO:42.

[0030] HLA-G has a typical 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 (β2M), which is shared with various other MHC I proteins.

[0031] 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 (e.g., 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). Unlike most other MHC class I molecules, HLA-G contains several free cysteine ​​residues. Boyson et al., Proc Nat Acad Sci USA, 99:16180 (2002), reported that recombinant soluble HLA-G5 can form disulfide-linked dimers with 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)).

[0032] HLA-G is primarily expressed on placental cytotrophoblasts. Several tumors (e.g., 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). Its expression has also been reported to be associated with pathologies such as inflammatory diseases, GvHD, and cancer. HLA-G expression has also been reported to be associated with poor cancer prognosis. Tumor cells evade host immune surveillance by inducing immune tolerance / suppression through HLA-G expression.

[0033] There are seven isoforms of HLA-G, three of which are secreted and four of which are membrane-bound. The most important functional isoforms of HLA-G include beta-2-microglobulin (β2M)-associated HLA-G1 and HLA-G5. However, the immune tolerance effects of these isoforms differ and depend on the form of the ligand (monomer, dimer) and the affinity of the ligand-receptor interaction.

[0034] HLA-G protein can be produced using standard molecular biology techniques. The nucleic acid sequence of HLA-G isoforms is known in the art. For example, see GenBank accession number AY359818.

[0035] HLA-G isotypes promote signaling through ILTs (Ig-like transcripts), particularly ILT2, ILT4, or a combination thereof.

[0036] 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, i.e., those containing cytoplasmic immunoreceptor tyrosine-dependent inhibitory motifs (ITIMs) and transducing inhibitory signals (ILT2, ILT3, ILT4, ILT5, and LIR8); (ii) activating, i.e., those containing short cytoplasmic tails and charged amino acid residues in their transmembrane domains (ILT1, ILT7, ILT8, and LIR6 alpha) and delivering activating signals via the cytoplasmic immunoreceptor tyrosine-dependent activation motifs (ITAMs) of the related common gamma chain of the Fc receptor; and (iii) ILT6, a soluble molecule lacking a transmembrane domain. Several recent studies have highlighted the immunoregulatory role of ILTs on the surface of antigen-presenting cells (APCs). The best-characterized immunoinhibitory receptors, ILT2, ILT3, and ILT4, are expressed on a wide range of immune cells, including monocytes, B cells, dendritic cells, plasmacytoid dendritic cells, and subsets of NK cells and T cells. ILT2 is expressed on a subset of T cells and has been shown to inhibit the activation and proliferation of these cells upon ligation (Colonna M. et al., J Immunol. 20011, 66:2514-2521, J Immunol 2000;165:3742-3755). ILT3 and ILT4 are upregulated by exposure of immature DCs to known immunosuppressive factors, including IL-10, vitamin D3, or suppressor CD8 T cells (Chang, CC, et al., Nat Immunol, 3:237-243 (2002)). The expression of ILTs on DCs is tightly regulated by inflammatory stimuli, cytokines, and growth factors, and is downregulated after DC activation (Ju, XS, et al., Gene, 331:159-164 (2004)).Expression of the ILT2 and ILT4 receptors is highly regulated by histone acetylation, which contributes to tightly regulated gene expression only in the myeloid lineage of cells (Nakajima, H., J Immunol, 171:6611-6620 (2003)).

[0037] Binding (activation) 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 the Suciu-Foca 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 several inhibitory ILT receptors is HLA-G. HLA-G potentially plays a role in maternal-fetal tolerance and in tumor cell evasion from immune recognition and destruction (Hunt, J. et al., Faseb J, 19:681-693 (2005)). Regulation of DC function by HLA-G-ILT interactions is most likely an important pathway in DC biology. It has been confirmed that human monocyte-derived DCs highly expressing ILT2 and ILT4 receptors still maintain a stable tolerogenic-like phenotype (CD80 low, CD86 low, HLA-DR low) that has the potential to induce T cell anergy when treated with HLA-G and stimulated with allogeneic T cells (Ristich, V., et al., Eur J Immunol, 35:1133-1142 (2005)). Furthermore, HLA-G interaction with DCs highly expressing ILT2 and ILT4 receptors resulted in the downregulation of several 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, since in vivo T cell responses to specific antigens were reduced in animals lacking GILT after targeted gene disruption (Marie, M., et al., Science, 294:1361-1365 (2001)). HLA-G / ILT interactions on DCs may interfere with the assembly and transport of MHC class II molecules to the cell surface, resulting in less efficient presentation or expression of structurally abnormal MHC class II molecules. HLA-G was confirmed 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)).

[0038] Another receptor for HLA-G is KIR2DL4, which 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 EOLong. [A published errata appears in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093; Ponte, M. et al., PNASUSA 96 (1999) 5674). KIR2DL4 (also called 2DL4) is a KIR family member (also called CD158d) that shares structural features with both activating and inhibitory receptors (Selvakumar, A. et al., Tissue Antigens 48 (1996) 285). 2DL4 has a cytoplasmic ITIM, suggesting an inhibitory function, and a positively charged amino acid in the transmembrane region, a characteristic feature of activating KIRs. Unlike other clonally distributed KIRs, 2DL4 is transcribed by all NK cells (Valiante, NM et al. Immunity 7 (1997) 739; Cantoni, C. et al. Eur J Immunol 28 (1998) 1980; Rajagopalan, S. and EOLong. [A published errata appears in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093).

[0039] HLA-G has also been shown to interact with CD8 on cytotoxic T cells (Sanders et al., J. Exp. Med., 174 (1991), 737-740) and induce CD95-mediated apoptosis in activated CD8-positive cytotoxic T cells (Fournel et al., J. Immun., 164 (2000), 6100-6104). This mechanism of elimination of cytotoxic T cells has been reported as one of the mechanisms for immune evasion and tolerance induction in pregnancy, inflammatory diseases, and cancer (Amodio G. et al., Tissue Antigens, 84 (2014), 255-263).

[0040] The term "inhibits ILT2 binding to HLA-G on JEG-3 cells (ATCC HTB36)" refers to inhibition of the binding interaction of (recombinant) ILT2 in the assay described, for example, in Example 5 of WO 2022 / 129120.

[0041] As used herein, the terms "first," "second," or "third," as used with respect to Fab molecules, etc., are used for ease of distinction when more than one of each type of moiety is present. The use of these terms is not intended to confer a particular order or orientation of the bispecific antibody unless otherwise specified.

[0042] As used herein, the term "valency" refers to the presence of a specified number of antigen-binding sites in an antibody. Thus, the term "monovalent binding to an antigen" refers to the presence of one (and no more than one) antigen-binding site in the antibody that is specific for the antigen.

[0043] 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.

[0044] 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 the structure of a native antibody.

[0045] As used herein, the term "T cell bispecific antibody (TCB)" or "T cell activating bispecific antibody" refers to a particular type of multispecific antibody, a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an activating, invariant component of the T cell receptor (TCR) complex, such as CD3, to retarget the T cell to kill the target cell. The terms "anti-HLA-G / anti-CD3 antibody," "anti-HLA-G / anti-CD3 bispecific antibody," "HLA-G TCB," and "anti-HLA-G TCB" are used interchangeably herein to describe multispecific antibodies, particularly bispecific antibodies, in which one binding specificity is for HLA-G and the other is for CD3. Examples of bispecific antibody formats that may be useful for generating anti-HLA-G / anti-CD3 antibodies include so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("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 stabilizing attachment (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)).Certain T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8)(2016)e1203498.

[0046] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, 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. For a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having extended in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). 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 some embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).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.

[0047] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain 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, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. When used herein in reference to a variable region sequence, "Kabat numbering" refers to the numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0048] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as "Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) pp. 647-660) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by referring to "Kabat EU index numbering" in this case.

[0049] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain whose sequence is hypervariable and which determines antigen-binding specificity, e.g., a "complementarity-determining region" (CDR). Generally, antibodies contain six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include the following: (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 present 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)); and (c) Antigen contact sites present 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)).

[0050] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR nomenclature may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature.

[0051] "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 are usually arranged in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0052] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which 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.

[0053] 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").

[0054] As used herein, the term "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 one another), i.e., a crossover Fab molecule refers to a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1 in the N-terminal to C-terminal direction), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL in the N-terminal to C-terminal direction). 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.

[0055] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain (VH-CH1 from N to C-terminus) composed of a heavy chain variable domain and a constant domain, and a light chain (VL-CL from N to C-terminus) composed of a light chain variable domain and a constant domain.

[0056] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

[0057] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing 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 human IgG heavy chain Fc region is usually defined to stretch from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain a full-length heavy chain or a truncated variant of the full-length 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) of the Fc region, or the C-terminal glycine (Gly446) and lysine (K447), may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is according to the EU numbering system, also known 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 also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a 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, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0058] A "modification that promotes association of 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 association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form homodimers. 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 Fc domain subunits) that are desired to associate, which 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 not be identical in the sense that the additional components (e.g., antigen-binding moieties) fused to each of the subunits are not the same. In some embodiments, a modification that promotes association includes an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In certain embodiments, the association-promoting modifications comprise separate amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0059] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0060] "Percent (%) amino acid sequence identity" 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 to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package version 36.3.8c or later, with the BLOSUM50 comparison matrix. The FASTA program package is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml and is described in W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; W.R. Pearson (1996), "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et al., (1997) Genomics 46:24-36.Alternatively, sequences can be compared using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch(global protein:protein) program and default options (BLOSUM50, open:-10, ext:-2, Ktup=2), ensuring global rather than local alignment. The percent amino acid identity is shown in the output alignment header.

[0061] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0062] "Decreased binding," e.g., decreased binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured, e.g., by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0063] By "fused" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0064] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n (SEQ ID NO: 62), (SG4) n (SEQ ID NO: 63) or G4 (SG4) n(SEQ ID NO: 64) peptide linker, where "n" is generally 1 to 10, typically 2 to 4, and particularly 2. Peptide linkers of particular interest are GG, GGS, GSGS (SEQ ID NO: 61), (G4S) (SEQ ID NO: 56), (G4S)2 (SEQ ID NO: 57), (G4S)3 (SEQ ID NO: 58), (G4S)4 (SEQ ID NO: 59) and GGGGSGGGGG (SEQ ID NO: 60), more particularly (G4S)2 (SEQ ID NO: 57).

[0065] In one aspect the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen-binding moiety that specifically binds to CD3, a second antigen-binding moiety that specifically binds to HLA-G, in particular HLA-G expressed on JEG3 cells (ATCC No. HTB36) and inhibits ILT2 binding to HLA-G expressed on JEG-3 cells (ATCC No. HTB36), and optionally a third antigen-binding moiety.

[0066] In another aspect, the first antigen-binding portion comprises a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, and a CDR-H3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6.

[0067] In a further aspect, the second antigen-binding moiety and (if present) the third antigen-binding moiety comprise a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 9, a CDR-H2 of SEQ ID NO: 10 and a CDR-H3 of SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 13 and a CDR-L3 of SEQ ID NO: 14.

[0068] In a particular embodiment, the anti-HLA-G / anti-CD3 bispecific antibody comprises: (i) a first antigen-binding portion that specifically binds to CD3 and comprises a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, and a CDR-H3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6; (ii) a second antigen-binding portion that specifically binds to HLA-G and comprises a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 9, a CDR-H2 of SEQ ID NO: 10, and a CDR-H3 of SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 13, and a CDR-L3 of SEQ ID NO: 14, and optionally a third antigen-binding portion.

[0069] In one aspect, the first antigen-binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0070] In one embodiment, the first antigen binding portion comprises the heavy chain variable region sequence of SEQ ID NO:7 and the light chain variable region sequence of SEQ ID NO:8.

[0071] In one aspect, the second antigen-binding portion and (if present) the third antigen-binding portion comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0072] In one aspect, the second antigen-binding portion and (if present) the third antigen-binding portion comprise the heavy chain variable region sequence of SEQ ID NO:15 and the light chain variable region sequence of SEQ ID NO:16.

[0073] In some embodiments, the first antigen-binding moiety, the second antigen-binding moiety, and / or the third antigen-binding moiety (if present) are Fab molecules. In some embodiments, the first antigen-binding moiety is a crossover Fab molecule in which the variable or constant regions of the Fab light chain and the Fab heavy chain have been swapped. In such embodiments, the second antigen-binding moiety and the third antigen-binding moiety (if present) are preferably conventional Fab molecules.

[0074] In some embodiments, wherein the first antigen-binding portion, the second antigen-binding portion, and / or the third antigen-binding portion (if present) of the bispecific antibody are all Fab molecules, and in one of the antigen-binding portions (particularly the first antigen-binding portion), the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other: i) in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (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 by a negatively charged amino acid (Kabat EU index numbering), or ii) the amino acid at position 124 in the constant domain CL of the second and / or third antigen-binding moiety (if present) has been substituted by a positively charged amino acid (Kabat numbering), and the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the second and / or third antigen-binding moiety (if present) has been substituted by a negatively charged amino acid (Kabat EU index numbering).

[0075] Bispecific antibodies do not contain both of the modifications described in i) and ii): the constant domains CL and CH1 of the antigen-binding moiety having a VH / VL exchange are not replaced by each other (i.e. remain unexchanged).

[0076] In a more specific embodiment, i) 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), or ii) in the constant domain CL of the second antigen-binding moiety and / or the third antigen-binding moiety (if present) 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 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0077] 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 and third antigen-binding moieties, 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).

[0078] In a further aspect, in the constant domain CL of the second and / or third antigen-binding moiety (if present) the amino acid at position 124 is substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CH1 of the second and / or third antigen-binding moiety (if present) the amino acid at position 147 is substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0079] In a preferred aspect, in the constant domain CL of the second and / or third antigen-binding moiety (if present), the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (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 the second and / or third antigen-binding moiety (if present), 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).

[0080] In one embodiment, in the constant domain CL of the second and / or third antigen-binding moiety (if present) there is a substitution of the amino acid at position 124 by lysine (K) (numbering according to Kabat) and the amino acid at position 123 by lysine (K) (numbering according to Kabat); and in the constant domain CHI of the second and / or third antigen-binding moiety (if present) there is a substitution of the amino acid at position 147 by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 by glutamic acid (E) (numbering according to Kabat EU index).

[0081] In one embodiment, in the constant domain CL of the second and / or third antigen-binding moiety (if present) there is a substitution of the amino acid at position 124 with a lysine (K) (numbering according to Kabat) and the amino acid at position 123 with an arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second and / or third antigen-binding moiety (if present) there is a substitution of the amino acid at position 147 with a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 with a glutamic acid (E) (numbering according to Kabat EU index).

[0082] In a particular aspect, when the amino acid substitutions according to the above aspects are made in the constant domain CL and the constant domain CH1 of the second and / or third antigen-binding moiety (if present), the constant domain CL of the second and / or third antigen-binding moiety (if present) is of the kappa isotype.

[0083] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are fused to each other, optionally via a peptide linker.

[0084] In some embodiments, each of the first and second antigen-binding moieties is a Fab molecule, and (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.

[0085] In some embodiments, the anti-HLA-G / anti-CD3 bispecific antibody provides monovalent binding to CD3.

[0086] In certain embodiments, an anti-HLA-G / anti-CD3 bispecific antibody comprises a single antigen-binding portion that specifically binds CD3 and two antigen-binding portions that specifically bind HLA-G. Accordingly, in some embodiments, an anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen-binding portion, particularly a Fab molecule, more particularly a conventional Fab molecule, that specifically binds HLA-G. The third antigen-binding portion can incorporate all of the features described above for the second antigen-binding portion (e.g., CDR sequences, variable region sequences, and / or amino acid substitutions in the constant region), alone or in combination. In some embodiments, the third antigen-binding portion is identical to the second antigen-binding portion (e.g., is also a conventional Fab molecule and comprises the same amino acid sequence).

[0087] In certain embodiments, the anti-HLA-G / anti-CD3 bispecific antibody further comprises an Fc domain composed of a first subunit and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In certain embodiments, 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 in vivo Fab arm exchange of IgG4 antibodies (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 a particularly preferred embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is set forth in SEQ ID NO: 43.

[0088] In some embodiments in which the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, 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, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0089] In some embodiments in which each of the first antigen-binding moiety, the second antigen-binding moiety, and, if present, the third antigen-binding moiety, is a Fab molecule, (a) (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and (b) if present, the third antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0090] In certain embodiments, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is the CH3 domain. Thus, in one embodiment, the modification is located within the CH3 domain of the Fc domain.

[0091] In a specific embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes 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. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 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 protrusion ("knob") on the interface of a first polypeptide and a corresponding cavity ("hole") on the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Complementary cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0092] Thus, in some embodiments, an amino acid residue in the CH3 domain of a first subunit of an Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of a second subunit, and an amino acid residue in the CH3 domain of a second subunit of an Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can be positioned within the protrusion in the CH3 domain of the first subunit. Preferably, the 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). Preferably, the 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). The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0093] In a specific such embodiment, 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 tryptophan residue (T366S), and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, 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). In a preferred embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0094] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or decrease effector function.

[0095] In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0096] Typically, the same one or more amino acid substitutions are present in each of the two subunits of the Fc domain. In one embodiment, the one or more amino acid substitutions reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the one or more amino acid substitutions 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.

[0097] In one aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to Kabat EU index). In a more specific aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to Kabat EU index). In some aspects, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In such aspects, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to Kabat EU index). In one aspect, 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 aspect, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular aspect, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more specific aspect, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA," "PGLALA," or "LALAPG"). Specifically, in a preferred embodiment, each subunit of the Fc domain contains the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) in each of the first and second subunits of the Fc domain (Kabat EU index numbering).In such embodiments, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain.

[0098] In a preferred embodiment, the anti-HLA-G / anti-CD3 bispecific antibody comprises: (i) a first antigen-binding moiety that specifically binds to CD3, the first antigen-binding moiety comprising a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, and a CDR-H3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6, wherein the first antigen-binding moiety is a crossover Fab molecule in which either the variable or constant regions of the Fab light chain and the Fab heavy chain have been swapped, particularly the variable regions; (ii) second and third antigen-binding moieties that specifically bind to HLA-G, the second and third antigen-binding moieties comprising a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 9, a CDR-H2 of SEQ ID NO: 10, and a CDR-H3 of SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 13, and a CDR-L3 of SEQ ID NO: 14, wherein the second and third antigen-binding moieties are each Fab molecules, particularly conventional Fab molecules; (iii) an Fc domain composed of a first subunit and a second subunit; A 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, a 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, and a third antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0099] In one aspect, the first antigen-binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0100] In one embodiment, the first antigen binding portion comprises the heavy chain variable region sequence of SEQ ID NO:7 and the light chain variable region sequence of SEQ ID NO:8.

[0101] In one aspect, the second and third antigen-binding portions comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0102] In one aspect, the second and third antigen binding portions comprise a heavy chain variable region of SEQ ID NO:15 and a light chain variable region of SEQ ID NO:16.

[0103] The Fc domain according to the above embodiment may comprise, either alone or in combination, all of the features described above in relation to the Fc domain.

[0104] In one embodiment, the antigen-binding portion and the Fc region are fused to each other by a peptide linker, in particular the peptide linkers in SEQ ID NOs: 56-64.

[0105] In one embodiment, in the constant domain CL of the second and third Fab molecules of (ii) 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), in particular by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second and third Fab molecules of (ii) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to EU index of Kabat) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to EU index of Kabat).

[0106] In one embodiment the anti-HLA-G / anti-CD3 bispecific antibody comprises a polypeptide (in particular two polypeptides) comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 46, a polypeptide comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 47, a polypeptide comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 48, and a polypeptide comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 49.

[0107] In one embodiment the anti-HLA-G / anti-CD3 bispecific antibody comprises a polypeptide (in particular two polypeptides) comprising the sequence of SEQ ID NO: 46, a polypeptide comprising the sequence of SEQ ID NO: 47, a polypeptide comprising the sequence of SEQ ID NO: 48 and a polypeptide comprising the sequence of SEQ ID NO: 49.

[0108] The anti-HLA-G / anti-CD3 bispecific antibodies herein are used in combination with a 4-1BB (CD137) agonist.

[0109] As used herein, the terms "4-1BB" or "CD137" refer to native 4-1BB from vertebrates, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed 4-1BB and 4-1BB resulting from intracellular processing. The term also encompasses naturally occurring variants of 4-1BB, such as splice variants or allelic variants. The amino acid sequence of human 4-1BB is shown in UniProt accession number Q07011 (accession version 185).

[0110] "4-1BBL" or "4-1BB ligand" or "CD137L" is a member of the costimulatory TNF ligand family and can costimulate T cell proliferation and cytokine production. Costimulatory TNF family ligands costimulate TCR signaling by interacting with their corresponding TNF receptors, which recruit TNFR-associated factors (TRAFs) and initiate a signaling cascade leading to T cell activation. 4-1BBL is a type II transmembrane protein. Intact or intact 4-1BBL, having the amino acid sequence set forth in UniProt accession number P41273 (registration version 153), has been described to form trimers on the surface of cells. Trimer formation is enabled by a specific motif in the ectodomain of 4-1BBL. This motif is referred to herein as the "trimerization region." Amino acids 50 to 254 of the human 4-1BBL sequence (SEQ ID NO: 55) form the ectodomain of 4-1BBL, but even fragments of it can form trimers.

[0111] An "ectodomain" is a domain of a membrane protein that extends into the extracellular space (i.e., the space outside the cell), also called the "extracellular domain." The ectodomain of 4-1BBL as defined herein refers not only to the part of the 4-1BBL protein, in particular the human 4-1BBL protein (UniProt accession number P41273 (registration version 153)), that extends into the extracellular space (extracellular domain), but also includes shorter parts or fragments thereof that are responsible for trimerization and binding to the corresponding receptor 4-1BB.

[0112] Thus, the term "ectodomain of 4-1BBL or a fragment thereof" refers to the extracellular domain of 4-1BBL or a portion thereof capable of binding to 4-1BB and having the ability to trimerize. In a specific embodiment of the present invention, the term "ectodomain of 4-1BBL or a fragment thereof" refers to a polypeptide having amino acids selected from SEQ ID NO: 25 (amino acids 71-254 of human 4-1BBL), SEQ ID NO: 26 (amino acids 85-254 of human 4-1BBL), SEQ ID NO: 27 (amino acids 80-254 of human 4-1BBL), SEQ ID NO: 28 (amino acids 52-254 of human 4-1BBL), SEQ ID NO: 29 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 30 (amino acids 85-248 of human 4-1BBL), SEQ ID NO: 31 (amino acids 80-248 of human 4-1BBL), and SEQ ID NO: 32 (amino acids 52-248 of human 4-1BBL).

[0113] As used herein, the term "antigen-binding molecule" broadly refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules include antibodies, antibody fragments, and antigen-binding protein scaffolds.

[0114] "Fibroblast activation protein (FAP)," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses not only "full-length," unprocessed FAPs, but also any form of FAP resulting from intracellular processing. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. The amino acid sequence of human FAP is set forth in UniProt (www.uniprot.org) accession number Q12884 (registration version 197). The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. As used herein, antigen-binding moieties that bind to FAPs preferably bind to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described, for example, in WO 2012 / 020006.

[0115] Particularly useful 4-1BB (CD137) agonists for use in the present invention are described, for example, in WO 2016 / 075278 or WO 2016 / 156291, which are incorporated by reference in their entireties.

[0116] In one aspect, the 4-1BB (CD137) agonist comprises 4-1BBL (particularly human 4-1BBL) or a fragment thereof, particularly the 4-1BBL ectodomain or a fragment thereof. In one aspect, the 4-1BB (CD137) agonist comprises the three ectodomains of 4-1BBL or fragments thereof (i.e., the first, second, and third ectodomains of 4-1BBL or fragments thereof). In one aspect, the 4-1BBL ectodomain or fragment thereof comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32. In one aspect, the ectodomain of 4-1BBL, or a fragment thereof, comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. In one aspect, the ectodomain of 4-1BBL, or a fragment thereof, comprises the amino acid sequence of SEQ ID NO: 29. In one aspect, the ectodomain of 4-1BBL, or a fragment thereof, consists of the amino acid sequence of SEQ ID NO: 29.

[0117] In a particular aspect, the 4-1BB (CD137) agonist is a molecule comprising the three ectodomains of 4-1BBL or fragments thereof, wherein the ectodomains of 4-1BBL or fragments thereof comprise (or consist of) the amino acid sequence of SEQ ID NO:29.

[0118] In one aspect, the 4-1BB (CD137) agonist comprises three ectodomains of 4-1BBL or fragments thereof (i.e., the first, second, and third ectodomains of 4-1BBL or fragments thereof), wherein the first ectodomain of 4-1BBL or fragment thereof and the second ectodomain of 4-1BBL or fragment thereof are fused to each other, optionally via a peptide linker (i.e., the first and second ectodomains of 4-1BBL or fragments thereof are on the same polypeptide), and the third ectodomain of 4-1BBL or fragment thereof is not fused to the first or second ectodomains of 4-1BBL or fragments thereof (i.e., the third ectodomain of 4-1BBL or fragment thereof is on a separate polypeptide from the first and second ectodomains of 4-1BBL or fragments thereof).

[0119] In one embodiment, the 4-1BB (CD137) agonist is a molecule comprising a first polypeptide comprising the first and second ectodomains of 4-1BBL or fragments thereof, and a second polypeptide comprising the third ectodomain of 4-1BBL or fragments thereof. In one embodiment, the first and second ectodomains of 4-1BBL or fragments thereof are fused via a peptide linker, particularly a (G4S)2 peptide linker. In one embodiment, the first and second polypeptides are linked by a disulfide bond. In one embodiment, the first polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 37. In one embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 37. In one embodiment, the second polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In one embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO:29.

[0120] In further embodiments, the 4-1BB (CD137) agonist comprises an antigen-binding portion. In particular embodiments, the 4-1BB (CD137) agonist comprises an antigen-binding portion that specifically binds to a tumor-associated antigen, particularly a tumor stromal antigen (i.e., a tumor stromal-associated antigen), and more particularly a tumor fibroblast antigen (i.e., an antigen expressed on cancer-associated fibroblasts).

[0121] In a preferred embodiment, the antigen-binding portion specifically binds to fibroblast activation protein (FAP), particularly human FAP.

[0122] In one embodiment, the antigen-binding portion is a Fab molecule, particularly a conventional Fab molecule.

[0123] Thus, in a particular embodiment, the 4-1BB (CD137) agonist is an antigen-binding molecule comprising the three ectodomains of 4-1BBL or fragments thereof and at least one antigen-binding portion that specifically binds to a tumor-associated antigen, in particular an antigen-binding portion that specifically binds to a FAP.

[0124] In one aspect, an antigen-binding portion that specifically binds to a FAP comprises a heavy chain variable region (VH) comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 17, a CDR-H2 of SEQ ID NO: 18, and a CDR-H3 of SEQ ID NO: 19, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 20, a CDR-L2 of SEQ ID NO: 21, and a CDR-L3 of SEQ ID NO: 22.

[0125] In one embodiment, an antigen-binding portion that specifically binds to a FAP comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:23 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:24.

[0126] In one embodiment, the antigen binding portion that specifically binds to a FAP comprises the heavy chain variable region sequence of SEQ ID NO:23 and the light chain variable region sequence of SEQ ID NO:24.

[0127] In a further aspect, the 4-1BB (CD137) agonist comprises an Fc domain composed of a first and a second subunit.

[0128] The Fc domain contained in the 4-1BB (CD137) agonist may incorporate all of the features described herein above in relation to the Fc domain contained in the anti-HLA-G / anti-CD3 bispecific antibody, either alone or in combination.

[0129] In particular, in one embodiment, the Fc domain comprised in the 4-1BB (CD137) agonist is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In a more particular embodiment, the Fc domain is a human Fc domain. In a particularly preferred embodiment, the Fc domain is a human IgG1 Fc domain.

[0130] In certain embodiments, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain (e.g., a "knob-into-hole" modification), as described herein above in connection with anti-HLA-G / anti-CD3 bispecific antibodies.

[0131] In further particular embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or decrease effector function (e.g., the "P329G LALA," "PGLALA," or "LALAPG" amino acid substitutions) as described herein above in connection with anti-HLA-G / anti-CD3 bispecific antibodies.

[0132] In certain preferred embodiments, the Fc domain comprised in the 4-1BB (CD137) agonist is a human IgG1 Fc domain, and each subunit of the Fc domain contains the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering).

[0133] In one aspect, the 4-1BB (CD137) agonist is (i) the three ectodomains of 4-1BBL or fragments thereof; (ii) an antigen-binding portion that specifically binds to a FAP, and in particular an Fab molecule; (iii) an Fc domain composed of a first and a second subunit, the Fc domain comprising a modification that promotes association of the first and second subunits of the Fc domain, in particular, and / or one or more amino acid substitutions that reduce binding to an Fc receptor and / or reduce effector function; and an antigen-binding molecule comprising:

[0134] In certain embodiments, the 4-1BB (CD137) agonist is (i) the first, second and third ectodomains of 4-1BBL or fragments thereof; (ii) an antigen-binding portion that specifically binds to a FAP, wherein the antigen-binding portion is a Fab molecule; and (iii) an Fc domain composed of a first and a second subunit, the Fc domain comprising a modification that specifically promotes association of the first and second subunits of the Fc domain and / or one or more amino acid substitutions that reduce binding to an Fc receptor and / or reduce effector function; (iv) CL domain and CH1 domain and the antigen-binding molecule comprises: (a) a first polypeptide, (a1) a first ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of a second ectodomain of 4-1BBL or a fragment thereof; (a2) the second ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of the CL domain; (a3) a CL domain fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit); (a4) one of the subunits of the Fc domain (e.g., the first subunit); a first polypeptide comprising: (b) a second polypeptide, (b1) the third ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of the CH1 domain; (b2) CH1 domain and and a second polypeptide comprising: (c) a third polypeptide, (c1) a heavy chain of a Fab molecule fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the second subunit); (c2) one of the subunits of the Fc domain (e.g., the second subunit) and a third polypeptide comprising: (d) a fourth polypeptide comprising the light chain of a Fab molecule; and It consists of:

[0135] Fusion between various domains of the antigen-binding molecule is preferably via a peptide linker, which may also comprise (part of) an immunoglobulin hinge region or consist of the same. In particular, the fusion between the first ectodomain of 4-1BBL or a fragment thereof and the second ectodomain of 4-1BBL or a fragment thereof is via a peptide linker, in particular a(G4S)2 linker (SEQ ID NO: 57).

[0136] In one embodiment, the first and second polypeptides are linked to each other by a disulfide bond, particularly a disulfide bond between the CL domain and the CH1 domain.

[0137] In one aspect, in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (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 CH1 domain of the second polypeptide, 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).

[0138] In one embodiment, in the CL domain of the first polypeptide, the amino acid at position 124 is substituted by lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) (Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering).

[0139] In a particular embodiment, in the CL domain of the first polypeptide, the amino acid at position 124 is substituted by lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted by arginine (R) (Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering).

[0140] In a particular embodiment, when amino acid substitutions according to the above embodiments are made in the CL and CH1 domains of the first and second polypeptides, the CL domain of the first polypeptide is of the kappa isotype.

[0141] In one embodiment, the CL domain is a human CL domain, particularly a human CL domain of the kappa isotype. In a further embodiment, the CH1 domain is a human CH1 domain, particularly a human CH1 domain of the gamma isotype, most particularly a human CH1 domain of the gamma isotype.

[0142] In one aspect, the first polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:51.

[0143] In one embodiment, the second polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:52.

[0144] In one embodiment, the third polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:53.

[0145] In one embodiment, the fourth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:54.

[0146] In one embodiment, the 4-1BB (CD137) agonist is an antigen-binding molecule comprising: a first polypeptide 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: 51; a second polypeptide 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: 52; a third polypeptide 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: 53; and a fourth polypeptide 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: 54.

[0147] In one aspect, the 4-1BB (CD137) agonist comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 51, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 52, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 54.

[0148] In an alternative embodiment, the 4-1BB agonist may be an anti-4-1BB antibody, in particular an anti-FAP / anti-4-1BB bispecific antibody.

[0149] The term "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Non-limiting examples of cancer include blood cancers such as leukemia, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, bile duct cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, skin cancer, squamous cell carcinoma, sarcoma, bone cancer, and kidney cancer. Other cell proliferative disorders include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included.

[0150] In some embodiments of the anti-HLA-G / anti-CD3 bispecific antibodies, 4-1BB (CD137) agonists, methods, uses, and kits of the present invention, the cancer is a solid tumor cancer. By "solid tumor cancer" is meant a malignant tumor that forms a discrete tumor mass (including tumor metastases) in a specific location within a patient's body, such as a sarcoma or carcinoma (as opposed to, for example, blood cancers such as leukemia, which do not generally form solid tumors). Non-limiting examples of solid tumor cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, skin cancer, squamous cell carcinoma, bone cancer, liver cancer, and kidney cancer (renal carcinoma). Other solid tumor cancers contemplated in the context of the present invention include, but are not limited to, tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, muscle, spleen, thoracic region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included.

[0151] In one aspect, the cancer is a cancer selected from the group consisting of renal cell carcinoma, colorectal cancer, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC), head and neck cancer, bladder cancer, esophageal cancer, skin cancer, soft tissue cancer, gastric cancer, cervical cancer, and ovarian cancer. In one aspect, the cancer is a cancer selected from the group consisting of renal cell carcinoma, colorectal cancer, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0152] In some embodiments, the cancer is an HLA-G-positive cancer. "HLA-G-positive cancer" or "HLA-G-expressing cancer" refers to a cancer characterized by expression or overexpression of HLA-G in cancer cells. HLA-G expression can be determined, for example, by quantitative real-time PCR (measuring HLA-G mRNA levels), immunohistochemistry (IHC), or Western blot assay. In one embodiment, the cancer expresses HLA-G. In one embodiment, the cancer expresses HLA-G in at least 20%, preferably at least 50% or at least 80% of tumor cells, as measured by immunohistochemistry (IHC) using an antibody specific for HLA-G. In one embodiment, the HLA-G-expressing cancer is selected from renal cell carcinoma, colorectal cancer, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0153] In some embodiments, the cancer comprises cells (e.g., fibroblasts) that express a FAP. In some embodiments, the cancer expresses a FAP, particularly in the tumor stroma.

[0154] As used herein, a "patient," "subject," or "individual" refers to any single human subject who is experiencing or has experienced one or more signs, symptoms, or other indicators of cancer and is eligible for treatment. In some embodiments, the patient has cancer or has been diagnosed with cancer. The patient may or may not have been previously treated with an anti-HLA-G / anti-CD3 bispecific antibody or another drug. In certain embodiments, the patient has not been previously treated with an anti-HLA-G / anti-CD3 bispecific antibody. The patient may have been treated with a therapy including one or more drugs other than an anti-HLA-G / anti-CD3 bispecific antibody before anti-HLA-G / anti-CD3 bispecific antibody therapy is initiated.

[0155] As used herein, "treatment" (and its grammatical variants, e.g., "treat" or "treating") refers to a clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be carried out 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 the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the condition, and remission or improved prognosis.

[0156] The anti-HLA-G / anti-CD3 bispecific antibody and the 4-1BB (CD137) agonist are administered in effective amounts.

[0157] An "effective amount" of a medicament, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0158] In some embodiments of the anti-HLA-G / anti-CD3 bispecific antibody, 4-1BB (CD137) agonist, methods, uses, or kits described above and herein, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist results in an increase in the number of tumor-infiltrating immune cells, particularly the number of CD45+ cells, at the cancer site. In one embodiment, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist results in an increase in the number of T cells, particularly the number of cytotoxic T cells and / or CD4+ T cells, at the cancer site. In one embodiment, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist results in the activation of T cells, particularly cytotoxic T cells, particularly at the cancer site. Activation can include T cell proliferation, T cell differentiation, cytokine secretion by T cells, release of cytotoxic effector molecules from T cells, cytotoxic activity of T cells, and expression of activation markers by T cells. In some embodiments of the anti-HLA-G / anti-CD3 bispecific antibodies, 4-1BB (CD137) agonists, methods, uses, or kits described above and herein, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibodies and 4-1BB (CD137) agonists results in increased activation of T cells, particularly cytotoxic T cells, particularly at the site of cancer, compared to treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibodies alone. In certain embodiments, activation includes cytotoxic activity of T cells (particularly lysis of cancer cells), such as release of granzyme B and / or secretion of cytokines (particularly IL-2, TNF-α, and / or interferon-γ) by T cells. In some embodiments of the anti-HLA-G / anti-CD3 bispecific antibody, 4-1BB (CD137) agonist, methods, uses, or kits described above and herein, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist results in a decrease in macrophage infiltration of tumors, particularly with macrophages that are CD68+. Said macrophage infiltration may include a decrease in macrophage-mediated T-cell suppression and / or an increase in T-cell activation.

[0159] In some embodiments of the anti-HLA-G / anti-CD3 bispecific antibody, 4-1BB (CD137) agonist, methods, uses, or kits described above and herein, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist results in increased differentiation of naive T cells into memory T cells, particularly at the site of cancer, compared to treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody alone. In one embodiment, differentiation is detected by measuring CD45RA expression, e.g., using flow cytometry.

[0160] In some embodiments of the anti-HLA-G / anti-CD3 bispecific antibody, 4-1BB (CD137) agonist, methods, uses, or kits described above and herein, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist may result in a response in an individual. In some embodiments, the response may be a complete remission. In some embodiments, the response may be a sustained response after cessation of treatment. In some embodiments, the response may be a complete remission that is sustained after cessation of treatment. In other embodiments, the response may be a partial remission. In some embodiments, the response may be a partial remission that is sustained after cessation of treatment. In some embodiments, treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist may improve the response compared to treatment with or administration of the anti-HLA-G / anti-CD3 bispecific antibody alone (i.e., without the 4-1BB (CD137) agonist).

[0161] In some embodiments, treatment with or administration of an anti-HLA-G / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist may increase the response rate in a patient population compared to a corresponding patient population treated with an anti-HLA-G / anti-CD3 bispecific antibody alone (i.e., without a 4-1BB (CD137) agonist).

[0162] The combination therapy of the present invention involves the administration of an anti-HLA-G / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist.

[0163] As used herein, "combination" (and grammatical variations thereof, such as "combine" or "combining") encompasses the combination of an anti-HLA-G / anti-CD3 bispecific antibody according to the invention with a 4-1BB (CD137) agonist, wherein the anti-HLA-G / anti-CD3 bispecific antibody and the 4-1BB (CD137) agonist are in the same or different containers, in the same or different pharmaceutical formulations, administered simultaneously or separately, administered simultaneously or sequentially (in any order), and administered by the same or different routes, provided that the anti-HLA-G / anti-CD3 bispecific antibody and the 4-1BB (CD137) agonist are capable of simultaneously exerting their biological effects in the body. For example, "combining" an anti-HLA-G / anti-CD3 bispecific antibody according to the invention with a 4-1BB (CD137) agonist means administering first the anti-HLA-G / anti-CD3 bispecific antibody in a particular pharmaceutical formulation, followed by the 4-1BB (CD137) agonist in a different pharmaceutical formulation, or vice versa.

[0164] The anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist may be administered in any suitable manner known in the art. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist are administered sequentially (at different times). In another embodiment, the anti-HLA-G / anti-CD3 bispecific antibody and 4-1BB (CD137) agonist are administered simultaneously (at the same time). Without wishing to be bound by theory, it may be advantageous to administer the 4-1BB (CD137) agonist prior to and / or simultaneously with the anti-HLA-G / anti-CD3 bispecific antibody. In some embodiments, the anti-HLA-G / anti-CD3 bispecific antibody is in a separate composition from the 4-1BB (CD137) agonist. In some embodiments, the anti-HLA-G / anti-CD3 bispecific antibody is in the same composition as the 4-1BB (CD137) agonist.

[0165] The anti-HLA-G / anti-CD3 bispecific antibody and the 4-1BB (CD137) agonist can be administered by any suitable route, and may be administered by the same route or by different routes. In some embodiments, the anti-HLA-G / anti-CD3 bispecific antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by injection, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In particular embodiments, the anti-HLA-G / anti-CD3 bispecific antibody is administered intravenously. In some embodiments, the 4-1BB (CD137) agonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by injection, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In particular embodiments, the 4-1BB (CD137) agonist is administered intravenously. Effective amounts of anti-HLA-G / anti-CD3 bispecific antibodies and 4-1BB (CD137) agonists can be administered for the prevention or treatment of diseases. The appropriate route of administration and dosage of the anti-HLA-G / anti-CD3 bispecific antibodies and / or 4-1BB (CD137) agonists can be determined based on the type of disease being treated, the type of anti-HLA-G / anti-CD3 bispecific antibody, the type of 4-1BB (CD137) agonist, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician. Administration can be by any appropriate route, for example, injection, such as intravenous or subcutaneous injection, depending on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion. The anti-HLA-G / anti-CD3 bispecific antibody and the 4-1BB (CD137) agonist are suitably administered to the patient at one time or over a series of treatments.

[0166] The combination of the present invention can be used alone or in combination with other drugs for therapeutic purposes. For example, the combination of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-cancer agent, such as a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis. The combination of the present invention can also be combined with radiation therapy.

[0167] The kits provided herein typically include one or more containers and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition used alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-HLA-G / anti-CD3 bispecific antibody used in the combination of the invention. Another active agent is a 4-1BB (CD137) agonist used in the combination of the invention, which may be in the same composition and container as the bispecific antibody or may be provided in a different composition and container. The label or package insert indicates that the one or more compositions are used to treat a selected condition, such as cancer.

[0168] In one aspect, the present invention provides kits intended for the treatment of cancer, the kit comprising, in the same or separate containers, (a) an anti-HLA-G / anti-CD3 bispecific antibody and (b) a 4-1BB (CD137) agonist, and optionally (c) a package insert comprising printed instructions directing the use of the combination treatment as a method for treating cancer. Furthermore, the kit comprises: (a) a first container having contained therein a composition comprising the anti-HLA-G / anti-CD3 bispecific antibody; (b) a second container having contained therein a composition comprising the 4-1BB (CD137) agonist; and, optionally, (c) a third container having contained therein a composition comprising an additional cytotoxic or other therapeutic agent. Kits of these aspects of the present invention may further comprise a package insert indicating that the composition can be used to treat cancer. Alternatively, or additionally, the kit may further comprise a third (or fourth) container containing a pharmaceutically acceptable buffer (e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution). It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0169] Amino acid sequence TIFF2026508605000001.tif180148TIFF2026508605000002.tif240148TIFF2026508605000003.tif238148 TIFF2026508605000004.tif238148TIFF2026508605000005.tif239148TIFF2026508605000006.tif239148 [Brief explanation of the drawings]

[0170] [Figure 1]Figure 1A is a schematic diagram of the HLA-G-targeting T cell bispecific (TCB) antibody molecule ("HLA-G TCB") used in the examples. The molecule contains a single antigen-binding moiety for CD3, two antigen-binding moieties for HLA-G, and an Fc domain. Figure 1B is a schematic diagram of the FAP-targeting 4-1BB (CD137) agonist ("FAP4-1BBL") used in the examples. The molecule contains a 4-1BBL ectodomain trimer, an antigen-binding moiety for FAP, and an Fc domain. Black dots: modifications in the Fc domain that promote heterodimerization. *: oppositely charged amino acids introduced into the CH and CL domains. [Figure 2] This figure shows the protocol for an in vivo efficacy experiment of HLA-G TCB (P1AF7977) in combination with FAP4-1BBL (P1AD5195) against HLA-G TCB (P1AF7977) in BC004 PDX-bearing fully humanized NSG mice. The table below the treatment scheme indicates which subgroups of mice received which treatment. Animals receiving histidine buffer (vehicle) were used as negative controls. After tumors reached an average volume of approximately 200-300 mm, mice were randomized into five groups and administered one of the following: A) histidine buffer (vehicle) as a control; B) HLA-G-TCB (0.5 mg / kg intravenously once weekly); C) HLA-G-TCB (0.05 mg / kg intravenously once weekly); D) HLA-G-TCB (0.5 mg / kg intravenously once weekly) + FAP4-1-BBL (1 mg / kg intravenously every 2 weeks); E) HLA-G-TCB (0.05 mg / kg intravenously once weekly) + FAP4-1-BBL (1 mg / kg intravenously every 2 weeks). This experiment is described in Example 1. [Figure 3A]Figures 3A and 3B show the efficacy of an anti-HLA-G / anti-CD3 bispecific antibody (P1AF7977) at two different doses (0.05 mg / kg and 0.5 mg / kg) alone or in combination with FAP4-1BBL (at 1 mg / kg) in the BC004 PDX mouse model, which has FAP-expressing stroma. The figures show tumor growth curves (expressed as tumor volume) for mice receiving the indicated treatments (see Figure 2 for treatment scheme). Figure 3A shows an overlay of all data on a single graph, while Figure 3B shows the data obtained for each animal on separate graphs for the different treatment groups. [Figure 3B] See legend to Figure 3A. [Figure 4] Figure 2 shows quantification of cellular and molecular immune components associated with the antitumor effects of treatment in the experiment described in Figure 2. Flow cytometry analysis of the indicated immune populations in tumors was performed 9 days after randomization (i.e., 2 days after the second injection). The number of immune cells infiltrating the tumor is shown as a percentage of viable cells relative to CD45+ cells and as absolute numbers (counts / mg). The numbers of CD3+ T cells and CD68+ macrophages are shown as absolute numbers (counts per mg) and ratios per immune cell (CD45+). [Figure 5]Figure 1 shows the protocol for an in vivo efficacy study of HLA-G TCB (P1AF7977) in combination with FAP4-1BBL (P1AD5195) against HLA-G TCB (P1AF7977) in CR5044-bearing fully humanized NSG mice. The table below the treatment scheme indicates which subgroup of mice received which treatment / combination. Animals receiving histidine buffer (vehicle) were used as negative controls. After tumors reached an average volume of approximately 200-300 mm, mice were randomized into five groups and administered one of the following: A) histidine buffer (vehicle) as a control; B) HLA-G-TCB (2.5 mg / kg intravenously once weekly); C) HLA-G-TCB (0.5 mg / kg intravenously once weekly); D) HLA-G-TCB (0.1 mg / kg intravenously once weekly); or E) HLA-G-TCB (0.5 mg / kg intravenously once weekly) plus FAP4-1-BBL (1 mg / kg intravenously every 2 weeks). This experiment is described in Example 2. [Figure 6A] Figures 6A and 6B show the efficacy of a bispecific anti-HLA-G / anti-CD3 bispecific antibody (P1AF7977) alone at three different doses (0.1 mg / kg, 0.5 mg / kg, and 2.5 mg / kg) or in combination with 1 mg / kg FAP4-1-BBL at a concentration of 0.5 mg / kg in a CR5044 PDX mouse model (FAP4-1BBL-treated) with FAP-expressing stroma. The figures show tumor growth curves (expressed as tumor volume) for mice receiving the indicated treatments. Figure 6A shows an overlay of all data on a single graph, while Figure 6B shows the data obtained for each animal on separate graphs for different treatment schedules. [Figure 6B] See legend to Figure 6A. [Figure 7]Figure 5 shows quantification of cellular immune components associated with the antitumor effects of treatment in the experiment described in Figure 5. Flow cytometry analysis of the indicated immune populations in the tumor was performed 9 days after randomization (i.e., 2 days after the second injection). Immune cell numbers are presented as a percentage of viable cells and absolute numbers (counts per mg) for CD45+ cells. Numbers of CD4+ T cells, CD8+ T cells, Tregs (FoxP3+), NK cells (CD56+), and macrophages (CD68+) are presented as absolute numbers (counts per mg), with macrophages also presented as a percentage of immune cells (CD45+). Granzyme B (GZMB) expression on NK cells is presented as a percentage of CD56+ cells. Animals administered histidine buffer (vehicle) were used as negative controls. [Figure 8] Quantification of soluble CD25 release (indicative of T cell activation) associated with the antitumor effect of treatment in the experiment described in Figure 5 is shown. Analysis of the indicated soluble CD25 levels in serum samples from CR5044 tumor-bearing mice was performed 6 hours after the first and fourth treatment cycles. Animals receiving histidine buffer (vehicle) served as negative controls. [Example]

[0171] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced given the general description provided above.

[0172] Example 1 - Antitumor activity of HLA-G TCB monotherapy and in combination with FAP4-1BBL in the BC004 PDX model in humanized NSG mice

[0173] To monitor tumor growth inhibition (TGI) induced by either the anti-HLA-G / anti-CD3 bispecific antibody (hereinafter also referred to as "P1AF7977") alone or the combination of the anti-HLA-G / anti-CD3 bispecific antibody and FAP4-1BBL (hereinafter also referred to as "P1AD5195"), female 3-week-old NSG mice were irradiated (140 cGy) and immunized with 9 × 104 Humanized NSG mice were humanized by intravenous injection of 1000 CD34+ umbilical cord blood cells per mouse. The sequences of the molecules used in this experiment are listed in the table below. After reaching a blood human immune infiltrate of greater than 25% (as indicated by human CD45+ cells), the humanized NSG mice were transported to Roche and maintained for 5 days to allow them to acclimate to their new environment. In accordance with relevant guidelines (GV-Solas, Felasa, TierschG), the mice were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark cycle daily. Continuous health monitoring was performed daily. The experimental study protocol was reviewed and approved by the local government (ROB-55.2-2532.Vet_03-16-10).

[0174] For assays in the BC004 PDX model, the human breast cancer PDX model BC004 was purchased from OncoTest (Freiburg, Germany). Tumor fragments were digested with collagenase D (catalog no. 11088882001, Sigma-Aldrich, Minneapolis, MI) and DNase I (catalog no. 10104159001, Sigma-Aldrich) for 20 minutes to prepare a single-cell suspension. Cell number and viability were determined using a ViCell counter (Beckman Coulter, Brea, CA). On day 0 ("d0"), 2 x 10 cells were inoculated into the intramammary fat pad of humanized mice in a total volume of 20 μL of phosphate-buffered saline. 6 BC004 cells were injected. The tumors were approximately 200-300 mm 3 Once an average volume (= "dx") was reached, the mice were randomized into different treatment groups. The first group of mice received histidine buffer (vehicle) as a control. All molecules were prepared fresh in 20 mM histidine, 140 mM NaCl, pH 6.0 before injection and administered intravenously (IV) at the doses and schedules shown in Figure 2. HLA-G TCB was injected weekly, and FAP4-1BBL, where applicable, was injected every two weeks.

[0175] (Table) Amino acid sequences of molecules used in the examples TIFF2026508605000007.tif26143

[0176] Animals were monitored daily for clinical signs and adverse effects. Tumor volumes were measured by caliper and body weights were monitored twice weekly.

[0177] Treatment with HLA-G TCB resulted in a dose-dependent antitumor response in BC004 tumor-bearing animals. At a weekly dose of 0.05 mg / kg, slight but statistically insignificant tumor growth inhibition was observed, whereas at a weekly dose of 0.5 mg / kg, tumor volume reduction was much more pronounced (Figure 3). Compared with HLA-G TCB monotherapy, tumor growth inhibition was clearly increased after HLA-G TCB therapy combined with FAP4-1BBL. In the BC004 model, a 0.05 mg / kg dose of HLA-G TCB combined with FAP4-1BBL resulted in significantly greater tumor volume reduction than HLA-G TCB alone at a 0.05 mg / kg dose. The combination of 0.5 mg / kg HLA-G TCB and FAP4-1BBL resulted in all mice being tumor-free. Graphs were generated using GraphPad Prism Software. Time-to-event analysis was performed using the internal tool DOPSa (based on the R software). Test groups were compared using the Log-Rank test, and p values ​​were corrected for multiple testing using the Bonferroni-Holm method. Significant changes compared to the control group (vehicle) with p<0.05 are indicated with an asterisk (*p<0.05, **p<0.01, ***p<0.001).

[0178] To evaluate the effects of HLA-G TCB treatment as monotherapy and combination therapy on immune PD changes, flow cytometry analysis was performed on day 9 of treatment ("dx+9," 2 days after the second HLA-G TCB injection). Tumors were harvested from four mice per group and processed to obtain single-cell suspensions. Samples were disrupted using manual scissors and a Miltenyi Gentle MACS instrument, then digested in an enzyme mixture containing DNAse I (catalog no. 10104159001, Sigma-Aldrich) and collagenase D (catalog no. 11088882001, Sigma-Aldrich). After digestion, the tissue mixture was filtered through 100 and 70 μm filters and resuspended in a single-cell suspension in a buffer containing a cocktail of fluorescently labeled antibodies for detection of immune cells and tumor targets. μ-stained cells were analyzed on a fluorescence-activated cell sorting (FACS) Fortessa device running Diva software. Raw data were analyzed using FlowJo software and a predefined gating strategy to identify tumor-infiltrating lymphocytes.

[0179] Immunopharmacodynamic (ImmunoPD) findings confirmed a dose-dependent immune cell infiltration overall (as indicated by CD45+ cells) and specifically for tumor T cells (CD3+ cells), with a slight upregulation of HLA-G in tumors across treatment groups and in T cells after treatment with 0.5 mg / kg HLA-G TCB as monotherapy (Figure 4). Human macrophage (CD68+) levels were significantly lower in tumors from mice treated with the combination of HLA-G TCB and FAP4-1BBL than in tumors from mice treated with monotherapy. Because tumor-associated macrophages are known to suppress T cell function, a low macrophage content in tumors may result in reduced T cell suppression and, therefore, sustained T cell activation.

[0180] The results obtained provide preclinical evidence that the therapeutic response to an anti-HLA-G / anti-CD3 bispecific antibody can be enhanced by combination with FAP4-1BBL.

[0181] Example 2 - Antitumor activity of HLA-G TCB monotherapy and in combination with FAP4-1BBL in the CR5044 PDX model in humanized NSG mice

[0182] To monitor tumor growth inhibition induced by either the anti-HLA-G / anti-CD3 bispecific antibody alone or the combination of the anti-HLA-G / anti-CD3 bispecific antibody and FAP4-1BBL, 3-week-old female NSG mice were irradiated (140 cGy) and immunized with 9 × 10 mAbs at Jackson Laboratories on day 0 (= "d0"). 4 Humanized NSG mice were humanized by intravenous injection of 1000 CD34+ umbilical cord blood cells / mouse. After reaching a blood human immune infiltrate (human CD45) of greater than 25%, the humanized NSG mice were transported to Roche and maintained for 5 days to allow them to acclimate to their new environment. In accordance with relevant guidelines (GV-Solas, Felasa, TierschG), mice were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark cycle daily. Continuous health monitoring was performed daily. The experimental study protocol was reviewed and approved by the local authority (ROB-55.2-2532.Vet_03-20-170).

[0183] For the CR5044 PDX model, tumor cell suspension of the human colorectal cancer PDX model CR5044 was purchased from CrownBio (San Diego, USA) and 1 × 10 cells were inoculated into the right flank of mice in 100 μL of histidine buffer and Matrigel (1:1). 6 The cells were injected subcutaneously. The tumors were approximately 200-300 mm 3 Once the tumors reached an average volume of 1000 mg / kg, mice were randomized into different treatment groups based on tumor volume (= "dx"). The first group of mice received histidine buffer (vehicle) as a control. All molecules were freshly prepared in 20 mM histidine, 140 mM NaCl, pH 6.0 before injection and administered intravenously (IV) at the doses and schedules shown in Figure 5. HLA-G TCB was injected weekly, and FAP4-1BBL was injected every two weeks. The administered molecules had the same sequences as those described in Example 1.

[0184] Animals were monitored daily for clinical signs and adverse effects. Tumor volumes were measured by caliper and body weights were monitored twice weekly. Animal termination criteria were visible disease (shaggy hair, arched back, respiratory distress, movement disorder), weight loss (≥25% within 7 days after the first treatment or ≥20% on the 7th day after treatment) or tumor size (diameter ≥2 cm, volume ≥4000 mm). 3 Animals were sacrificed according to the termination criteria or at the end of the experiment.

[0185] In the CR5044 PDX model, minimal antitumor activity after HLA-G TCB monotherapy was measured for treatment with 2.5 mg / kg and 0.5 mg / kg HLA-G TCB. In comparison, TGI was clearly increased after combination therapy with HLA-G TCB and FAP4-1BBL, and the response was more uniform compared to HLA-G TCB monotherapy (Figure 6). Graphs were generated using GraphPad Prism Software. Time-to-event analysis was performed using the internal tool DOPSa (based on R software). Test groups were compared using the Log-Rank test, and p values ​​were corrected for multiple testing using the Bonferroni-Holm method. Significant changes compared to the control group (vehicle) at p<0.05 are indicated with an asterisk (*p<0.05, **p<0.01, ***p<0.001).

[0186] To evaluate the effect of HLA-G TCB treatment on immune PD changes as monotherapy and in combination with FAP4-1BBL, flow cytometry analysis was performed on day 9 of treatment ("dx+9," 2 days after the second injection). Tumors from four mice per group were harvested and processed to obtain single-cell suspensions. Samples were disrupted using manual scissors and a Miltenyi Gentle MACS instrument, then digested in an enzyme mixture containing DNAse I (catalog no. 10104159001, Sigma-Aldrich) and collagenase D (catalog no. 11088882001, Sigma-Aldrich). After digestion, the tissue mixture was filtered through 100 and 70 μm filters and resuspended in a single-cell suspension in a buffer containing a cocktail of fluorescently labeled antibodies for detection of immune cells and tumor targets. μ-stained cells were analyzed on a fluorescence-activated cell sorting (FACS) Fortessa device running Diva software. Raw data were analyzed using FlowJo software and a predefined gating strategy to identify tumor-infiltrating lymphocytes. Immunopharmacodynamic analysis confirmed immune cell infiltration (e.g., CD45+, CD4+, and CD8+ T cells, Tregs, and NK cells) in tumors after P1AF7977 monotherapy and combination therapy with FAP4-1BBL (Figure 7). In tumors from mice treated with HLA-G TCB in combination with FAP4-1BBL, the levels of macrophages (CD68+) were significantly lower than in tumors from mice treated with 0.5 mg / kg P1AF7977 monotherapy, indicating a potential reduction in macrophage-mediated suppression of T cell activation.

[0187] Soluble CD25 levels were measured in 50 μL serum samples (1:5 dilution) at two different time points (6 h after the first and fourth treatment cycles, respectively) using a human CD25 / interleukin (IL)-2R alpha kit (catalog number LXSAHM-01, R&D Systems, Minneapolis, MN). Soluble CD25 levels increased in the serum of CR5044 tumor-bearing mice after HLA-G TCB monotherapy and in combination with P1AD5195, indicating T cell activation during treatment (Figure 8). In the combination treatment group, elevated serum CD25 levels were observed for a long period of time, even after the fourth HLA-G TCB treatment cycle, indicating sustained T cell activation (Figure 8, left).

[0188] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the illustrations and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. 1. An anti-HLA-G / anti-CD3 bispecific antibody for use in the treatment of cancer in an individual, said treatment comprising administration of said anti-HLA-G / anti-CD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

2. 1. A 4-1BB (CD137) agonist for use in the treatment of cancer in an individual, said treatment comprising administration of said 4-1BB (CD137) agonist in combination with an anti-HLA-G / anti-CD3 bispecific antibody.

3. 1. Use of an anti-HLA-G / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of cancer in an individual, said treatment comprising administration of said anti-HLA-G / anti-CD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

4. 1. Use of a 4-1BB (CD137) agonist in the manufacture of a medicament for the treatment of cancer in an individual, said treatment comprising administration of said 4-1BB (CD137) agonist in combination with an anti-HLA-G / anti-CD3 bispecific antibody.

5. A method for treating cancer in an individual, comprising administering to said individual an anti-HLA-G / anti-CD3 bispecific antibody and a 4-1BB (CD137) agonist.

6. 1. A kit comprising a first medicament comprising an anti-HLA-G / anti-CD3 bispecific antibody and a second medicament comprising a 4-1BB (CD137) agonist, optionally further comprising a package insert containing instructions for administering the first medicament in combination with the second medicament to treat cancer in an individual.

7. the anti-HLA-G / anti-CD3 bispecific antibody (i) a first antigen-binding portion that specifically binds to CD3 and comprises a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, and a CDR-H3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6; (ii) a second antigen-binding portion that specifically binds to HLA-G and comprises a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 9, a CDR-H2 of SEQ ID NO: 10, and a CDR-H3 of SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 13, and a CDR-L3 of SEQ ID NO: 14; Including, 10. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to any one of the preceding claims.

8. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to any one of the preceding claims, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen-binding portion that specifically binds to HLA-G and / or an Fc domain composed of a first subunit and a second subunit.

9. the anti-HLA-G / anti-CD3 bispecific antibody (i) a first antigen-binding moiety that specifically binds to CD3, the first antigen-binding moiety comprising a heavy chain variable region comprising a heavy chain CDR (CDR-H) 1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 2, and a CDR-H3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR (CDR-L) 4 of SEQ ID NO: 4, a CDR-L2 of SEQ ID NO: 5, and a CDR-L3 of SEQ ID NO: 6, the first antigen-binding moiety being a crossover Fab molecule in which either the variable region or the constant region of the Fab light chain and the Fab heavy chain have been exchanged; (ii) a second antigen-binding portion and a third antigen-binding portion that specifically bind to HLA-G, each of which comprises a heavy chain variable region comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 9, a CDR-H2 of SEQ ID NO: 10, and a CDR-H3 of SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 12, a CDR-L2 of SEQ ID NO: 13, and a CDR-L3 of SEQ ID NO: 14, and which are Fab molecules, particularly conventional Fab molecules; (iii) an Fc domain composed of a first subunit and a second subunit; Including, the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain, the first antigen-binding portion is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the third antigen-binding portion is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

10. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to any one of the preceding claims.

10. the first antigen-binding portion of the anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8; and / or the second antigen-binding portion and (if present) the third antigen-binding portion of the anti-HLA-G / anti-CD3 bispecific antibody comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; 10. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to any one of the preceding claims.

11. the first antigen-binding portion of the anti-HLA-G / anti-CD3 bispecific antibody is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged; and wherein the second antigen-binding portion and, if present, the third antigen-binding portion of said anti-HLA-G / anti-CD3 bispecific antibody are conventional Fab molecules in which, in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (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, 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), 10. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to any one of the preceding claims.

12. the Fc domain of said anti-HLA-G / anti-CD3 bispecific antibody comprises a modification that promotes association of the first and second subunits of said Fc domain; and / or the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or reduce effector function; 10. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to any one of the preceding claims.

13. 10. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to any one of the preceding claims, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises: a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 46; a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47; a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 48; and a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

49.

14. 10. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to any one of the preceding claims, wherein the 4-1BB (CD137) agonist comprises the ectodomain of 4-1BBL or a fragment thereof, in particular the three ectodomains of 4-1BBL or a fragment thereof.

15. 10. The anti-HLA-G / anti-CD3 bispecific antibody for use, 4-1BB (CD137) agonist for use, use, method, or kit according to any one of the preceding claims, wherein the ectodomain of 4-1BBL or a fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, in particular an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

29.

16. 10. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to any one of the preceding claims, wherein the 4-1BB (CD137) agonist comprises an antigen-binding portion that specifically binds to a tumor-associated antigen, in particular fibroblast activation protein (FAP).

17. 10. The anti-HLA-G / anti-CD3 bispecific antibody for use, 4-1BB (CD137) agonist for use, use, method, or kit according to any one of the preceding claims, wherein the antigen-binding portion that specifically binds to FAP comprises a heavy chain variable region (VH) comprising a heavy chain CDR (CDR-H)1 of SEQ ID NO: 17, a CDR-H2 of SEQ ID NO: 18, and a CDR-H3 of SEQ ID NO: 19, and a light chain variable region comprising a light chain CDR (CDR-L)1 of SEQ ID NO: 20, a CDR-L2 of SEQ ID NO: 21, and a CDR-L3 of SEQ ID NO:

22.

18. 20. The anti-HLA-G / anti-CD3 bispecific antibody for use, 4-1BB (CD137) agonist for use, use, method, or kit according to any one of the preceding claims, wherein the antigen-binding portion that specifically binds to FAP comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

24.

19. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to any one of the preceding claims, wherein the 4-1BB (CD137) agonist comprises an Fc domain composed of a first subunit and a second subunit.

20. the Fc domain of said 4-1BB (CD137) agonist comprises a modification that promotes association of a first subunit with a second subunit of said Fc domain; and / or the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or reduce effector function; 10. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to any one of the preceding claims.

21. The 4-1BB (CD137) agonist (i) the first, second and third ectodomains of 4-1BBL or fragments thereof; (ii) an antigen-binding portion that specifically binds to FAP, wherein the antigen-binding portion is a Fab molecule; (iii) an Fc domain composed of a first subunit and a second subunit; (iv) a CL domain and a CH1 domain; an antigen-binding molecule comprising The antigen-binding molecule (a) (a1) the first ectodomain of 4-1BBL or a fragment thereof, which is fused at its C-terminus to the N-terminus of the second ectodomain of 4-1BBL or a fragment thereof; (a2) the second ectodomain of 4-1BBL or a fragment thereof, wherein the second ectodomain of 4-1BBL or a fragment thereof is fused at its C-terminus to the N-terminus of the CL domain; (a3) the CL domain, wherein the CL domain is fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit); and (a4) one of the subunits of the Fc domain (e.g., the first subunit) a first polypeptide comprising: (b) (b1) the third ectodomain of 4-1BBL or a fragment thereof, wherein the third ectodomain of 4-1BBL or a fragment thereof is fused at its C-terminus to the N-terminus of the CH1 domain; and (b2) the CH1 domain and a second polypeptide comprising: (c) (c1) a heavy chain of the Fab molecule, the heavy chain being fused at its C-terminus to the N-terminus of the other of the subunits of the Fc domain (e.g., the second subunit); and (c2) the other of the subunits of the Fc domain (e.g., the second subunit) and a third polypeptide comprising: (d) a fourth polypeptide comprising the light chain of said Fab molecule; and It consists of 10. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to any one of the preceding claims.

22. in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering); and and wherein in the CH1 domain of said second polypeptide, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering); 22. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to claim 21.

23. the 4-1BB (CD137) agonist first polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:51; the 4-1BB (CD137) agonist second polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52; the 4-1BB (CD137) agonist third polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:53; and the 4-1BB (CD137) agonist fourth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:54; 23. An anti-HLA-G / anti-CD3 bispecific antibody for use, a 4-1BB (CD137) agonist for use, a use, a method, or a kit according to claim 21 or claim 22.

24. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to any one of claims 1 to 13, wherein the 4-1BB (CD137) agonist is an anti-4-1BB antibody, in particular an anti-FAP / anti-4-1BB bispecific antibody.

25. 10. The anti-HLA-G / anti-CD3 bispecific antibody for use, 4-1BB (CD137) agonist for use, use, method, or kit according to any one of the preceding claims, wherein the cancer is an HLA-G positive cancer.

26. 10. The anti-HLA-G / anti-CD3 bispecific antibody for use, the 4-1BB (CD137) agonist for use, the use, the method, or the kit according to any one of the preceding claims, wherein the cancer is a cancer selected from the group consisting of lung cancer, head and neck cancer, bladder cancer, esophageal cancer, skin cancer, soft tissue cancer, gastric cancer, cervical cancer, and ovarian cancer.

27. The invention as herein described.

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