Combination therapy with PD1-LAG3 bispecific antibodies and HLA-g t cell bispecific antibodies

Abstract

The present invention relates to combination therapies employing an anti-PD1 / anti-LAG3 bispecific antibody and an HLA-G T cell activation bispecific antibody, the use of these combination therapies for the treatment of cancer and methods of using the combination therapies.

Description

TECHNICAL FIELD

[0001] The present invention relates to combination therapies employing PD1-LAG3 bispecific antibodies and HLA-G T cell activating bispecific antibodies, the use of these combination therapies for the treatment of cancer and methods of using these combination therapies. BACKGROUND

[0002] T cell activating bispecific antibodies are a promising class of cancer therapeutics designed to arm cytotoxic T cells against tumor cells. Such antibodies bind simultaneously to CD3 on T cells and an antigen expressed on tumor cells leading to activation of the T cells and subsequent lysis of the tumor cells.

[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 class I nonclassical family of molecules. This class I molecule is a heterodimer consisting of one heavy chain and one light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane but can also be shed / secreted. HLA-G is mainly expressed on cytotrophoblasts in the placenta. Several tumors, including pancreatic, breast, skin, colorectal, gastric and ovarian cancer, 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). There are also reports that this expression is associated with pathological conditions such as inflammatory diseases, GvHD and cancer. Expression of HLA-G has been reported to be associated with poor prognosis in cancer. Tumor cells induce immune tolerance / suppression to evade host immune surveillance via HLA-G expression.

[0004] HLA-G shares high homology (>98%) with other MHC I molecules, making it difficult to generate truly HLA-G specific antibodies that are not cross-reactive with other MHC I molecules. Due to the high polymorphism and high homology of the HLA family, many antibodies lack a truly specific HLA-G binding property and often also bind or cross-react with other HLA family members (as MHC complex with ß2M or in its ß2M free form) or they do not inhibit the binding of the HLA-G ß2M MHC complex to its receptors ILT2 and / or ILT4 at all (and are considered non-antagonistic antibodies). Antibodies that specifically bind to HLA-G are described in WO 2019 / 202040, WO 2019 / 202041 and WO 2022129120. These documents also describe T cell bispecific antibodies comprising a binding moiety that specifically binds to HLA-G.

[0005] The anti-tumor activity of HLA-G-TCB has been demonstrated in vitro using different HLA-G positive tumor cell lines and in vivo using a mouse model. HLA-G-TCB induced T cell activation, IFNy secretion and cytotoxicity showed a dose-dependent manner and correlated with the HLA-G density on the cell surface as well as the percentage of HLA-G positive cells. In line with the mode of action of TCB, tumor growth inhibition was accompanied by an increase in cytokine secretion (including IFNy), tumor T cell infiltration and activation, as reflected by increased expression of T cell activation markers including CD69, CD25 and granzyme B. Assessment of HLA-G expression on tumor cell lines upon IFNy stimulation in vitro as well as in PDX (patient-derived xenograft) tumors treated with HLA-G TCB indicated that HLA-G expression can be upregulated upon IFNy stimulation and TCB treatment, respectively, and can positively influence the in vivo anti-tumor activity of HLA-G-TCB.

[0006] T cell exhaustion can pose a challenge if the redirected T cells do not efficiently recognize and destroy the targeted tumor cells, leading to a reduced efficacy of T cell directed immunotherapy. Exhausted T cells are characterized by sustained expression of the inhibitory molecule PD1 (programmed cell death protein 1) and it has been found that blocking the PD1 and PD-L1 (PD-1 ligand) interaction can reverse T cell exhaustion and restore antigen-specific T cell responses. However, targeting the PD1-PD-L1 pathway alone does not always lead to a reversal of T cell exhaustion, possibly due to resistance mechanisms, immunosuppressive activity of MDSCs and / or regulatory T cells.

[0007] Lymphocyte activation gene-3 (LAG3 or CD223) was originally discovered in an experiment designed to selectively isolate molecules expressed in an IL-2 dependent NK cell line (Triebel F et al., Cancer Lett. 235 (2006), 147-153). LAG3 is a unique transmembrane protein that shares structural homology with CD4 with four extracellular immunoglobulin superfamily-like domains (D1-D4). The membrane distal IgG domain contains a short amino acid sequence, a so-called extra loop, not found in other IgG superfamily proteins. The intracellular domain contains a unique amino acid sequence (KIEELE, SEQ ID NO: 105) that is essential for LAG3 to negatively affect T cell function. LAG3 can be cleaved by metalloproteases at the connecting peptide (CP) to generate a soluble form that is detectable in serum. Like CD4, the LAG3 protein binds to MHC class II molecules, but with higher affinity and at a different site than CD4 (Huard et al. Proc. Natl. Acad. Sci. USA 94 (1997), 5744-5749). LAG3 is expressed by T cells, B cells, NK cells and plasmacytoid dendritic cells (pDC) and is upregulated upon T cell activation. It modulates T cell function as well as T cell homeostasis. A subpopulation of anergized or functionally impaired conventional T cells express LAG3. LAG3 + T cells are enriched at tumor sites and during chronic viral infections (Sierro et al. Expert Opin. Ther. Targets 15 (2011), 91-101). LAG3 has been shown to play a role in CD8 T cell exhaustion (Blackburn et al. Nature Immunol. 10 (2009), 29-37). Therefore, there is a need for antibodies that are able to antagonize the activity of LAG3 and can be used to generate and restore an immune response against tumors.

[0008] By targeting both PD1 and LAG3 on dysfunctional tumor-specific T lymphocytes, PD1-LAG3 aims to restore effective anti-tumor immune responses and provide survival benefits to more cancer patients compared to currently available checkpoint inhibitors. By preferentially targeting dysfunctional T cells co-expressing PD1 / LAG3 and potentially reducing LAG3-expressing Tregs in the tumor microenvironment, PD1-LAG3 BsAb can avoid re-activation of Treg-mediated immune suppression while restoring anti-tumor immune responses.

[0009] There is a need for new combination therapies using two or more bispecific antibodies to improve immunotherapy by additive stimulation that cannot be achieved with a single molecule to overcome the effects caused by lack of co-stimulation during T cell bispecific antibody-mediated cancer immunotherapy, such as poor persistence of T cell responses. SUMMARY

[0010] The present invention relates to combination therapies employing an anti-HLA-G / anti-CD3 bispecific antibody and a bispecific antibody comprising a first antigen binding domain that specifically binds to Programmed cell death protein 1 (PD1) and a second antigen binding domain that specifically binds to Lymphocyte-activation gene-3 (LAG3). It has been found that the anti-PD1 / anti-LAG3 bispecific antibodies described herein are superior to anti-PD1 antibodies, in particular when used in combination with an anti-HLA-G / anti-CD3 bispecific antibody, as they provide better selectivity and efficacy. These anti-PD1 / anti-LAG3 bispecific antibodies are further characterized in that they show reduced sink effect (as shown by reduced internalization by T cells), they preferentially bind to conventional T cells but not to Tregs and are able to restore T cell effector function from Treg suppression, they show increased tumor-specific T cell effector function and increased tumor eradication in vivo. Based on these properties, they are advantageous for use in combination with T cell bispecific antibodies, in particular anti-HLA-G / anti-CD3 bispecific antibodies.

[0011] Described herein is an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, in particular HLA-G-expressing cancer, wherein the anti-HLA-G / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody.

[0012] The present application provides an anti-HLA-G / anti-CD3 bispecific antibody for use in a method as defined hereinbefore, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to Programmed cell death protein 1 (PD1) and a second antigen binding domain that specifically binds to Lymphocyte-activating gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1 comprises

[0013] a VH domain comprising

[0014] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0015] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and

[0016] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0017] a VL domain comprising

[0018] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4;

[0019] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0020] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0021] In one aspect, there is provided an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, in particular cancer expressing HLA-G, wherein the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition, or separately in two or more different compositions.

[0022] Further, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, in particular HLA-G-expressing cancer, is provided, wherein the anti-HLA-G / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, and wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain, which is an IgG Fc domain, in particular an IgGl Fc domain or an IgG4 Fc domain, and wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular to an Fcy receptor. More particularly, the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain of the human IgGl subclass, which Fc domain contains the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

[0023] In one aspect, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method as described in the preceding text is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, which second antigen binding domain comprises

[0024] (a) a VH domain, which VH domain comprises

[0025] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0026] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and

[0027] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and

[0028] a VL domain, which VL domain comprises

[0029] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14;

[0030] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and

[0031] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or

[0032] (b) a VH domain, which VH domain comprises

[0033] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19;

[0034] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and

[0035] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21 ; and

[0036] a VL domain comprising

[0037] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22;

[0038] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and

[0039] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0040] In another aspect, there is provided an anti-HLA-G / anti-CD3 bispecific antibody for use in a method as disclosed herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to PD1, the first antigen binding domain comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0041] In a further aspect, there is provided an anti-HLA-G / anti-CD3 bispecific antibody for use as described herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising

[0042] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or

[0043] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0044] In another aspect, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method as described herein is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to PD1, the first antigen binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, and

[0045] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising the amino acid sequence of SEQ ID NO: 28, or

[0046] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29; and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, or

[0047] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31; and a VL domain comprising the amino acid sequence of SEQ ID NO: 32, or

[0048] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 33; and a VL domain comprising the amino acid sequence of SEQ ID NO: 34, or

[0049] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 64; and a VL domain comprising the amino acid sequence of SEQ ID NO: 65.

[0050] In another aspect, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method as disclosed herein is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to PD1, the first antigen binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, and

[0051] a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0052] a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0053] In a further aspect, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, in particular HLA-G expressing cancer, is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1 and a Fab fragment that specifically binds to LAG3. In one aspect, the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1, wherein the variable domains VL and VH are replaced by each other, such that VL is part of the heavy chain and VH is part of the light chain.

[0054] In another aspect, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method as previously disclosed is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises monovalent binding to PD-1 and monovalent binding to LAG3.

[0055] In a further aspect, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method as previously disclosed is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody is a humanized antibody or a chimeric antibody. In particular, the anti-PD1 / anti-LAG3 bispecific antibody is a humanized antibody. Further, an anti-PD1 / anti-LAG3 bispecific antibody as previously described is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain comprising a modification that promotes association of a first subunit with a second subunit of the Fc domain. In one aspect, an anti-PD1 / anti-LAG3 bispecific antibody is provided, wherein the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole according to the knob-in-hole approach. Specifically, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), while the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index).

[0056] In a particular aspect, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, in particular HLA-G expressing cancer, is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises

[0057] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37; and a second light chain comprising the amino acid sequence of SEQ ID NO: 38; or

[0058] (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 39; and a second light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0059] More particularly, the anti-PD1 / anti-LAG3 bispecific antibody comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37; and a second light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0060] Further, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, in particular cancer expressing HLA-G, is provided, wherein the anti-HLA-G / anti-CD3 bispecific antibody is for use in combination with an anti-PD1 / anti-LAG3 bispecific antibody, and wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to HLA-G, the first antigen binding domain comprising a heavy chain variable region (V H CD3) and a light chain variable region (V L CD3), and the second antigen binding domain comprising a heavy chain variable region (V H HLA-G) and a light chain variable region (V L HLA-G).

[0061] In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain that specifically binds to CD3, the first antigen binding domain comprising

[0062] a heavy chain variable region (V H CD3) comprising a CDR-H1 sequence of SEQ ID NO: 41, a CDR-H2 sequence of SEQ ID NO: 42, and a CDR-H3 sequence of SEQ ID NO: 43; and / or

[0063] a heavy chain variable region (V L CD3) comprising a CDR-H1 sequence of SEQ ID NO: 44, a CDR-H2 sequence of SEQ ID NO: 45, and a CDR-H3 sequence of SEQ ID NO: 46.

[0064] More particularly, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain that specifically binds to CD3, the first antigen binding domain comprising a heavy chain variable region (V H CD3) comprising the amino acid sequence of SEQ ID NO: 47; and / or a light chain variable region (V L CD3) comprising the amino acid sequence of SEQ ID NO: 48.

[0065] In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, in particular HLA-G expressing cancer, comprises a second antigen binding domain that specifically binds to HLA-G, the second antigen binding domain comprising

[0066] a heavy chain variable region (V H HLA-G) comprising a CDR-H1 sequence of SEQ ID NO: 49, a CDR-H2 sequence of SEQ ID NO: 50, and a CDR-H3 sequence of SEQ ID NO: 51 ; and / or

[0067] a light chain variable region (V L HLA-G) comprising a CDR-L1 sequence of SEQ ID NO: 52, a CDR-L2 sequence of SEQ ID NO: 53, and a CDR-L3 sequence of SEQ ID NO: 54.

[0068] In particular, the second antigen binding domain comprises a heavy chain variable region (V H HLA-G) comprising the amino acid sequence of SEQ ID NO: 55; and / or a light chain variable region (V LIn a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody for use in treating cancer, in particular cancer expressing HLA-G, comprises a third antigen binding domain that binds to HLA-G. In another aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

[0069] In one aspect, the method is provided, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first Fab fragment that specifically binds to CD3 and a second and optional third Fab fragment that specifically binds to HLA-G, which first Fab fragment comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 47 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48; and which second and optional third Fab fragment comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 55 and a VL domain comprising the amino acid sequence of SEQ ID NO: 56.

[0070] In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody for use in treating cancer, in particular cancer expressing HLA-G, is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, and wherein the combination is administered at intervals of about one to three weeks.

[0071] In one further aspect, a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in treating cancer, in particular cancer expressing HLA-G, is provided, wherein the treatment comprises administering the composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with a composition comprising an anti-HLA-G / anti-CD3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen binding domain that specifically binds to lymphocyte-activation gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1 comprises a VH domain comprising

[0072] (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0073] (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 2; and

[0074] (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 3; and

[0075] a VL domain comprising

[0076] (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 4;

[0077] (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 5; and

[0078] (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 6.

[0079] In one aspect, the composition comprises an anti-PD1 / anti-LAG3 bispecific antibody comprising a first antigen binding domain that specifically binds to PD1, the first antigen binding domain comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0080] In one further aspect, the composition comprises an anti-PD1 / anti-LAG3 bispecific antibody comprising a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising

[0081] (a) a VH domain, the VH domain comprising

[0082] (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 11;

[0083] (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 12; and

[0084] (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 13; and

[0085] a VL domain comprising

[0086] (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 14;

[0087] (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 15; and

[0088] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or

[0089] (b) a VH domain comprising

[0090] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19;

[0091] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and

[0092] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21 ; and

[0093] a VL domain comprising

[0094] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22;

[0095] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and

[0096] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0097] In one aspect, the composition comprises an anti-PD1 / anti-LAG3 bispecific antibody comprising an antigen binding domain that specifically binds to LAG3, the antigen binding domain comprising

[0098] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or

[0099] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0100] In one particular aspect, the composition comprises an anti-PD1 / anti-LAG3 bispecific antibody comprising

[0101] a first Fab fragment that specifically binds to PD1, the first Fab fragment comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, and

[0102] a second Fab fragment that specifically binds to LAG3, the second Fab fragment comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0103] Further, a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in the treatment of cancer, in particular cancer expressing HLA-G, is provided, wherein the treatment comprises administering the composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with a composition comprising an anti-HLA-G / anti-CD3 bispecific antibody, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain and a second antigen binding domain, the first antigen binding domain comprising a heavy chain variable region (V H CD3) and a light chain variable region (V L CD3), and the second antigen binding domain comprising a heavy chain variable region (V H HLA-G) and a light chain variable region (V L HLA-G).

[0104] In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain comprising a heavy chain variable region (V H CD3) comprising the CDR-H1 sequence of SEQ ID NO: 41, the CDR-H2 sequence of SEQ ID NO: 42, and the CDR-H3 sequence of SEQ ID NO: 43; and / or a light chain variable region (V L CD3) comprising the CDR-L1 sequence of SEQ ID NO: 44, the CDR-L2 sequence of SEQ ID NO: 45, and the CDR-L3 sequence of SEQ ID NO: 46. More particularly, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain comprising a heavy chain variable region (V H CD3) comprising the amino acid sequence of SEQ ID NO: 47; and / or a light chain variable region (V L CD3) comprising the amino acid sequence of SEQ ID NO: 48. In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen binding domain comprising a heavy chain variable region (V Ha heavy chain variable region (VH) comprising a CDR-H1 sequence of SEQ ID NO: 49, a CDR-H2 sequence of SEQ ID NO: 50, and a CDR-H3 sequence of SEQ ID NO: 51 ; and / or a light chain variable region (VL) comprising a CDR-L1 sequence of SEQ ID NO: 52, a CDR-L2 sequence of SEQ ID NO: 53, and a CDR-L3 sequence of SEQ ID NO: 54. In particular, the second antigen binding domain comprises: a VH domain comprising an amino acid sequence of SEQ ID NO: 55, and a VL domain comprising an amino acid sequence of SEQ ID NO: 56. L a heavy chain variable region (VH) comprising a CDR-H1 sequence of SEQ ID NO: 49, a CDR-H2 sequence of SEQ ID NO: 50, and a CDR-H3 sequence of SEQ ID NO: 51 ; and / or a light chain variable region (VL) comprising a CDR-L1 sequence of SEQ ID NO: 52, a CDR-L2 sequence of SEQ ID NO: 53, and a CDR-L3 sequence of SEQ ID NO: 54. In particular, the second antigen binding domain comprises: a VH domain comprising an amino acid sequence of SEQ ID NO: 55, and a VL domain comprising an amino acid sequence of SEQ ID NO: 56. H a heavy chain variable region (VH) comprising a CDR-H1 sequence of SEQ ID NO: 49, a CDR-H2 sequence of SEQ ID NO: 50, and a CDR-H3 sequence of SEQ ID NO: 51 ; and / or a light chain variable region (VL) comprising a CDR-L1 sequence of SEQ ID NO: 52, a CDR-L2 sequence of SEQ ID NO: 53, and a CDR-L3 sequence of SEQ ID NO: 54. In particular, the second antigen binding domain comprises: a VH domain comprising an amino acid sequence of SEQ ID NO: 55, and a VL domain comprising an amino acid sequence of SEQ ID NO: 56. L a heavy chain variable region (VH) comprising a CDR-H1 sequence of SEQ ID NO: 49, a CDR-H2 sequence of SEQ ID NO: 50, and a CDR-H3 sequence of SEQ ID NO: 51 ; and / or a light chain variable region (VL) comprising a CDR-L1 sequence of SEQ ID NO: 52, a CDR-L2 sequence of SEQ ID NO: 53, and a CDR-L3 sequence of SEQ ID NO: 54. In particular, the second antigen binding domain comprises: a VH domain comprising an amino acid sequence of SEQ ID NO: 55, and a VL domain comprising an amino acid sequence of SEQ ID NO: 56.

[0105] In a further aspect, a pharmaceutical product is provided, comprising

[0106] (A) a first composition comprising, as active ingredient, an anti-HLA-G / anti-CD3 bispecific antibody and a pharmaceutical carrier; and

[0107] (B) a second composition comprising, as active ingredient, an anti-PD1 / anti-LAG3 bispecific antibody and a pharmaceutical carrier,

[0108] for the combined, sequential or simultaneous treatment of a disease, in particular a cancer, in particular a cancer expressing HLA-G.

[0109] In another aspect, there is provided a pharmaceutical composition comprising an anti-HLA-G / anti-CD3 bispecific antibody in combination with an anti-PD1 / anti-LAG3 bispecific antibody for use in the treatment of a disease, in particular a cancer, in particular a cancer expressing HLA-G, in particular a disease selected from the group consisting of lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the naso-pharynx, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancers, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including any of the foregoing in a refractory form, or a combination of one or more of the foregoing cancers, in combination, sequentially or simultaneously, in particular for the treatment of solid tumors, including but not limited to renal cell carcinoma, colorectal cancer, non-small cell lung cancer and pancreatic ductal adenocarcinoma (PDAC).

[0110] In another aspect, there is provided the use of an anti-HLA-G / anti-CD3 bispecific antibody in combination with an anti-PD1 / anti-LAG3 bispecific antibody for the manufacture of a medicament for the treatment of or to delay progression of a proliferative disease, in particular for the treatment of a cancer, in particular a cancer expressing HLA-G, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to Programmed Cell Death 1 (PD1) and a second antigen binding domain that specifically binds to Lymphocyte-Activation Gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1 comprises

[0111] (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0112] (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and

[0113] (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0114] a VL domain comprising

[0115] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4;

[0116] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0117] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0118] In another aspect, the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising

[0119] (a) a VH domain comprising

[0120] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0121] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and

[0122] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and

[0123] a VL domain comprising

[0124] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14;

[0125] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and

[0126] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or

[0127] (b) a VH domain comprising

[0128] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19;

[0129] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and

[0130] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21 ; and

[0131] a VL domain comprising

[0132] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22;

[0133] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and

[0134] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0135] In another aspect, there is provided use of an anti-HLA-G / anti-CD3 bispecific antibody in combination with an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for treating or delaying progression of a proliferative disease, in particular treating a cancer, in particular a HLA-G expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first Fab fragment that specifically binds to PD1 and a second Fab fragment that specifically binds to LAG3, the first Fab fragment comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 9, and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and the second Fab fragment comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0136] In a further aspect, there is provided a method of treating a cancer, in particular a HLA-G expressing cancer, in a subject, the method comprising administering to the subject an effective amount of an anti-HLA-G / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to Programmed Cell Death Protein 1 (PD1) and a second antigen binding domain that specifically binds to Lymphocyte-Activating Gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1

[0137] comprising: a VH domain comprising

[0138] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0139] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and

[0140] (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 3; and

[0141] a VL domain comprising

[0142] (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 4;

[0143] (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 5; and

[0144] (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 6.

[0145] In one aspect, the method is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising

[0146] (a) a VH domain comprising

[0147] (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 11 ;

[0148] (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 12; and

[0149] (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 13; and

[0150] a VL domain comprising

[0151] (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 14;

[0152] (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 15; and

[0153] (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 16; or

[0154] (b) a VH domain comprising

[0155] (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 19;

[0156] (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 20; and

[0157] (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 21; and

[0158] VL domain, comprising

[0159] (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 22;

[0160] (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 23; and

[0161] (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 24.

[0162] In another aspect, a method for treating cancer is provided, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1 and a second Fab fragment that specifically binds to LAG3, the first Fab fragment comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 9, and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and the second Fab fragment comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0163] In one aspect, a method for treating cancer is provided, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to HLA-G, the first antigen binding domain comprises a heavy chain variable region (V H CD3) and a light chain variable region (V L CD3), and the second antigen binding domain comprises a heavy chain variable region (V H HLA-G) and a light chain variable region (V L HLA-G). In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain comprising: a heavy chain variable region (V H CD3) comprising a CDR-H1 sequence of SEQ ID NO: 41, a CDR-H2 sequence of SEQ ID NO: 42, and a CDR-H3 sequence of SEQ ID NO: 43; and / or a light chain variable region (V LCD3), which comprises a CDR-L1 sequence of SEQ ID NO: 44, a CDR-L2 sequence of SEQ ID NO: 45, and a CDR-L3 sequence of SEQ ID NO: 46. More specifically, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen-binding domain comprising: a heavy chain variable region (V H CD3), which comprises the amino acid sequence of SEQ ID NO: 47; and / or a light chain variable region (V L CD3), which comprises the amino acid sequence of SEQ ID NO: 48. In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen-binding domain, the second antigen-binding domain comprising: a heavy chain variable region (V H HLA-G), which comprises the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50 and the CDR-H3 sequence of SEQ ID NO: 51; and / or a light chain variable region (V L HLA-G), comprising a CDR-L1 sequence of SEQ ID NO: 52, a CDR-L2 sequence of SEQ ID NO: 53, and a CDR-L3 sequence of SEQ ID NO: 54. In particular, the second antigen-binding domain comprises: a heavy chain variable region (V H HLA-G), comprising the amino acid sequence of SEQ ID NO: 55; and / or a light chain variable region (V L HLA-G) comprising the amino acid sequence of SEQ ID NO: 56. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to HLA-G. In particular, the first antigen-binding domain that specifically binds to CD3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 47 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48; and the second (and optional third) antigen-binding domain that specifically binds to HLA-G comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 55 and a VL domain comprising the amino acid sequence of SEQ ID NO: 56. In another aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function.

[0164] In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody is administered together with the anti-PD1 / anti-LAG3 bispecific antibody in a single composition, or administered separately in two or more different compositions. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously. In another aspect, the anti-HLA-G / anti-CD3 bispecific antibody is administered simultaneously with, prior to, or after the anti-PD1 / anti-LAG3 bispecific antibody.

[0165] In any of the above aspects, the subject is preferably a mammal, in particular a human. BRIEF DESCRIPTION OF DRAWINGS

[0166] Figures 1A and IB are schematic representations of the specific anti-HLA-G / anti-CD3 bispecific antibody (Figure 1A) and the specific anti-PD1 / anti-LAG3 bispecific antibody (Figure IB) as used in the examples. These molecules are described in more detail in Example 1. In Figure 1A, an exemplary bispecific anti-HLA-G / anti-CD3 antibody in 2+1 format is shown (designated HLA-G TCB). This molecule comprises a single antigen binding moiety against CD3, two antigen binding moieties against HLA-G, and an Fc domain. Figure IB shows an anti-PD1 / anti-LAG3 bispecific antibody in 1+1 CrossMab format, wherein the PD1 binding domain comprises a crossFab with VH / VL domain exchange, and the LAG3 binding domain comprises CH1 and CK domains with amino acid mutations to support correct pairing (“charged variants”). The Fc part of both antibodies shown here includes a knob-in-hole mutation (shown by black circles) as well as the amino acid mutations L234A, L235A, and P329G, which almost completely eliminate Fc receptor binding to human IgGl Fc domains.

[0167] Figure 2 shows a comparison of the regimen of in vivo efficacy study #1173 of HLA-G TCB (P1AD7977) as monotherapy and in combination with PD1-LAG3 bispecific antibody (P1AA0927) or with PD-L1 antibody (P1AE0828) to PD-L1 antibody alone in fully humanized NSG mice carrying BC004 PDX (patient-derived xenograft). 2 x 10 6BC004 cells were injected into the mammary fat pad (IMFP) of humanized NSG mice. 3 After the average volume of , mice were randomly divided into five groups to receive: A) histidine buffer (vehicle) as a control; B) HLA-G-TCB (0.5 mg / kg, once weekly, intravenously); C) anti-PD-L1 antibody (10 mg / kg, once weekly, intravenously); D) HLA-G-TCB (0.5 mg / kg, once weekly, intravenously) + anti-PD-L1 antibody (10 mg / kg, once weekly, intravenously); E) HLA-G-TCB (0.5 mg / kg, once weekly, intravenously) + PD1-LAG3 bispecific Ab (3 mg / kg, once weekly, intravenously). The subgroups of mice receiving different treatment combinations are defined in the table below the protocol. This experiment was described in Example 2.

[0168] Figure 3 shows the results of Study #1173. Tumor volume was measured twice weekly with calipers. Figure 3A shows a direct comparison of the results for all subgroups over the time period from Day 34 to Day 65. Data are presented as tumor volume (median + / - IQR). In Figure 3B, tumor volume measurements (mm) for each individual animal over the time period from Day 34 to Day 65 are shown. 3 ), showing uniformity of antitumor responses across various subgroups.

[0169] Figure 4 shows the scheme of in vivo efficacy study #1655 of HLAG TCB (P1AD7977) as monotherapy and in combination with two different concentrations (1.5 mg / kg or 3 mg / kg) of PD1-LAG3 bispecific antibodies or with a combination of (P1AA0927) PD1 antibody (P1AA6975) + anti-LAG3 antibody (P1AD8676) in fully humanized NSG mice bearing BC004 PDX (patient-derived xenografts). 2 × 10 6 BC004 cells were injected into the mammary fat pad (IMFP) of humanized NSG mice. 3After the average volume of the tumors was determined, the mice were randomized into 5 groups to receive: A) histidine buffer (vehicle) as control; B) HLA-G-TCB (0.5 mg / kg, once a week, intravenously), C) HLA-G-TCB (0.5 mg / kg, once a week, intravenously) and anti-PD1-LAG3 antibodies (3 mg / kg, once a week, intravenously), D) HLA-G-TCB (0.5 mg / kg, once a week, intravenously) and anti-PD1-LAG3 bispecific antibodies (1.5 mg / kg, once a week, intravenously), E) HLA-G-TCB (0.5 mg / kg, once a week, intravenously) + anti-PD1 Ab (1.5 mg / kg, once a week, intravenously) + anti-LAG3 antibodies (1.5 mg / kg, once a week, intravenously). Subgroups of mice receiving different treatment combinations are defined in the table below the protocol. The experiment was performed as described in Example 2.

[0170] Figure 5 shows the results of study #1655. Tumor volume was measured with calipers 2 times per week. Figure 5A shows a direct comparison of all subgroup results in the time period from day 35 to day 60. Data is shown as tumor volume (median + / - IQR). In Figure 5B, the individual tumor volume measurements (mm 3 ) of each individual animal in the time period from day 35 to day 60 are shown, demonstrating the homogeneity of the anti-tumor response in the various subgroups.

[0171] Figure 6 shows the results of the immunopharmacodynamic analysis performed in study #1655. For this, flow cytometry analysis was performed on single cell suspensions obtained by harvesting and processing tumors and staining them with a cocktail of fluorescently labeled antibodies to detect immune cell markers on treatment day 17 and on day 32 after the end of treatment. Figure 6A shows the level of immune cell infiltration (as indicated by the number of CD45+ cells) in the different treatment groups on treatment day 17 and on day 32. Figure 6B shows the level of T cell infiltration (as indicated by the number of CD3+ cells) and the level of T cell activation (as indicated by granzyme B expression as percentage of CD8+ T cells) in the different treatment groups on treatment day 17 and on day 32. DETAILED DESCRIPTION

[0172] DEFINITIONS

[0173] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will include the plural and vice versa.

[0174] The term "antibody", as used herein, is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0175] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the mAb, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb of a mAb preparation is directed against a single determinant on the antigen.

[0176] The term "monospecific" antibody, as used herein, denotes an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The term "bispecific" means an antibody which is able to bind specifically to at least two different antigenic determinants, e.g., two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) bind to different antigens or different epitopes on the same antigen. The bispecific antibody is in 1+1 format. Other bispecific antibody formats are in 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or in 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, a bispecific antibody comprises two antigen binding sites, each of the two antigen binding sites having specificity for a different antigenic determinant.

[0177] The term "valency" as used in the present application denotes that a specified number of binding domains are present in an antigen binding molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" denote that two binding domains, four binding domains and six binding domains, respectively, are present in an antigen binding molecule. Bispecific antibodies according to the present application are at least "bivalent" and can be "trivalent" or "multivalent" (e.g. "tetravalent" or "hexavalent"). In a particular aspect, the antibodies of the present application have two or more binding sites and are bispecific. That is, the antibodies can be bispecific even in the presence of more than two binding sites, i.e. the antibodies are trivalent or multivalent.

[0178] The terms "full-length antibody" and "intact antibody" and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of about 150000 Daltons, composed of two light chains and two heavy chains that are linked by disulfide bonds. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

[0179] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments and single domain antibodies. For a review of certain 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 discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding domains that can 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 comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Pat. No. 6,248,516 Bl). Additionally, antibody fragments comprise single-chain polypeptides characterized by having the features of a VH domain, i.e. capable of assembling with a VL domain into a functional antigen-binding site; or having the features of a VL domain, i.e. capable of assembling with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody. Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of an intact antibody, as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

[0180] Papain digestion of intact antibodies produces two identical antigen binding fragments, called "Fab" fragments, each containing a heavy chain variable domain and a light chain variable domain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Accordingly, as used herein, the term "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a light chain constant domain (CL), as well as a VH domain and a first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear free thiol groups. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.

[0181] The term "cross Fab fragment" or "xFab fragment" or "cross-over Fab fragment" refers to a Fab fragment, wherein the variable or constant regions of the heavy and light chain are exchanged. Two different chain compositions of the cross-over Fab molecule are possible and comprised in the bispecific antibodies of the present application: In one aspect, the variable regions of the Fab heavy and light chain are exchanged, i.e. the cross-over Fab molecule comprises a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), as well as a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). This cross-over Fab molecule is also referred to as CrossFab (VLVH) . In another aspect, when the constant regions of the Fab heavy and light chain are exchanged, the cross-over Fab molecule comprises a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), as well as a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1). This cross-over Fab molecule is also referred to as CrossFab (CLCH1) .

[0182] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by forming interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0183] "Exchanged single-chain Fab fragments" or "x-scFabs" are polypeptides composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form an antigen-binding domain that specifically binds to an antigen, and wherein the linker is a polypeptide of at least 30 amino acids. Furthermore, these x-scFab molecules can be further stabilized by forming interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0184] A "single-chain variable fragment (scFv)" is a fragment of the heavy chain variable region (V H ) and light chain variable region (V L ) fusion proteins, connected by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and can be V H The N-terminus of LC-terminal end, or vice versa. Despite removal of the constant regions and introduction of the linker, the protein retains the specificity of the original antibody. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides which are characterized by having a VH domain, i.e. are capable of assembling together with a VL domain to a functional antigen binding site; or a VL domain, i.e. are capable of assembling together with a VH domain to a functional antigen binding site, thereby providing the antigen binding properties of a full-length antibody.

[0185] "Scaffold antigen binding proteins" are known in the art, for example fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see for example Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one aspect of the application, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (Evibody), Lipocalins (Anticalins), Protein A derived molecules such as the Z domain of Protein A (Affibodies), A domains (Avimers / MegaBodies), serum transferrin (Transbodies); designed ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAb), variable domains of antibody heavy chains (nanobodies, aVH), V NAR fragments, fibronectin (AdNectin), C-type lectin domains (Tetranectins); variable domains of new antigen receptor beta-lactamase (V NARFragments), human gamma-crystallins or ubiquitin proteins (Affilin molecules); kunitz type domains of human protease inhibitors, microbodies such as proteins from the knottin family, peptide aptamers and fibronectin (adnectins). CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a CD28 family receptor expressed primarily on CD4+ T cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to antibody CDRs can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies (e.g. US7166697B1). Evibodies are approximately the same size as isolated variable regions of antibodies (e.g. domain antibodies). For further details see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bile pigments, retinoids and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of a cone-shaped structure that can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details see Biochim Biophys Acta 1482:337-350 (2000), US7250297B1 and US20070224633. Affibodies are scaffolds derived from protein A of Staphylococcus aureus that can be engineered to bind antigens. The domain consists of a three-helix bundle of about 58 amino acids. Libraries have been created by randomization of surface residues. For further details see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818A1. Avimers are multi-domain proteins derived from the A domain scaffold family. The native domain of about 35 amino acids adopts a defined disulphide-bonded structure. Diversity is created by natural variation exhibited by the recombination A domain family. For further details see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrins are monomeric serum transport glycoproteins.Transferrin can be engineered to bind different target antigens by inserting peptide sequences in the allowed surface loops. An example of an engineered transferrin scaffold includes the Transbody. For further details see J. Biol. Chem 274, 24066-24073 (1999). Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrins, a family of proteins that mediate attachment of integral membrane proteins to the cellular scaffold. Individual ankyrin repeats are 33-residue motifs consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and the beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (affinity maturation approach). For further details see J. Mol. Biol. 332, 489-503 (2003); PNAS 100(4), 1700-1705 (2003); and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

[0186] Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of an antibody heavy chain of Camelidae (nanobody or VHH). In addition, the term single domain antibody comprises an autologous human heavy chain variable domain (aVH) or a VHH derived from sharks (VNAR or VHH). H H fragment). In addition, the term single domain antibody comprises an autologous human heavy chain variable domain (aVH) or a VHH derived from sharks (VNAR or VHH). NARFragments. Fibronectin can be engineered to bind the scaffold of an antigen. Adnectins consist of the backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). The three loops at one end of the beta- sandwich can be engineered to enable the Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, usually thioredoxin A (TrxA), containing constrained variable peptide loops inserted at the active site. For further details see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins containing 3-4 cysteine bridges, 25-50 amino acids in length, examples of which include KalataBI and conotoxin, and knottins. Microproteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For further details of engineered knottin domains see WO2008098796.

[0187] An "antigen binding molecule that binds the same epitope as the reference molecule" with reference to a reference molecule means an antigen binding molecule that blocks the binding of the reference molecule to its antigen by 50% or more in a competition assay, and conversely, the reference molecule blocks the binding of the antigen binding molecule to its antigen by 50% or more in a competition assay.

[0188] As used herein, the term "antigen binding domain" or "antigen binding site" refers to the part of an antigen binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen binding domain" refers to the part of an antibody that comprises the region that specifically binds to and is complementary to a part or all of an antigen. In the case of larger antigens, the antigen binding molecule can bind only to a particular part of the antigen, which is referred to as an epitope. The antigen binding domain can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding domain is capable of binding to its antigen and blocking or partially blocking the function of the antigen. Antigen binding domains that specifically bind to PD1 or LAG3 include antibodies and fragments thereof as further defined herein. In addition, the antigen binding domain can include a scaffold antigen binding protein, such as a designed repeat protein or a binding domain based on a designed repeat domain (see, e.g., WO 2002 / 020565).

[0189] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids) to which an antigen binding moiety binds, thereby forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, proteins used as antigens herein can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human protein. When referring to a particular protein herein, the term encompasses "full-length," unprocessed protein, as well as any form of the protein that results from cellular processing. The term is also intended to encompass naturally occurring variants of the protein, e.g., splice variants or allelic variants.

[0190] "Specific binding" refers to binding that is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar 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 traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, for example, as measured by SPR, the degree of binding of an antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. In certain embodiments, the molecule bound to the antigen has a dissociation constant (K D ) is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -7 M or lower, such as 10 -7 M to 10 -13 M, for example 10 -9 to 10 -13 M).

[0191] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (k off and k on ). 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 conventional methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0192] As used herein, the term "high affinity" refers to an antibody that has 10 −9 M or smaller, and even more specifically 10 −10 M or smaller K D The term "low affinity" refers to antibodies with K DFor 10 -8 or higher.

[0193] An "affinity matured" antibody refers to an antibody with one or more alterations which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody which does not possess such alterations. Typically, such alterations are found in the HVRs of the antibody.

[0194] The terms "HLA-G", "human HLA-G", "HLAG" when used herein refer to HLA-G human major histocompatibility complex class I G, also known as human leukocyte antigen G (HLA-G) (exemplary SEQ ID NO: 61). Typically, HLA-G forms a MHC class I complex with beta 2 microglobulin (B2M or beta 2m). In one embodiment, HLA-G refers to the MHC class I complex of HLA-G with beta 2 microglobulin. In a preferred embodiment, HLA-G refers to the cell surface bound MHC class I complex of HLA-G and beta 2 microglobulin, also known as HLA-G1 (see e.g. WO 2022 / 129120 Al, Figure 1 and Blasschitz et al., Molecular Human Reproduction, 11 (2005) 699-710, especially Figure 1).

[0195] As used herein, an antibody (monospecific antibody, multispecific antibody or bispecific antibody) or antigen binding domain "binds to", "binds specifically to", "binds to" or is "anti-HLA-G" if it binds to the (human) HLA-G antigen or extracellular domain (ECD) thereof with a K -8 value of 5.0 x 10 D value of 5.0 x 10 D value of 5.0 x 10 D value of 5.0 x 10 -8 value of 5.0 x 10 -13 value of 5.0 x 10 In one embodiment, the antibody binds to the HLA-G ß2M MHC I complex comprising SEQ ID NO: 106.

[0196] Binding affinity is determined with standard binding assays such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), e.g. using a construct comprising the HLA-G extracellular domain (e.g. in its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined with standard binding assays, wherein an exemplary soluble HLA-G comprising an MHC class I complex comprising SEQ ID NO: 106 is used.

[0197] HLA-G has a regular MHC I fold and is composed of two chains: Chain 1 is composed of three domains: al, a2, and a3. The al and a2 domains form a peptide binding groove flanked by two a helices. Similar to other MHC I proteins, small molecule peptides (about 9-mer) can bind to this groove. Chain 2 is the b2 microglobulin (b2M), which is shared with various other MHC I proteins.

[0198] HLA-G can form functionally active complex oligomeric structures (Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729). A disulfide-linked dimer is formed between Cys 42 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. in Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729, Allan D.S., 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 has several free cysteine residues. Boyson et al., Proc Nat Acad Sci USA, 99: 16180 (2002) reported that a recombinant soluble form of HLA-G5 can form disulfide-linked dimers via intermolecular Cys42-Cys42 disulfide bonds. 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)).

[0199] HLA-G is mainly expressed on cytotrophoblasts in the placenta. Several tumors, including pancreatic cancer, breast cancer, skin cancer, colorectal cancer, gastric cancer, and ovarian cancer, 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). There are also reports that this expression is associated with pathological conditions such as inflammatory diseases, GvHD, and cancer. Expression of HLA-G has been reported to be associated with poor prognosis of cancer. Tumor cells induce immune tolerance / suppression to evade host immune surveillance via HLA-G expression.

[0200] For HLA-G, there are 7 isoforms, of which 3 are secreted and 4 are membrane-bound. The most important functional isoforms of HLA-G include HLA-G1 and HLA-G5 associated with ß2M. However, the tolerogenic immune effects of these isoforms differ and depend on the form of the ligand (monomer, dimer) and the affinity of the ligand-receptor interaction.

[0201] HLA-G proteins can be produced using standard molecular biology techniques. The nucleic acid sequences of HLA-G isoforms are known in the art. See, e.g., GenBank Accession No. AY359818.

[0202] HLA-G isoform forms promote signaling through ILTs (Ig-like transcripts), particularly ILT2, ILT4, or a combination thereof.

[0203] ILT stands for Ig-type activating and inhibitory receptors that are involved in the regulation of immune cell activation and control of immune cell function (Borges, L. et al., Curr Top Microbial Immunol, 244: 123-136 (1999)). ILTs are divided into three groups: (i) inhibitory ILTs, which contain a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and transmit inhibitory signals (ILT2, ILT3, ILT4, ILT5, and LIR8); (ii) activating ILTs, which contain a short cytoplasmic tail and charged amino acid residues in the transmembrane domain (ILT1, ILT7, ILT8, and LIR6a) and deliver activating signals through the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of the associated common gamma chain of Fc receptors; and (iii) the soluble molecule ILT6, which lacks a transmembrane domain. Recent studies have highlighted the immunoregulatory role of ILTs on the surface of antigen-presenting cells (APCs). ILT2, ILT3, and ILT4 receptors are the most well-characterized immunoinhibitory receptors, which are expressed on a variety of immune cells, including monocytes, B cells, dendritic cells, plasmacytoid dendritic cells, and subsets of NK and T cells. ILT2 is expressed on subsets of T cells and has been shown to inhibit the activation and proliferation of these cells upon ligation (Colonna M. et al., J Immunol. 2001 1, 66:2514-2521, J Immunol 2000; 165:3742-3755). ILT3 and ILT4 are upregulated by exposing immature DCs to known immunosuppressive factors, including IL-10, vitamin D3, or suppressor CD8 T cells (Chang, C. C. et al., Nat Immunol, 3:237-243 (2002)). Expression of ILTs on DCs is tightly controlled by inflammatory stimuli, cytokines, and growth factors, and is downregulated upon DC activation (Ju, X. S. et al., Gene, 331:159-164 (2004)). Expression of ILT2 and ILT4 receptors is tightly regulated by histone acetylation, which contributes to the tight control of gene expression that occurs only in myeloid lineage cells (Nakajima, H., J Immunol, 171:6611-6620 (2003)).

[0204] Involvement of the inhibitory receptors ILT2 and ILT4 alters the cytokine and chemokine secretion / release profile of monocytes and can inhibit Fc receptor signaling (Colonna, M., et al. J Leukoc Biol, 66:375-381 (1999)). The role and function of ILT3 on DCs has been precisely described by the 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), competing 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 plays a potential role in the mechanisms of maternal-fetal tolerance and tumor cell evasion of immune recognition and destruction (Hunt, J. S. et al., Faseb J, 19:681-693 (2005)). Modulation of DC function by HLA-G-ILT interactions is most likely an important pathway in the biological mechanisms of DCs. Human monocyte-derived DCs expressing high levels of ILT2 and ILT4 receptors, when treated with HLA-G and stimulated with allogeneic T cells, still retain the potential to induce T cell anergy (CD80low, CD86lowand HLA-DRlow) (Ristich, V. et al., Eur J Immunol, 35:1133-1142 (2005)). In addition, HLA-G interaction with DCs expressing high levels of ILT2 and ILT4 receptors results in down-regulation of several genes involved in the MHC class II presentation pathway. Lysosomal thiol reductase IFN-γ inducible lysosomal thiol reductase (GILT), which is abundantly expressed by professional APCs, is greatly reduced in HLA-G-modified DCs. DC expression of GILT can affect the repertoire of priming CD4+ T cells, as the response of T cells to a selected antigen in vivo is decreased in animals lacking GILT after targeted gene disruption (Marie, M. et al., Science, 294:1361-1365 (2001)). HLA-G / ILT interactions on DCs interfere with MHC class II molecule assembly and transport to the cell surface, which can result in presentation or expression of structurally abnormal MHC class II molecules or decreased efficiency.It has been determined that HLA-G significantly reduces the transcription of invariant chain (CD74), HLA-DMA and HLA-DMB genes on highly expressed ILT inhibitory receptors of human monocyte-derived DCs (Ristich, V. et al. Eur J Immunol 35: 1133-1142 (2005)).

[0205] Another receptor for HLA-G is KIR2DL4, as KIR2DL4 binds to cells expressing HLA-G (US2003232051; Cantoni, C. et al. Eur J Immunol 28 (1998) 1980; Rajagopalan, S. and E. O. Long. [Correction published in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093; Ponte, M. et al. PNAS USA 96 (1999) 5674). KIR2DL4 (also known as 2DL4) is a member of the KIR family (also known as CD158d), sharing structural features with both activating and inhibitory receptors (Selvakumar, A. et al. Tissue Antigens 48 (1996) 285). 2DL4 has a cytoplasmic ITIM, indicating an inhibitory function, and has positively charged amino acids in the transmembrane region, which is a typical feature of activating KIRs. Unlike other clonally distributed KIRs, 2DL4 is transcribed by all NK cells (Valiante, N. M. et al. Immunity 7 (1997) 739; Cantoni, C. et al. Eur J Immunol 28 (1998) 1980; Rajagopalan, S. and E. O. Long. [Correction published in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093).

[0206] The term “inhibiting the binding of ILT2 to HLA-G on JEG-3 cells (ATCC HTB36)” refers to inhibiting the binding interaction of (recombinant) ILT2 in the determination as described in Example 5 of WO2022 / 129120.

[0207] The term "reducing" (and grammatical variations thereof, such as the verb or gerund form), e.g., the reduction of B cell numbers or cytokine release, refers to a decrease in the corresponding number, as measured by an appropriate method known in the art. For the sake of clarity, the term also includes a reduction to zero (or below the detection limit of the analytical method), i.e., complete abrogation or elimination. In contrast, "increasing" refers to an increase in the corresponding number.

[0208] As used herein, "T cell antigen" refers to an antigenic determinant present on the surface of a T lymphocyte, in particular a cytotoxic T lymphocyte.

[0209] As used herein, "T cell activating therapeutic agent" refers to a therapeutic agent capable of inducing T cell activation in a subject, in particular a therapeutic agent designed to induce T cell activation in a subject. Examples of T cell activating therapeutic agents include bispecific antibodies that specifically bind an activating T cell antigen (referred to as "T cell bispecific antibodies" or "TCB") (such as CD3) and a target cell antigen (such as HLA-G). Other examples include chimeric antigen receptors (CARs) comprising a T cell activating domain and an antigen binding moiety that specifically binds to a target cell antigen (such as HLA-G).

[0210] As used herein, "activating T cell antigen" refers to an antigenic determinant expressed by a T lymphocyte, in particular a cytotoxic T lymphocyte, which is capable of inducing or enhancing T cell activation upon interaction with an antigen binding molecule. In particular, the interaction of the antigen binding molecule with the activating T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. An exemplary activating T cell antigen is CD3.

[0211] The term "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CD3, as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, e.g., splice variants or allelic variants. In one embodiment, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3 epsilon). The amino acid sequence of human CD3 epsilon is set forth in UniProt (www.uniprot.org) accession no. P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO: 66. The amino acid sequence of Macaca fascicularis CD3 epsilon is set forth in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 67.

[0212] The terms "bispecific antibody comprising a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to HLA-G," "bispecific antibody that specifically binds to CD3 and HLA-G," "bispecific antibody specific for CD3 and HLA-G," or "anti-HLA-G / anti-CD3 bispecific antibody," "HLA-G CD3 TCB," or "HLA-G TCB" are used interchangeably herein and refer to a bispecific antibody that is capable of binding to CD3 and HLA-G with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3 and HLA-G.

[0213] The term "PD1", also known as Programmed cell death protein 1, is a type I membrane protein consisting of 288 amino acids, first described in 1992 (Ishida et al., EMBO J., 11 (1992), 3887-3895). PD1 is a member of the extended CD28 / CTLA-4 family of T cell regulators and has two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein structure includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which suggests that PD1 negatively regulates TCR signaling. This is consistent with the binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 upon ligand binding. While PD1 is not expressed on naive T cells, it is upregulated upon T cell receptor (TCR)-mediated activation and is observed on both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells have a dysfunctional phenotype and are unable to respond appropriately. Despite the relatively broad expression pattern of PD1, its most important role can be as a co-inhibitory receptor on T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Thus, current therapeutic approaches focus on blocking the interaction of PD1 with its ligands to enhance T cell responses. The terms "Programmed Death 1", "Programmed Cell Death 1", "Protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1", and "hPD-I" are used interchangeably and include variants, isoforms, species homologs of human PD1, as well as analogs having at least one common epitope with PD-1. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org) accession number Q15116 (SEQ ID NO:68).

[0214] The terms "anti-PD1 antibody" and "antibody comprising an antigen binding domain that binds PD1" refer to an antibody that is capable of binding PD1, particularly a PD1 polypeptide expressed on a cell surface, with sufficient affinity that the antibody is useful as a diagnostic and / or therapeutic agent for targeting PD1. In one aspect, the extent of binding of an anti-PD1 antibody to an unrelated, non-PD1 protein is less than about 10% of the binding of the antibody to PD1, for example, as measured by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance assays, e.g., using a Biacore® system. In certain aspects, an antigen binding protein that binds to human PD1 has a binding affinity (K D Value of < 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10 -8 M or lower, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In a preferred embodiment, the respective K D Value is determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human PD1 (PD1-ECD) to obtain the PD1 binding affinity. The term "anti-PD1 antibody" also encompasses bispecific antibodies that are capable of binding PD1 and a second antigen.

[0215] In one particular aspect, the anti-PD1 antibody is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), PDR001 (spartalizumab), SHR1210 (camrelizumab), MEDI-0680 (AMP-514), REGN2810, and BGB-108. In a particular aspect, the anti-PD1 antibody is pembrolizumab or an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 76. Pembrolizumab (Merck), also known as MK-3475, Merck 3475, Keytruda, SCH-900475, and KEYTRUDA®, is an anti-PD1 antibody described in WO 2009 / 114335 (CAS Registry Number 1374853-91-4). In a particular aspect, the anti-PD1 antibody is nivolumab or an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 77 and a light chain comprising the amino acid sequence of SEQ ID NO: 78. Nivolumab (CAS Registry Number: 946414-94-4, Bristol-Myers Squibb / Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD1 antibody described in WO 2006 / 121168 (CAS Registry Number 946414-94-4). In another particular aspect, the anti-PD1 antibody comprises a heavy chain variable domain VH comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain VL comprising the amino acid sequence of SEQ ID NO: 8; or a humanized variant thereof. In a particular aspect, the anti-PD1 antibody comprises a heavy chain variable domain VH comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain VL comprising the amino acid sequence of SEQ ID NO: 10.

[0216] As used herein, unless otherwise indicated, the term "LAG3" or "Lag-3" or "lymphocyte activation gene-3" or "CD223" refers to any native LAG3 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed LAG3, as well as any form of LAG3 resulting from intracellular processing. The term also encompasses naturally occurring variants of LAG3, such as splice variants or allelic variants. In a preferred embodiment, the term "LAG3" refers to human LAG3. The amino acid sequence of an exemplary processed (without signal sequence) LAG3 is set forth in SEQ ID NO:69. The amino acid sequence of an exemplary extracellular domain (ECD) LAG3 is set forth in SEQ ID NO:70.

[0217] The terms "anti-LAG3 antibody" and "antibody that binds to LAG3" refer to an antibody that binds to LAG3 with sufficient affinity to allow for use as a diagnostic and / or therapeutic agent targeting LAG3. In one aspect, the anti-LAG3 antibody binds to an unrelated, non-LAG3 protein to less than about 10% of the extent of binding of the antibody to LAG3, e.g., as measured by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to LAG3 has a p-value of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., ≤ 10 -8 M or lower, such as 10 -8 M to 10 -13 M, for example 10 -9 to 10 -13 The dissociation constant (K D In certain aspects, the anti-LAG3 antibody binds to an epitope of LAG3 that is conserved among LAG3 from different species. In a preferred embodiment, "anti-LAG3 antibody," "antibody that specifically binds to human LAG3," and "antibody that specifically binds to human LAG3" refer to antibodies that specifically bind to the human LAG3 antigen or its extracellular domain (ECD) with a binding affinity of K. D The value is 1.0 x 10 -8 mol / l or less, in one embodiment, K D The value is 1.0 x 10 -9 mol / l or less, in one embodiment, K Da value of 1.0 x 10 -9 a value of 1.0 x 10 -13 mol / l. In this context, binding affinity is determined using standard binding assays, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), for example using the LAG3 extracellular domain. The term "anti-LAG3 antibody" also encompasses bispecific antibodies capable of binding LAG3 and a second antigen. In one aspect, the anti-LAG3 antibody is relatlimab or BMS-986016, or an antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 28. In a particular aspect, the anti-LAG3 antibody comprises a heavy chain variable domain VH comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain VL comprising the amino acid sequence of SEQ ID NO: 18, or a heavy chain variable domain VH comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable domain VL comprising the amino acid sequence of SEQ ID NO: 26, or a heavy chain variable domain VH comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable domain VL comprising the amino acid sequence of SEQ ID NO: 65.

[0218] The terms "bispecific antibody that comprises a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3," "bispecific antibody that specifically binds to PD1 and LAG3," "bispecific antibody that has specificity for PD1 and LAG3," or "anti-PD1 / anti-LAG3 (bispecific) antibody" are used interchangeably herein and refer to a bispecific antibody that is capable of binding to PD1 and LAG3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD1 and LAG3.

[0219] A "blocking" antibody or an "antagonist" antibody is one that inhibits or reduces a biological activity of an antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits a biological activity of an antigen. For example, the bispecific antibodies of the application block signaling through PD1 and LAG3, thereby restoring functional responses (e.g., proliferation, cytokine production, target cell killing) by T cells from a dysfunctional state to antigen stimulation.

[0220] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved with binding the antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain can be sufficient to confer antigen-binding specificity.

[0221] The term "hypervariable region" or "HVR," as used herein, refers to each of the regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, e.g., the "complementarity determining regions" ("CDRs"). Generally, an antibody comprises 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:

[0222] (a) the hypervariable loops that exist between 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));

[0223] (b) the CDRs that occur 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

[0224] (c) the antigen contacts that occur 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)).

[0225] Unless otherwise indicated, CDRs are determined according to the method described by Kabat et al. (supra). Those skilled in the art will appreciate that CDR designations can also be determined according to the method described by Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system.

[0226] The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof, refers to the numbering system used in the compilation of antibody heavy chain variable domains or light chain variable domains in Kabat et al. Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat numbering) and insertions of residues after residue 82 of a heavy chain FR (e.g., residues 82a, 82b, and 82c, etc. according to Kabat numbering). The Kabat numbering of residues for a given antibody can be determined by alignment of the antibody sequence with the homologous regions of the "standard" Kabat numbered sequence. Typically, native four-chain antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).

[0227] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain are typically comprised of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following sequence in VHand (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0228] A "recipient human framework" for purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. A recipient human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence as that human immunoglobulin framework or human consensus framework, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, a VL recipient human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0229] The term "chimeric" antibody refers to an antibody in which the heavy and / or light chain is part of the antibody is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0230] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0231] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to a humanized antibody that has been subjected to humanization. Other forms of "humanized antibodies" encompassed by the present application are antibodies in which the constant region has been additionally modified or changed relative to that of the original antibody to generate properties in accordance with the application, especially properties with respect to C1q binding and / or Fc receptor (FcR) binding.

[0232] A "human" antibody is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes a human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0233] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the mAb, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb of a mAb preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the mAbs to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage -display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0234] The term "Fc domain" or "Fc region" herein is used to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In particular, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present. The amino acid sequence of the heavy chain is always presented with the C-terminal lysine, however, variants without the C-terminal lysine are included in the present application.

[0235] The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The "CH2 domain" of a human IgG Fc region generally extends from about amino acid residue 231 to about amino acid residue 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises a stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG). The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced "knob" ("knuckle") in one chain and a corresponding introduced "hole" ("hole") in the other chain; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0236] "Knob-into-hole" technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this approach involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity in order to promote formation of a heterodimer and impede formation of a homodimer. The protuberance is constructed by substituting a small amino acid side chain (e.g., alanine or threonine) for a large amino acid side chain from the interface of the first polypeptide. A compensating cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide by substituting a small amino acid side chain (e.g., alanine or threonine) for a large amino acid side chain. The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptide, e.g., by site-specific mutagenesis or by peptide synthesis. In a particular embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another particular embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bond between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0237] "Region identical with the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants having modifications that result in substitutions, additions or deletions but that do not substantially decrease the ability of the immunoglobulin to mediate effector functions, such as antibody-dependent cellular cytotoxicity. For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantially losing biological function. Such variants can be selected according to general rules known in the art so as to have minimal influence on activity (see, e.g., Bowie, J. U. et al., Science 247: 1306-10 (1990)).

[0238] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq 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 receptor), and B cell activation.

[0239] An "activating Fc receptor" is an Fc receptor that, upon engagement of the Fc region of an antibody, causes signaling events that stimulate the cell bearing the receptor to perform an effector function. Activating Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89). A particular activating Fc receptor is human FcyRIIIa (see UniProt accession number P08637, version 141).

[0240] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G4S) n , (SG4) n or G4(SG4) n peptide linker, wherein "n" is typically a number between 1 to 10 (typically between 2 to 4, in particular 2). Peptide linkers of particular interest are (G4S) (SEQ ID NO: 71), (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72), (G4S)3 (SEQ ID NO: 73) and (G4S)4 (SEQ ID NO: 74), more particularly (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72).

[0241] "fusion to" or "linked to" means that components (e.g., antigen binding domain and FC domain) are connected by a peptide bond, either directly or via one or more peptide linkers.

[0242] The term "amino acid" as used in the present application denotes the group of naturally occurring carboxylic alpha-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0243] "Percentage (%) amino acid sequence identity" with respect to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the amino acid residues of the candidate sequences with the amino acid residues of the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance using publicly available computer software such as BLAST, BLAST-2, ALIGN. SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, the sequence comparison computer program ALIGN-2 is used to calculate the percent amino acid sequence identity. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not variable. In cases where ALIGN-2 is employed for amino acid sequence comparisons, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A has or comprises a certain percent amino acid sequence identity to a given amino acid sequence B) is calculated as follows:

[0244] 100 times the fraction X / Y

[0245] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0246] In certain aspects, amino acid sequence variants of the bispecific antibodies of the invention provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the bispecific antibody. Amino acid sequence variants of the bispecific antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding. Sites of interest for substitution mutagenesis include HVRs and framework (FR). Conservative substitutions are provided in Table A under the heading "Preferred Substitutions" and are further described below with reference to amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into the target molecule and the product screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0247] Amino acids can be grouped according to common side chain properties:

[0248] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0249] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0250] (3) Acidic: Asp, Glu;

[0251] (4) Basic: His, Lys, Arg;

[0252] (5) Residues affecting chain orientation: Gly, Pro;

[0253] (6) Aromatic: Trp, Tyr, Phe.

[0254] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0255] Table A

[0256]

[0257] The term "amino acid sequence variants" includes substantial variants wherein there are amino acid substitutions in one or more hypervariable region residues of a parent antigen binding molecule (e.g., a humanized or human antibody). Generally, one or more of the resulting variants selected for further study will have alterations (e.g., improvements) in certain biological properties relative to the parent antigen binding molecule (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antigen binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, e.g., using phage display based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antigen binding molecules are displayed on phage and screened for a particular biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antigen binding molecule to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs. Methods which can be used to identify antibody residues or regions which can be targeted for mutagenesis are referred to as "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by alanine or a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interactions of the antibody with antigen are affected. Further substitutions can be introduced at the amino acid positions which have shown sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of the antigen-antigen binding molecule complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened for appropriate binding affinity.

[0258] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include bispecific antibodies with an N-terminal methionyl residue. Other insertional variants of the molecule include fusion to the N- or C-terminus of the bispecific antibody to a polypeptide which increases the serum half-life of the bispecific antibody.

[0259] In certain aspects, the bispecific antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation variants of the molecules can be conveniently produced by altering the amino acid sequence such that one or more glycosylation sites are created or removed, for example, the carbohydrate attached to an Fc domain can be altered. Native antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides that are typically linked to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the bispecific antibodies of the application can be made in order to create variants with certain improved properties. In one aspect, variants of the bispecific antibodies are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. Such fucosylation variants can have improved ADCC function, see e.g. US Patent Publication Nos. US 2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Other variants of the bispecific antibodies of the application include variants with bisected oligosaccharides, e.g., in which the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants can have reduced fucosylation and / or improved ADCC function, see e.g. WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants can have improved CDC function and are described in, e.g., WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0260] In certain aspects, cysteine engineered variants of the bispecific antibodies of the application, e.g., "thioMAbs," in which one or more residues are substituted with cysteine residues are desirable. In particular embodiments, the substituted residues occur at accessible sites of the molecule. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat Numbering) of the light chain; A118 (EU Numbering) of the heavy chain; and S400 (EU Numbering) of the heavy chain Fc region. Cysteine engineered antigen binding molecules can be formed as described, e.g., in U.S. Patent No. 7,521,541.

[0261] In certain aspects, the bispecific antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known to the art and readily available to the skilled worker. Suitable moieties for derivatization of the antibody include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), co-polymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextraf, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxpane, ethylene / maleic acid copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. The polymer can have any mean molecular weight and can or can not have branching. The number of polymers attached to the antibody can vary and, if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the bispecific antibody derivative will be used for therapy under defined conditions, etc.

[0262] In another aspect, conjugates of antibodies and non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is described by Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells, but heat the non-proteinaceous moiety to a temperature that kills cells in the vicinity of the antibody-non-proteinaceous moiety.

[0263] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0264] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), a viral-derived RNA, or a plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., amide bond, such as present in peptide nucleic acid (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0265] With respect to an "isolated" nucleic acid molecule or polynucleotide, it is meant a nucleic acid molecule, DNA or RNA, which has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide comprised in a vector is considered isolated for the purpose of the present application. Further examples of an isolated polynucleotide include a recombinant polynucleotide maintained in a heterologous host cell or a purified (partially or substantially purified) polynucleotide in solution. An isolated polynucleotide includes a polynucleotide molecule that is contained in the cell in which it normally would be found but is present outside the chromosome or in a different location in the chromosome than it normally would be found. An isolated RNA molecule includes in vivo or in vitro RNA transcripts of the present application, as well as positive and negative sense and double-stranded forms. An isolated polynucleotide or nucleic acid according to the present application further includes such molecules produced by synthesis. In addition, the polynucleotide or nucleic acid can be or can include regulatory elements, such as a promoter, ribosome binding site, or transcriptional terminator.

[0266] A nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the invention refers to a polynucleotide having a sequence that differs from the reference sequence by no more than five point mutations per each 100 nucleotides of the reference polynucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total number of nucleotides in the reference sequence can be inserted into the reference sequence. These alterations in the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the invention will be determined within the context of the particular polynucleotide sequence and the criteria used to determine such identity. As used herein, a "point mutation" is a change in a single nucleotide in a polynucleotide sequence. As used herein, a "deletion" is a change in a polynucleotide sequence in which one or more nucleotides are absent. As used herein, an "insertion" is a change in a polynucleotide sequence in which one or more nucleotides are present that are not present in the reference sequence. As used herein, a "substitution" is a change in a polynucleotide sequence in which one or more nucleotides are replaced by different nucleotides.

[0267] The term "expression cassette" refers to a polynucleotide generated recombinantly or synthetically, having a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassettes of the present invention comprise a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0268] The terms "vector" or "expression vector" are synonymous with "expression construct" and refer to a DNA molecule that serves to introduce a particular gene into a target cell with which it is operably associated and directs expression of the gene. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that become incorporated into the genome of a host cell into which they have been introduced. The expression vectors of the present invention comprise an expression cassette. Expression vectors allow for the transcription of large quantities of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vectors of the present invention comprise an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0269] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny can not be identical to the parent cell from which they were derived, having for example mutations not present in the original cell. Included are progeny from original transformants that have been screened or selected for increased or decreased biological activity, for example. Host cells are any type of cellular system that can be used to produce the bispecific antigen binding molecules of the application. In particular, host cells are prokaryotic or eukaryotic host cells. Host cells include cultured cells, e.g., cultured mammalian cells such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells included in a transgenic animal, transgenic plant, or cultured plant or animal tissue.

[0270] An "effective amount" of an agent refers to the amount necessary to effect a desired physiological change in the cells or tissues to which it is administered.

[0271] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, decreases, delays, minimizes, or prevents adverse effects of a disease.

[0272] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0273] The term "pharmaceutical composition" refers to a preparation which is in a form suitable for administration into a subject and which is not biologically unacceptable in that it does not contain additional ingredients which are unacceptable for the subject to which the formulation is to be administered.

[0274] A "pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical composition other than the active ingredient, which is nontoxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizing agents, or preservatives.

[0275] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0276] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing or delaying the onset of disease, alleviating symptoms, diminishment of any direct or indirect pathological consequences, preventing metastasis, decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. In some embodiments, the molecules of the application are used to delay development of a disease or to slow the progression of a disease.

[0277] The term “cancer” as used herein includes lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the naso-pharynx, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancers, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. In one embodiment, the term cancer refers to a cancer that expresses HLA-G. In particular, the cancer that expresses HLA-G is selected from renal cell carcinoma, colorectal cancer, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0278] The term “expression of HLA-G” is intended to mean a significant level of expression of HLA-G on the cell surface of a cell, preferably from a tumor or cancer, preferably from a solid tumor. A patient having a “cancer that expresses HLA-G” can be determined by standard assays known in the art. For example, the expression of HLA-G antigen can be measured using immunohistochemistry (IHC) detection, FACS, or via corresponding mRNA detection based on PCR.

[0279] As used herein, the term "HLA-G-expressing cancer" or "HLA-G-positive cancer" refers to all cancers in which cancer cells show expression of HLA-G antigens. Preferably, HLA-G-expressing cancer as used herein refers to lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the naso-pharynx, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancers, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. In particular, the HLA-G-expressing cancer is selected from renal cell carcinoma, colorectal cancer, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0280] The term "method of treatment", "method of treating" or their equivalent, when applied to, for example, cancer, refers to a procedure or course of action intended to reduce or eliminate the number of cancer cells in a patient or to reduce the symptoms of the cancer. A "method of treating" cancer or another proliferative disease does not necessarily mean that the cancer cells or other disease will actually be eliminated, that the number of cells or the disease will actually be reduced, or that the cancer or other disease will actually be alleviated. Generally, a method of treating cancer is considered to initiate an overall beneficial course of action, even if the chances of success are low, taking into account the patient's history and estimated survival expectancy.

[0281] The terms "in combination," "co-administration," or "co-administered" mean that the anti-HLA-G / anti-CD3 bispecific antibody is administered with the anti-PD1 / anti-LAG3 bispecific antibody as two separate formulations (or as one single formulation). Co-administration can be simultaneous or sequential in any order, with the proviso that there is a period of time where both (or all) active agents exert their biological activities simultaneously. The anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered simultaneously or sequentially, for example, intravenously (i.v.) by continuous infusion (one for the anti-HLA-G / anti-CD3 bispecific antibody and one for the anti-PD1 / anti-LAG3 bispecific antibody). When the two therapeutic agents are co-administered sequentially, the dosing is either performed on the same day in two separate administrations or one drug is administered on day 1 and the second drug is co-administered between day 2 and day 7 (preferably between day 2 and day 4). Thus, the term "sequentially" means within 7 days after the administration of the first component (anti-HLA-G / anti-CD3 bispecific antibody or anti-PD1 / anti-LAG3 bispecific antibody), preferably within 4 days after the administration of the first component; and "simultaneously" means at the same time. The term "co-administration" with respect to the maintenance doses of the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody means that the maintenance doses can be co-administered either simultaneously, if the treatment cycle is applicable to both drugs, for example, every week. Or the anti-PD1 / anti-LAG3 bispecific antibody is administered every two weeks while the anti-HLA-G / anti-CD3 bispecific antibody is administered every three weeks. Or the maintenance doses are co-administered sequentially within one or several days.

[0282] Exemplary anti-HLA-G / anti-CD3 bispecific antibodies for use in the present application

[0283] It has been found that the combination of the anti-HLA-G / anti-CD3 bispecific antibody with the anti-PD1 / anti-LAG3 bispecific antibody shows greater efficacy in a tumor model in humanized mice than the anti-HLAG / anti-CD3 bispecific antibody monotherapy or the combination of the anti-HLAG / anti-CD3 bispecific antibody with the anti-PD1 antibody and the anti-LAG3 antibody. The data indicate that the combination with the anti-PD1 / anti-LAG3 bispecific antibody is superior to the combination with the anti-PD1 and / or anti-LAG3 monospecific antibodies as it provides better efficacy as shown by stronger tumor growth inhibition and better activation of T cells.

[0284] The present invention relates to anti-HLA-G / anti-CD3 bispecific antibodies and their use in combination with anti-PD1 / anti-LAG3 bispecific antibodies, in particular their use in methods for treating or delaying progression of a cancer expressing HLA-G. Anti-HLA-G / anti-CD3 bispecific antibodies as used herein are bispecific antibodies comprising a first antigen binding domain binding to CD3 and a second antigen binding domain binding to HLA-G. They thus target tumor cells expressing HLA-G.

[0285] Thus, anti-HLA-G / anti-CD3 bispecific antibodies as used herein comprise a first antigen binding domain comprising a heavy chain variable region (V H CD3) and a light chain variable region (V L CD3) specifically binding to CD3 and a second antigen binding domain comprising a heavy chain variable region (V H HLA-G) and a light chain variable region (V L HLA-G) specifically binding to HLA-G.

[0286] In a particular aspect, the anti-HLA-G / anti-CD3 bispecific antibody for use in combination comprises a first antigen binding domain comprising a heavy chain variable region (V H CD3) comprising a CDR-H1 sequence of SEQ ID NO: 41, a CDR-H2 sequence of SEQ ID NO: 42 and a CDR-H3 sequence of SEQ ID NO: 43; and / or a light chain variable region (V L CD3) comprising a CDR-L1 sequence of SEQ ID NO: 44, a CDR-L2 sequence of SEQ ID NO: 45 and a CDR-L3 sequence of SEQ ID NO: 46. More particularly, the anti-HLA-G / anti-CD3 bispecific comprises a first antigen binding domain comprising a heavy chain variable region (V H CD3) which is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 47; and / or a light chain variable region (V L CD3) which is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 48. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region (V HCD3), which comprises the amino acid sequence of SEQ ID NO: 47; and / or a light chain variable region (V L CD3), which comprises the amino acid sequence of SEQ ID NO:48.

[0287] In another aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen-binding domain that specifically binds to PD1, wherein the second antigen-binding domain comprises: a heavy chain variable region (V H HLA-G), which comprises the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50 and the CDR-H3 sequence of SEQ ID NO: 51; and / or a light chain variable region (V L HLA-G), comprising a CDR-L1 sequence of SEQ ID NO: 52, a CDR-L2 sequence of SEQ ID NO: 53, and a CDR-L3 sequence of SEQ ID NO: 54. More specifically, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen-binding domain comprising: a heavy chain variable region (V H HLA-G), which is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 55; and / or a light chain variable region (V L HLA-G) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 56. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen-binding domain comprising: a heavy chain variable region (V H HLA-G), comprising the amino acid sequence of SEQ ID NO: 55; and / or a light chain variable region (V L HLA-G), comprising the amino acid sequence of SEQ ID NO: 56. In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises an antigen-binding domain that specifically binds to CD3 and a second antigen-binding domain that specifically binds to HLA-G, the antigen-binding domain comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 47, and a VL domain comprising the amino acid sequence of SEQ ID NO: 48; and the second antigen-binding domain comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 55, and a VL domain comprising the amino acid sequence of SEQ ID NO: 56.

[0288] In another particular aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen binding domain that binds to HLA-G. In particular, the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen binding domain that comprises: a heavy chain variable region (V H HLA-G) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51; and / or a light chain variable region (V L HLA-G) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO: 54. More particularly, the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen binding domain that comprises: a heavy chain variable region (V H HLA-G) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 55; and / or a light chain variable region (V L HLA-G) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 56. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen binding domain that comprises: a heavy chain variable region (V H HLA-G) comprising the amino acid sequence of SEQ ID NO: 55; and / or a light chain variable region (V L HLA-G) comprising the amino acid sequence of SEQ ID NO: 56. In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises an antigen binding domain that specifically binds to CD3 and a second and optional third antigen binding domain that specifically binds to HLA-G, the antigen binding domain comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 47, and a VL domain comprising the amino acid sequence of SEQ ID NO: 48; and the second and optional third antigen binding domain comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 55, and a VL domain comprising the amino acid sequence of SEQ ID NO: 56.

[0289] In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody as used herein is a full-length antibody. In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody as used herein is a human IgG class antibody, particularly a human IgG1 class antibody.

[0290] In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody is a bispecific antibody, wherein the first antigen binding domain is a crossover Fab molecule in which the variable domain or the constant domain of the Fab heavy chain is exchanged with the variable domain or the constant domain of the Fab light chain, and the second antigen binding domain and the third antigen binding domain (if present) are conventional Fab molecules.

[0291] The Fab molecules can be fused directly to the Fc domain or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) (SEQ ID NO: 71), (G4S) or GGGGSGGGGS (SEQ ID NO: 72), (G4S) (SEQ ID NO: 73), and (G4S) (SEQ ID NO: 74), more particularly (G4S) or GGGGSGGGGS (SEQ ID NO: 72). A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules is (G4S). Another suitable linker comprises the sequence (G4S) (SEQ ID NO: 74). Additionally, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, in case of fusion of a Fab molecule to the N-terminus of an Fc domain subunit, the fusion can be performed via the immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0292] In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody is a bispecific antibody wherein (i) the second antigen binding domain 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 domain, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain (as shown in Figure 1A), or (ii) the first antigen binding domain 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 domain, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0293] In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function. In particular, the anti-HLA-G / anti-CD3 bispecific antibody comprises an IgGl Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (according to EU numbering).

[0294] In one particular aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 57, a second polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 58, a third polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 59, and a fourth polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 60. In yet another particular embodiment, the bispecific antibody comprises the first polypeptide sequence of SEQ ID NO: 57, the second polypeptide sequence of SEQ ID NO: 58, the third polypeptide sequence of SEQ ID NO: 59, and the fourth polypeptide sequence of SEQ ID NO: 60 (HLA-G TCB).

[0295] In another aspect, the anti-HLA-G / anti-CD3 bispecific antibody for use in combination comprises a first antigen binding domain comprising a heavy chain variable region (V H CD3) comprising the CDR-H1 sequence of SEQ ID NO: 83, the CDR-H2 sequence of SEQ ID NO: 84, and the CDR-H3 sequence of SEQ ID NO: 85; and / or a light chain variable region (V L CD3) comprising the CDR-L1 sequence of SEQ ID NO: 86, the CDR-L2 sequence of SEQ ID NO: 87, and the CDR-L3 sequence of SEQ ID NO: 88. More particularly, the anti-HLA-G / anti-CD3 bispecific comprises a first antigen binding domain comprising a heavy chain variable region (V H CD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 89; and / or a light chain variable region (V L CD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 90. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region (V H CD3) comprising the amino acid sequence of SEQ ID NO: 89; and / or a light chain variable region (V L CD3) comprising the amino acid sequence of SEQ ID NO: 90.

[0296] In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen binding domain comprising a heavy chain variable region (V H HLA-G) comprising the CDR-H1 sequence of SEQ ID NO: 91, the CDR-H2 sequence of SEQ ID NO: 92, and the CDR-H3 sequence of SEQ ID NO: 93; and / or a light chain variable region (V L HLA-G) comprising the CDR-L1 sequence of SEQ ID NO: 94, the CDR-L2 sequence of SEQ ID NO: 95, and the CDR-L3 sequence of SEQ ID NO: 96. More particularly, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen binding domain comprising a heavy chain variable region (V Han amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 97; and / or a light chain variable region (V L an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 98. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen binding domain comprising: a heavy chain variable region (V H an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 97; and / or a light chain variable region (V L an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 98.

[0297] In a particular aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 99, a second polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 100, a third polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 101, and a fourth polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 102. In yet another particular embodiment, the bispecific antibody comprises the first polypeptide sequence of SEQ ID NO: 99, the second polypeptide sequence of SEQ ID NO: 100, the third polypeptide sequence of SEQ ID NO: 101, and the fourth polypeptide sequence of SEQ ID NO: 102.

[0298] Particular anti-HLA-G / anti-CD3 bispecific antibodies are described in PCT Publication Nos. WO 2022 / 24024 or WO 2022 / 129120.

[0299] In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody can further comprise a BiTE® bispecific T cell engager.

[0300] Exemplary bispecific anti-PD1 / anti-LAG3 antibodies for use in the present application

[0301] For the combinations provided herein, novel bispecific antibodies comprising a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene-3 (LAG3) are used, which have particularly advantageous properties, such as manufacturability, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency, and reduced toxicity. Specific bispecific anti-PD1 / anti-LAG3 antibodies for use herein are described in WO 2018 / 185043 A1.

[0302] In certain aspects, a bispecific antibody comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3 is provided, which exhibits reduced internalization upon binding to the surface of T cells. Internalization represents a significant sink for the molecule, which can be degraded within hours, while the targeted receptor is rapidly re-expressed on the cell surface, ready to inhibit TCR signaling. In another aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3 is provided, which preferentially binds to conventional T cells over Tregs. This is advantageous, as targeting LAG3 on Tregs with blocking antibodies can be detrimental by increasing their suppressive function and ultimately masking the positive blocking effect on other T cells. In another aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3 is provided, which is capable of rescuing T cell effector function from Treg suppression. In another aspect, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which, when co-cultured with the tumor cell line ARH77, is capable of inducing granzyme B secretion by CD4 T cells, as shown in an assay provided herein. In another aspect, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which exhibits increased tumor-specific T cell effector function and / or enhanced T cell cytotoxicity. In another aspect, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds LAG3, which exhibits enhanced tumor eradication in vivo.

[0303] In one aspect, the present application provides an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the first antigen binding domain that specifically binds to PD1 comprises

[0304] a VH domain comprising

[0305] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 ;

[0306] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and

[0307] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0308] a VL domain, said VL domain comprising

[0309] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4;

[0310] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0311] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0312] In one aspect, the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain, which is an IgG, in particular an IgG1 Fc domain or an IgG4 Fc domain, and wherein the Fc domain has reduced or even abrogated effector function. Specifically, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular to an Fcy receptor.

[0313] In a further aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises an Fc domain, which is an IgG, in particular an IgG1 Fc domain or an IgG4 Fc domain, and wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular to an Fcy receptor.

[0314] In another aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination with an anti-CTLA4 antibody, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the second antigen binding domain that specifically binds to LAG3 comprises

[0315] (a) a VH domain comprising

[0316] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0317] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and

[0318] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and

[0319] a VL domain comprising

[0320] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14;

[0321] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and

[0322] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or

[0323] (b) a VH domain comprising

[0324] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19;

[0325] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and

[0326] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21 ; and

[0327] a VL domain comprising

[0328] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22;

[0329] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and

[0330] (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 24.

[0331] In a further aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the first antigen binding domain that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0332] In a further aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the first antigen binding domain that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0333] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or

[0334] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0335] In a further aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the first antigen binding domain that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0336] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising the amino acid sequence of SEQ ID NO: 28, or

[0337] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29; and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, or

[0338] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31 ; and a VL domain comprising the amino acid sequence of SEQ ID NO: 32, or

[0339] (d) a VH domain comprising the amino acid sequence of SEQ ID NO:33; and a VL domain comprising the amino acid sequence of SEQ ID NO:34, or

[0340] (e) a VH domain comprising the amino acid sequence of SEQ ID NO:64; and a VL domain comprising the amino acid sequence of SEQ ID NO:65.

[0341] In another aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the second antigen binding domain that specifically binds to LAG3 comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 81, and a VL domain comprising the amino acid sequence of SEQ ID NO: 82.

[0342] In another aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein

[0343] the first antigen binding domain that specifically binds to PD1 comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 9, and a VL domain comprising the amino acid sequence of SEQ ID NO: 10,

[0344] and the second antigen binding domain that specifically binds to LAG3 comprises: a VH domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL domain comprising the amino acid sequence of SEQ ID NO: 18; or a VH domain comprising the amino acid sequence of SEQ ID NO: 25, and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0345] In one aspect, the anti-PD1 / anti-LAG3 bispecific antibody of the application comprises a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, which first antigen binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and which second antigen binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0346] In a further aspect, the anti-PD1 / anti-LAG3 bispecific antibody of the application comprises a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, which first antigen binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and which second antigen binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0347] In a further aspect, the anti-PD1 / anti-LAG3 bispecific antibody comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3 is a human, a humanized or a chimeric antibody. In particular, the antibody is a humanized or a chimeric antibody.

[0348] In one aspect, the anti-PD1 / anti-LAG3 bispecific antibody comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3 is bivalent. This means that the bispecific antibody comprises one antigen binding domain that specifically binds to PD1 and one antigen binding domain that specifically binds to LAG3 (1+1 format).

[0349] In one aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises a Fc domain, a first Fab fragment comprising an antigen binding domain that specifically binds to PD1 and a second Fab fragment comprising an antigen binding domain that specifically binds to LAG3. In a particular aspect, in one of the Fab fragments, the variable domains VL and VH are replaced by each other, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In a particular aspect, in the first Fab fragment comprising the antigen binding domain that specifically binds to PD1, the variable domains VL and VH are replaced by each other.

[0350] In one aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody for use in combination, comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises

[0351] (a) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35; a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36;

[0352] a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 37; and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 38, or

[0353] (b) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35; a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36;

[0354] a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39; and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40.

[0355] More particularly, the bispecific antibody as used herein comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37; and a second light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0356] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0357] In certain aspects, an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 bispecific antibody is provided, wherein the bispecific antibody comprises an Fc domain comprising one or more amino acid modifications that reduce binding to an Fc receptor, in particular to an Fcy receptor, and reduce or eliminate effector function.

[0358] In certain aspects, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0359] The following section describes preferred aspects of the bispecific antigen binding molecules of the application comprising Fc domain modifications that reduce Fc receptor binding and / or effector function. In one aspect, the application relates to an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 bispecific antibody, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, in particular to an Fcy receptor. Specifically, the Fc domain belongs to the human IgGl subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index).

[0360] The Fc domain confers advantageous pharmacokinetic properties to the bispecific antibodies of the application, including a long serum half-life and a favorable tissue-blood partition ratio that contribute to good accumulation in target tissues. At the same time, however, it can lead to undesired targeting of the bispecific antibodies of the application to Fc receptor-expressing cells rather than to the preferred antigen-carrying cells. Therefore, in particular embodiments, the Fc domain of the bispecific antibodies of the application exhibits reduced binding affinity to Fc receptors and / or reduced effector function as compared to a native IgG Fc domain, in particular an IgGl Fc domain or an IgG4 Fc domain. More specifically, the Fc domain is an IgGl Fc domain.

[0361] In one such aspect, the Fc domain (or bispecific antigen binding molecule of the application comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to an Fc receptor as compared to a native IgGl Fc domain (or bispecific antigen binding molecule of the application comprising a native IgGl Fc domain); and / or the Fc domain (or bispecific antigen binding molecule of the application comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function as compared to a native IgGl Fc domain (or bispecific antigen binding molecule of the application comprising a native IgGl Fc domain). In one aspect, the Fc domain (or bispecific antigen binding molecule of the application comprising the Fc domain) does not significantly bind to an Fc receptor and / or induce an effector function. In a particular aspect, the Fc receptor is an Fc gamma receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fc gamma receptor, more specifically human Fc gamma RIIIa, Fc gamma RI, or Fc gamma RIIa, most specifically human Fc gamma RIIIa. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fc gamma receptor, more specifically human Fc gamma RIIIB. In one aspect, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a particular aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn) as compared to a native IgGl Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or bispecific antigen binding molecule of the application comprising the Fc domain) exhibits more than about 70%, specifically more than about 80%, more specifically more than about 90% of the binding affinity to FcRn as compared to a native IgGl Fc domain (or bispecific antigen binding molecule of the application comprising a native IgGl Fc domain).

[0362] In particular aspects, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function as compared to a non-engineered Fc domain. In particular aspects, the Fc domain of the bispecific antigen binding molecules of the application comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and / or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutation(s) reduce the binding affinity of the Fc domain to an Fc receptor. In another aspect, the amino acid mutation(s) reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In one aspect, the bispecific antigen binding molecules of the application comprising an engineered Fc domain exhibit less than 20%, specifically less than 10%, more specifically less than 5% of the binding affinity to an Fc receptor as compared to a bispecific antibody of the application comprising a non-engineered Fc domain. In particular aspects, the Fc receptor is an Fc gamma receptor. In other aspects, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an inhibitory Fc receptor. In particular aspects, the Fc receptor is an inhibitory human Fc gamma receptor, more specifically human Fc gamma RII B. In some aspects, the Fc receptor is an activating Fc receptor. In a particular aspect, the Fc receptor is an activating human Fc gamma receptor, more specifically human Fc gamma RIIIa, Fc gamma RI, or Fc gamma RIIa, most specifically human Fc gamma RIIIa. Preferably, binding to each of these receptors is reduced. In some aspects, the binding affinity to the complement component, in particular the specific binding affinity to Clq, is also reduced. In one aspect, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved when the Fc domain (or the bispecific antigen binding molecule of the application comprising said Fc domain) exhibits greater than about 70% of the binding affinity to FcRn of a non-engineered version of the Fc domain (or of the bispecific antigen binding molecule of the application comprising said non-engineered version of the Fc domain), i.e., the retention of the binding affinity of the Fc domain to said receptor is achieved. The Fc domain or the bispecific antigen binding molecule of the application comprising this Fc domain can exhibit greater than about 80% or even greater than about 90% of this affinity. In certain embodiments, the Fc domain of the bispecific antigen binding molecules of the application is engineered to have reduced effector function as compared to a non-engineered Fc domain.Reduced effector function can include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0363] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acids at positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, which has residues 265 and 297 substituted to alanine (U.S. Patent No. 7,332,581). Certain antibody variants with improved or diminished binding to FcRs are described. (e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0364] In one aspect of the application, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some aspects, the Fc domain comprises amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgGl Fc domain, particularly a human IgGl Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more particular aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and comprises an additional amino acid substitution selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions nearly completely abrogates Fc gamma receptor binding of a human IgGl Fc domain, as described in PCT Patent Application No. WO 2012 / 130831 Al. The document also describes methods of making such mutant Fc domains and methods for determining their properties, such as Fc receptor binding or effector function. Such antibodies are IgGl with mutations L234A and L235A or with mutations L234A, L235A, and P329G (numbering according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0365] In one aspect, the anti-PD1 / anti-LAG3 bispecific antibody and / or anti-HLA-G / anti-CD3 bispecific antibody comprises (all positions numbered according to the EU index of Kabat): (i) a homodimeric Fc region of the human IgGl subclass, optionally with mutations P329G, L234A, and L235A, or (ii) a homodimeric Fc region of the human IgG4 subclass, optionally with mutations P329G, S228P, and L235E, or (iii) a homodimeric Fc region of the human IgGl subclass, optionally with mutations P329G, L234A, L235A, I253A, H310A, and H435A, or optionally with mutations P329G, L234A, L235A, H310A, H433A, and Y436A, or (iv) a heterodimeric Fc region, where one Fc region polypeptide comprises mutation T366W and the other Fc region polypeptide comprises mutations T366S, L368A, and Y407V, or where one Fc region polypeptide comprises mutations T366W and Y349C and the other Fc region polypeptide comprises mutations T366S, L368A, Y407V, and S354C, or where one Fc region polypeptide comprises mutations T366W and S354C and the other Fc region polypeptide comprises mutations T366S, L368A, Y407V, and Y349C, or (v) a heterodimeric Fc region of the human IgGl subclass, where both Fc region polypeptides comprise mutations P329G, L234A, and L235A, and one Fc region polypeptide comprises mutation T366W and the other Fc region polypeptide comprises mutations T366S, L368A, and Y407V, or where one Fc region polypeptide comprises mutations T366W and Y349C and the other Fc region polypeptide comprises mutations T366S, L368A, Y407V, and S354C, or where one Fc region polypeptide comprises mutations T366W and S354C and the other Fc region polypeptide comprises mutations T366S, L368A, Y407V, and Y349C.

[0366] In one aspect, the Fc domain is an IgG4 Fc domain. In a more particular embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more particular embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E and S228P and P329G. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). Thus, in one aspect, a bispecific antibody is provided, comprising (all positions according to EU index of Kabat) a heterodimeric Fc region of the human IgG4 subclass, wherein both Fc region polypeptides comprise the mutations P329G, S228P and L235E and one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A and Y407V, or wherein one Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or wherein one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

[0367] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer, R.L. et al., J. Immunol. 117 (1976) 587-593 and Kim, J.K. et al., J. Immunol. 24 (1994) 2429-2434) are described in US 2005 / 0014934. Those antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). See also, Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94 / 29351 for additional examples of Fc region variants.

[0368] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as a BIAcore instrument (GE Healthcare), and Fc receptors such as can be obtained by recombinant expression. Suitable such binding assays are described herein. Alternatively, cell lines known to express particular Fc receptors (such as human NK cells expressing Fcyllla receptors) can be used to assess the binding affinity of an Fc domain or cell activating bispecific antigen binding molecule comprising an Fc domain to an Fc receptor. The effector function of an Fc domain, or a bispecific antibody of the application comprising an Fc domain, can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods can be used (see, for example, ACTI™ Non-Radioactive Cytotoxicity Assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cell toxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). ® Non-radioactive cytotoxicity assays (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0369] The following section describes preferred aspects of the bispecific antibodies of the application comprising Fc domain modifications that reduce Fc receptor binding and / or effector function. In one aspect, an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 bispecific antibody is provided, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor, in particular to an Fcy receptor. In another aspect, an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 bispecific antibody is provided, wherein the Fc domain comprises one or more amino acid substitutions that reduce effector function. In a particular aspect, the Fc domain belongs to the human IgGl subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

[0370] Fc domain modifications that promote heterodimerization

[0371] The bispecific antigen binding molecules as described herein comprise different antigen binding domains, fused to one or the other of the two subunits of the Fc domain, whereby the two subunits of the Fc domain can be comprised in two different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibodies of the application in recombinant production, it would be advantageous to introduce modifications in the Fc domain of the bispecific antigen binding molecules as described herein that promote the association of the desired polypeptides.

[0372] Thus, in a particular aspect, an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 antibody is provided, wherein the Fc domain comprises a modification that promotes the association of the first and second subunit of the Fc domain. The most widespread protein-protein interaction site between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect, the modification is in the CH3 domain of the Fc domain.

[0373] In a particular aspect, the modification is a so-called "knob-into-hole" modification which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Thus, the present application relates to a bispecific antibody comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding site that specifically binds to LAG3 and / or a bispecific antibody comprising a first antigen binding domain that specifically binds to CD3 and a second antigen binding site that specifically binds to HLA-G, wherein according to the knob-into-hole approach, the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole. In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).

[0374] The knob-into-hole technology is described, e.g., in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the approach involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity in order to promote the formation of a heterodimer and to impede the formation of a homodimer. The protuberance is constructed by substituting a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). The compensating cavity with the same or similar size as the protuberance is created in the interface of the second polypeptide by substituting a large amino acid side chain with a smaller one (e.g., alanine or threonine).

[0375] Accordingly, in one aspect, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecule of the application, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit in which the protuberance within the CH3 domain of the first subunit is positionable. The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptide, e.g., by site-specific mutagenesis or by peptide synthesis. In a specific aspect, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one aspect, additionally in the second subunit of the Fc domain, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0376] In yet a further aspect, additionally in the first subunit of the Fc domain, the serine residue at position 354 is replaced with a cysteine residue (S354C), and additionally in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C). Introduction of these two cysteine residues leads to the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a specific aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering in accordance with the Kabat EU index).

[0377] However, other knob-in-hole technologies as described in EP 1 870 459 can alternatively or additionally be used. In one embodiment, the multispecific antibody comprises the mutations R409D and K370E in the CH3 domain of the "knob chain" and D399K and E357K in the CH3 domain of the "hole chain" (numbering in accordance with the Kabat EU index).

[0378] In one aspect, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain" and mutations T366S, L368A and Y407V in the CH3 domain of the "hole chain", and in addition mutations R409D and K370E in the CH3 domain of the "knob chain" and mutations D399K and E357K in the CH3 domain of the "hole chain" (numbering according to Kabat EU index).

[0379] In one aspect, the bispecific antibody comprises mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains, or the multispecific antibody comprises mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains and in addition mutations R409D and K370E in the CH3 domain of the "knob chain" and mutations D399K and E357K in the CH3 domain of the "hole chain" (numbering according to Kabat EU index).

[0380] In an alternative aspect, the modification that promotes association of the first and second subunit of the Fc domain comprises a modification that mediates an electrostatic steering effect, as described, for example, in PCT publication WO 2009 / 089004. Generally, this approach involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, such that homodimer formation becomes electrostatically disfavored, but heterodimerization is electrostatically favored.

[0381] In addition to the "knob-in-hole technology", other techniques for modifying the CH3 domains of the heavy chains of a multispecific antibody to enforce heterodimerization are known in the art. These techniques, described, inter alia, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954 and WO 2013 / 096291, are considered herein as alternatives to the combination of the "knob-in-hole technology" with bispecific antibodies.

[0382] In one aspect, in the bispecific antibody, the method described in EP 1870459 is used to support heterodimerization of the first and second heavy chain of the multispecific antibody. This method is based on the introduction of oppositely charged charged amino acids at specific amino acid positions in the CH3 / CH3-domain-interface between the first and second heavy chain.

[0383] Thus, in this aspect of the tertiary structure of the multispecific antibody, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface between the respective antibody CH3 domains, wherein the respective amino acid sequence of the CH3 domain of the first heavy chain and the amino acid sequence of the CH3 domain of the second heavy chain each comprise a set of amino acids located within said interface in the tertiary structure of said antibody, wherein from said set of amino acids located in the interface in the CH3 domain of one heavy chain, a first amino acid is substituted by a positively charged amino acid, and from said set of amino acids located in the interface in the CH3 domain of the other heavy chain, a second amino acid is substituted by a negatively charged amino acid. The bispecific antibody according to this aspect is also referred to herein as "CH3(+ / -) engineered bispecific antibody" (wherein the abbreviation "+ / -" stands for oppositely charged amino acids introduced in the respective CH3 domains).

[0384] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acid is selected from K, R and H, and the negatively charged amino acid is selected from E or D.

[0385] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D.

[0386] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acid is K, and the negatively charged amino acid is E.

[0387] In one aspect, in the CH3(+ / -) engineered bispecific antibody, in the CH3 domain of one heavy chain, the amino acid R at position 409 is substituted by D and the amino acid K at position 399 is substituted by E, and in the CH3 domain of the other heavy chain, the amino acid D at position 357 is substituted by K and the amino acid E at position 399 is substituted by K (numbering according to Kabat EU index).

[0388] In one aspect, the heterodimerization of the first and second heavy chain of the multispecific antibody is supported using the methods described in WO 2013 / 157953. In one embodiment, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).

[0389] In another aspect, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). In addition, at least one of the following substitutions is comprised in the CH3 domain of the other heavy chain: the amino acid Y at position 349 is substituted by E, the amino acid Y at position 349 is substituted by D, and the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index). In one embodiment, the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).

[0390] In one aspect, the heterodimerization of the first and second heavy chain of the multispecific antibody is supported using the methods described in WO 2012 / 058768. In one aspect, in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment, in addition to the above substitutions, in the CH3 domain of the other heavy chain at least one of the amino acids originally at position 411 (T), 399 (D), 400 (S), 405 (F), 390 (N) and 392 (K) is substituted (numbering according to Kabat EU index). Preferred substitutions are:

[0391] - substitution of the amino acid T at position 411 (numbering according to Kabat EU index) by an amino acid selected from the group consisting of N, R, Q, K, D, E and W,

[0392] - substitution of the amino acid D at position 399 (numbering according to Kabat EU index) by an amino acid selected from the group consisting of R, W, Y and K,

[0393] - substitution of the amino acid S at position 400 (numbering according to Kabat EU index) by an amino acid selected from the group consisting of E, D, R and K,

[0394] - substitution of the amino acid F at position 405 (numbering according to Kabat EU index) by an amino acid selected from the group consisting of I, M, T, S, V and W;

[0395] - substitution of the amino acid N at position 390 (numbering according to Kabat EU index) by an amino acid selected from the group consisting of R, K and D; and

[0396] - substitution of the amino acid K at position 392 (numbering according to Kabat EU index) by an amino acid selected from the group consisting of V, M, R, L, F and E.

[0397] In another aspect, the bispecific antibody is engineered according to WO 2012 / 058768, i.e. in the CH3 domain of one heavy chain, the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by V and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment of the multispecific antibody, in the CH3 domain of one heavy chain, the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In a last of the above embodiments, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is substituted by E, the amino acid T at position 411 is substituted by E, the amino acid D at position 399 is substituted by R and the amino acid S at position 400 is substituted by R (numbering according to Kabat EU index).

[0398] In one aspect, the method described in WO 2011 / 143545 is used to support heterodimerization of the first and second heavy chain of the multispecific antibody. In one aspect, an amino acid modification is introduced at position 368 and / or 409 in the CH3 domain of both heavy chains (numbering according to Kabat EU index).

[0399] In one aspect, the method described in WO 2011 / 090762 is used to support heterodimerization of the first and second heavy chain of the bispecific antibody. WO 2011 / 090762 relates to amino acid modifications according to the "knobs-into-holes" (KiH) technology. In one embodiment, in the CH3 domain of one heavy chain the amino acid T at position 366 is replaced by W, while in the CH3 domain of the other heavy chain the amino acid Y at position 407 is replaced by A (numbering according to Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain the amino acid T at position 366 is replaced by Y, while in the CH3 domain of the other heavy chain the amino acid Y at position 407 is replaced by T (numbering according to Kabat EU index).

[0400] In one aspect, the heterodimerization of the first and second heavy chain of the bispecific antibody is supported using the methods described in WO 2009 / 089004. In one embodiment, in the CH3 domain of one heavy chain, the amino acid K or N at position 392 is substituted by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D), and in the CH3 domain of the other heavy chain, the amino acid D at position 399, the amino acid E or D at position 356 or the amino acid E at position 357 is substituted by a positively charged amino acid (in one embodiment by K or R, in a preferred embodiment by K, in a preferred embodiment the amino acid at position 399 or 356 is substituted by K) (numbering according to Kabat EU index). In another embodiment, in addition to the above substitutions, in the CH3 domain of one heavy chain, the amino acid K or R at position 409 is substituted by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D) (numbering according to Kabat EU index). In another aspect, in addition to or as an alternative to the above substitutions, in the CH3 domain of one heavy chain, the amino acid K at position 439 and / or the amino acid K at position 370 is independently from the other substituted by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D) (numbering according to Kabat EU index).

[0401] In one aspect, the heterodimerization of the first and second heavy chain of the multispecific antibody is supported using the methods described in WO 2007 / 147901. In one embodiment, in the CH3 domain of one heavy chain, the amino acid K at position 253 is substituted by E, the amino acid D at position 282 is substituted by K, and the amino acid K at position 322 is substituted by D, and in the CH3 domain of the other heavy chain, the amino acid D at position 239 is substituted by K, the amino acid E at position 240 is substituted by K, and the amino acid K at position 292 is substituted by D (numbering according to Kabat EU index).

[0402] The C-terminus of the heavy chain of the bispecific antibody as reported herein can be the complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG.

[0403] In one of all aspects as reported herein, the bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one embodiment of all aspects reported herein, the bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index).

[0404] Modifications in the Fab domain

[0405] In one aspect, an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 antibody is provided, wherein in one of the Fab fragments the variable domain VH is exchanged with VL or the constant domain CH1 is exchanged with CL. The bispecific antibodies are prepared according to the Crossmab technology.

[0406] Multispecific antibodies with domain swaps / exchanges in one binding arm (CrossMab VH-VL or CrossMab CH-CL) are described in detail in WO2009 / 080252, WO2009 / 080253 and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. They significantly reduce the side products resulting from mispairing of the light chain against the first antigen with the wrong heavy chain against the second antigen (compared to methods without such domain swaps).

[0407] In one particular aspect, an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 bispecific antibody is provided, wherein in one of the Fab fragments the variable domain VL is replaced by VH, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In one specific aspect, the bispecific antibody is a bispecific antibody, wherein in the first Fab fragment comprising an antigen binding domain specifically binding to PD1 the variable domains VL and VH are replaced by each other.

[0408] In another aspect, and to further improve correct pairing, the anti-PD1 / anti-LAG3 bispecific antibody and / or the anti-HLA-G / anti-CD3 antibody can comprise different charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the CH1 and CL domains, either in a cross-over or non-cross-over fashion. Such modifications are described, for example, in WO2015 / 150447, WO2016 / 020309 and PCT / EP2016 / 073408.

[0409] In a particular aspect, an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 bispecific antibody is provided, wherein in one of the Fab fragments the amino acid at position 124 in the constant domain CL is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index), and the amino acids at positions 147 and 213 in the constant domain CH1 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). In a particular aspect, the bispecific antibody is a bispecific antibody, wherein in the second Fab fragment comprising an antigen binding domain specifically binding to TIM3 the amino acid at position 124 in the constant domain CL is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index), and in the constant domain CH1 the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0410] In a particular aspect, there is provided an anti-PD1 / anti-LAG3 bispecific antibody and / or an anti-HLA-G / anti-CD3 antibody, wherein the amino acid at position 123 (EU numbering) in one of the CL domains has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been replaced by lysine (K), and wherein the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) in one of the CH1 domains have been replaced by glutamic acid (E). In a particular aspect, the bispecific antibody is one, wherein in the Fab fragment comprising the antigen binding domain specifically binding to LAG3, the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and the amino acid at position 124 (EU numbering) has been replaced by lysine (K), and wherein the amino acids at position 147 (EU numbering) and at position 213 (EU numbering) in one of the CH1 domains have been replaced by glutamic acid (E).

[0411] In another aspect, the bispecific antibody is a bivalent antibody comprising

[0412] a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and

[0413] b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, and wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other.

[0414] a) the antibody under a) does not contain the modification as reported under b), and the heavy chain and the light chain under a) are separate chains.

[0415] In the antibody under b), the variable light domain VL within the light chain is replaced by the variable heavy domain VH of the antibody, and the variable heavy domain VH within the heavy chain is replaced by the variable light domain VL of the antibody.

[0416] In one aspect, (i) in the constant domain CL of the first light chain under a), the amino acid at position 124 (numbering according to Kabat) is substituted by a positively charged amino acid, and wherein in the constant domain CH1 of the first heavy chain under a), the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negatively charged amino acid; or (ii) in the constant domain CL of the second light chain under b), the amino acid at position 124 (numbering according to Kabat) is substituted by a positively charged amino acid, and wherein in the constant domain CH1 of the second heavy chain under b), the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negatively charged amino acid.

[0417] In another aspect, (i) in the constant domain CL of the first light chain under a), the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (in a preferred embodiment, independently substituted by lysine (K) or arginine (R)), and wherein in the constant domain CH1 of the first heavy chain under a), the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index); or (ii) in the constant domain CL of the second light chain under b), the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (in a preferred embodiment, independently substituted by lysine (K) or arginine (R)), and wherein in the constant domain CH1 of the second heavy chain under b), the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0418] In one aspect, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted by K (numbering according to Kabat EU index).

[0419] In one aspect, in the constant domain CL of the second heavy chain, the amino acid at position 123 is substituted by R and the amino acid at position 124 is substituted by K (numbering according to Kabat EU index).

[0420] In one aspect, in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted by E (numbering according to Kabat EU index).

[0421] In one aspect, in the constant domain CL of the first light chain, the amino acids at positions 124 and 123 are substituted by K, and in the constant domain CH1 of the first heavy chain, the amino acids at positions 147 and 213 are substituted by E (numbering according to Kabat EU index).

[0422] In one aspect, in the constant domain CL of the first light chain, the amino acid at position 123 is substituted by R and the amino acid at position 124 is substituted by K, and in the constant domain CH1 of the first heavy chain, the amino acids at positions 147 and 213 are both substituted by E (numbering according to Kabat EU index).

[0423] In one aspect, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted by K, and wherein in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted by E; in the variable domain VL of the first light chain, the amino acid at position 38 is substituted by K; in the variable domain VH of the first heavy chain, the amino acid at position 39 is substituted by E; in the variable domain VL of the second heavy chain, the amino acid at position 38 is substituted by K; and in the variable domain VH of the second light chain, the amino acid at position 39 is substituted by E (numbering according to Kabat EU index).

[0424] In one aspect, the bispecific antibody is a bivalent antibody comprising

[0425] a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and

[0426] b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other, and wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

[0427] a) the antibody under a) does not contain the modification as reported under b), and the heavy and light chains under a) are separate chains. In the antibody under b), within the light chain the variable light domain VL is replaced by the variable heavy domain VH of the antibody, and the constant light domain CL is replaced by the constant heavy domain CHI of the antibody; within the heavy chain the variable heavy domain VH is replaced by the variable light domain VL of the antibody, and the constant heavy domain CHI is replaced by the constant light domain CL of the antibody.

[0428] In one aspect, the bispecific antibody is a diabody comprising

[0429] a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and

[0430] b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the constant domains CL and CHI of the second light chain and the second heavy chain are replaced by each other.

[0431] a) the antibody under a) does not contain the modification as reported under b), and the heavy and light chains under a) are separate chains. In the antibody under b), within the light chain the constant light domain CL is replaced by the constant heavy domain CHI of the antibody; and within the heavy chain the constant heavy domain CHI is replaced by the constant light domain CL of the antibody.

[0432] In one aspect, the bispecific antibody is a bispecific antibody comprising

[0433] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; and

[0434] b) one, two, three or four single-chain Fab fragments that specifically bind to a second antigen,

[0435] wherein the single-chain Fab fragments under b) are fused to the full-length antibody under a) via a peptide linker at the C-terminal or N-terminal end of the heavy or light chains of the full-length antibody.

[0436] In one aspect, one or two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminal end of the heavy or light chains of the full-length antibody.

[0437] In one aspect, one or two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminal end of the heavy chains of the full-length antibody.

[0438] In one aspect, one or two identical single-chain Fab (scFab) fragments binding to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of the light chain of the full-length antibody.

[0439] In one aspect, two identical single-chain Fab (scFab) fragments binding to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each heavy chain or light chain of the full-length antibody.

[0440] In one aspect, two identical single-chain Fab (scFab) fragments binding to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each heavy chain of the full-length antibody.

[0441] In one aspect, two identical single-chain Fab (scFab) fragments binding to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each light chain of the full-length antibody.

[0442] In one aspect, the bispecific antibody is a trivalent antibody comprising

[0443] a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains,

[0444] b) a first polypeptide consisting of:

[0445] ba) an antibody heavy chain variable domain (VH), or

[0446] bb) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1),

[0447] wherein the first polypeptide is fused at the N-terminus of its VH domain via a peptide linker to the C-terminus of one of the two heavy chains of the full-length antibody,

[0448] c) a second polypeptide consisting of:

[0449] ca) an antibody light chain variable domain (VL), or

[0450] cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL),

[0451] wherein the second polypeptide is fused at the N-terminus of the VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of the full-length antibody, and

[0452] wherein the antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen binding domain that specifically binds to the second antigen.

[0453] In one aspect, the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via interchain disulfide bridges by introducing a disulfide bond between

[0454] (i) position 44 of the heavy chain variable domain to position 100 of the light chain variable domain, or

[0455] (ii) position 105 of the heavy chain variable domain to position 43 of the light chain variable domain, or

[0456] (iii) position 101 of the heavy chain variable domain to position 100 of the light chain variable domain (always numbered according to Kabat EU index).

[0457] Techniques for introducing non-native disulfide bridges for stabilization are described, for example, in WO 94 / 029350; Rajagopal, V. et al., Prot. Eng. (1997) 1453-1459; Kobayashi, H. et al., Nucl. Med. Biol. 25 (1998) 387-393; and Schmidt, M. et al., Oncogene 18 (1999) 1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (always numbered according to Kabat). In one embodiment, trivalent bispecific antibodies without said optional disulfide stabilization between the variable domains VH and VL of the single chain Fab fragment are preferred.

[0458] In one aspect, the bispecific antibody is a trispecific or tetraspecific antibody

[0459] a) a first light chain and a first heavy chain of a full length antibody that specifically binds to a first antigen, and

[0460] b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody specifically binding to a second antigen, wherein the variable domains VL and VH are replaced by each other, and / or wherein the constant domains CL and CH1 are replaced by each other, and

[0461] c) wherein one to four antigen binding domains specifically binding to one or two further antigens (i.e. a third and / or a fourth antigen) are fused via a peptide linker to the C-terminus or N-terminus of the light chain or heavy chain of a) and / or b).

[0462] a) the antibody below does not contain modifications as reported under b) and the heavy chain and light chain under a) are separate chains.

[0463] In one aspect, the trispecific or tetraspecific antibody under c) comprises one or two antigen binding domains specifically binding to one or two further antigens.

[0464] In one aspect, the antigen binding domain is selected from the group consisting of a scFv fragment and a scFab fragment.

[0465] In one aspect, the antigen binding domain is a scFv fragment.

[0466] In one aspect, the antigen binding domain is a scFab fragment.

[0467] In one aspect, the antigen binding domain is fused to the C-terminus of the heavy chain under a) and / or b).

[0468] In one aspect, the trispecific or tetraspecific antibody under c) comprises one or two antigen binding domains specifically binding to another antigen.

[0469] In one aspect, the trispecific or tetraspecific antibody under c) comprises two identical antigen binding domains specifically binding to a third antigen. In a preferred embodiment, both of such two identical antigen binding domains are fused via the same peptide linker to the C-terminus of the heavy chain under a) and b). In a preferred embodiment, both of the two identical antigen binding domains are scFv fragments or scFab fragments.

[0470] In one aspect, the trispecific or tetraspecific antibody under c) comprises two antigen binding domains specifically binding to a third and a fourth antigen. In one embodiment, the two antigen binding domains are fused via the same peptide linker to the C-terminus of the heavy chain under a) and b). In a preferred embodiment, the two antigen binding domains are scFv fragments or scFab fragments.

[0471] In one aspect, the bispecific antibody is a bispecific tetravalent antibody comprising

[0472] a) two light chains and two heavy chains of an antibody that specifically binds to a first antigen (and comprises two Fab fragments),

[0473] b) two further Fab fragments of an antibody that specifically binds to a second antigen, wherein the further Fab fragments are each fused via a peptide linker to the C-terminus or the N-terminus of the heavy chain of a), and

[0474] wherein the following modifications are performed in the Fab fragments

[0475] (i) in the two Fab fragments of a), or in the two Fab fragments of b), the variable domains VL and VH are replaced by each other and / or the constant domains CL and CH1 are replaced by each other, or

[0476] (ii) in the two Fab fragments of a), the variable domains VL and VH are replaced by each other and the constant domains CL and CH1 are replaced by each other, and in the two Fab fragments of b), the variable domains VL and VH are replaced by each other or the constant domains CL and CH1 are replaced by each other, or

[0477] (iii) in the two Fab fragments of a), the variable domains VL and VH are replaced by each other or the constant domains CL and CH1 are replaced by each other, and in the two Fab fragments of b), the variable domains VL and VH are replaced by each other and the constant domains CL and CH1 are replaced by each other, or

[0478] (iv) in the two Fab fragments of a), the variable domains VL and VH are replaced by each other, and in the two Fab fragments of b), the constant domains CL and CH1 are replaced by each other, or

[0479] (v) in the two Fab fragments of a), the constant domains CL and CH1 are replaced by each other, and in the two Fab fragments of b), the variable domains VL and VH are replaced by each other.

[0480] In one aspect, the further Fab fragments are each fused via a peptide linker to the C-terminus of the heavy chain of a) or to the N-terminus of the heavy chain of a).

[0481] In one aspect, said additional Fab fragment is fused to the C-terminus of the heavy chain of a) via a peptide linker.

[0482] In one aspect, said additional Fab fragment is fused to the N-terminus of the heavy chain of a) via a peptide linker.

[0483] In one aspect, in the Fab fragments the following modifications are performed: in both Fab fragments of a) or in both Fab fragments of b), the variable domains VL and VH are replaced with each other and / or the constant domains CL and CH1 are replaced with each other.

[0484] In one aspect, the bispecific antibody is a tetravalent antibody comprising:

[0485] a) a (modified) heavy chain of a first antibody that specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein the N-terminus of a second VH-CH1 domain pair of said first antibody is fused to the C-terminus of said heavy chain via a peptide linker,

[0486] b) two light chains of the first antibody of a),

[0487] c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein the N-terminus of the second VH-CL domain pair of the second antibody is fused to the C-terminus of the heavy chain via a peptide linker, and

[0488] d) two (modified) light chains of the second antibody of c), each light chain comprising a CL-CH1 domain pair.

[0489] In one aspect, the bispecific antibody comprises

[0490] a) the heavy and light chains of a first full-length antibody that specifically binds to a first antigen, and

[0491] b) a heavy chain and a light chain of a second full-length antibody that specifically binds to a second antigen, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker.

[0492] The antibody under a) does not contain the modifications reported under b), and the heavy and light chains are separated chains.

[0493] In one aspect, the bispecific antibody comprises

[0494] a) a full-length antibody that specifically binds to a first antigen and is composed of two antibody heavy chains and two antibody light chains; and

[0495] b) an Fv fragment specifically binding to a second antigen, the Fv fragment comprising a VH2 domain and a VL2 domain, wherein the two domains are connected to each other via a disulfide bridge,

[0496] wherein only one of the VH2 domain or the VL2 domain is fused via a peptide linker to the heavy chain or the light chain of the full length antibody specifically binding to the first antigen.

[0497] In the bispecific antibody, the heavy chain and the light chain under a) are separate chains.

[0498] In one aspect, the other of the VH2 domain or the VL2 domain is not fused via a peptide linker to the heavy chain or the light chain of the full length antibody specifically binding to the first antigen.

[0499] In all aspects reported herein, the first light chain comprises a VL domain and a CL domain, and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

[0500] In one aspect, the bispecific antibody is a trivalent antibody comprising

[0501] a) two Fab fragments specifically binding to a first antigen,

[0502] b) one CrossFab fragment specifically binding to a second antigen, in which CrossFab fragment the CH1 and the CL domain are exchanged with each other,

[0503] c) one Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain,

[0504] wherein the C-terminus of the CH1 domain of the two Fab fragments is connected to the N-terminus of the heavy chain Fc region polypeptide, and wherein the C-terminus of the CL domain of the CrossFab fragment is connected to the N-terminus of the VH domain of one of the Fab fragments.

[0505] In one aspect, the bispecific antibody is a trivalent antibody comprising

[0506] a) two Fab fragments specifically binding to a first antigen,

[0507] b) one CrossFab fragment specifically binding to a second antigen, in which CrossFab fragment the CH1 and the CL domain are exchanged with each other,

[0508] c) an Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain,

[0509] wherein the C-terminus of the CH1 domain of the first Fab fragment is connected to the N-terminus of one of the heavy chain Fc region polypeptides, and the C-terminus of the CL domain of the CrossFab fragment is connected to the N-terminus of the other heavy chain Fc region polypeptide, and wherein the C-terminus of the CH1 domain of the second Fab fragment is connected to the N-terminus of the VH domain of the first Fab fragment or to the N-terminus of the VH domain of the CrossFab fragment.

[0510] In one aspect, the bispecific antibody comprises

[0511] a) a full length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; and

[0512] b) a Fab fragment that specifically binds to a second antigen, the Fab fragment comprising a VH2 domain and a VL2 domain that make up a heavy chain fragment and a light chain fragment, wherein within the light chain fragment the variable light domain VL2 is replaced by the variable heavy domain VH2 of the antibody and within the heavy chain fragment the variable heavy domain VH2 is replaced by the variable light domain VL2 of the antibody,

[0513] wherein the heavy chain Fab fragment is inserted between the CH1 domain of one of the heavy chains of the full length antibody and the corresponding Fc region of the full length antibody, and the N-terminus of the light chain Fab fragment is conjugated to the C-terminus of the light chain of the full length antibody that pairs with the heavy chain of the full length antibody into which the heavy chain Fab fragment has been inserted.

[0514] In one aspect, the bispecific antibody comprises

[0515] a) a full length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; and

[0516] b) a Fab fragment specifically binding to a second antigen, the Fab fragment comprising a VH2 domain and a VL2 domain constituting a heavy chain fragment and a light chain fragment, wherein within the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of the antibody and within the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of the antibody, and wherein the C-terminus of the heavy chain fragment of the Fab fragment is conjugated to the N-terminus of one of the heavy chains of the full length antibody and the C-terminus of the light chain fragment of the Fab fragment is conjugated to the N-terminus of the light chain of the full length antibody, the light chain of the full length antibody pairing with the heavy chain of the full length antibody to which the heavy chain fragment of the Fab fragment is conjugated to.

[0517] polynucleotide

[0518] Further, an isolated polynucleotide encoding a bispecific antibody or fragment thereof as described herein is provided.

[0519] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base (in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically denoted from 5' to 3'. In the present context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (in particular messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. A nucleic acid molecule can be linear or circular. Furthermore, the term nucleic acid molecule includes both the sense and the antisense strand, as well as single- and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present application in vitro and / or in vivo (e.g. in a host or patient). Such DNA (e.g. cDNA) or RNA (e.g. mRNA) vectors can be unmodified or modified. For example, the mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject in vivo to produce the antibody in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0520] An "isolated" polynucleotide refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated polynucleotide includes a nucleic acid molecule contained in cells that typically contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0521] An isolated polynucleotide encoding a bispecific antibody of the application can be expressed as a single polynucleotide that encodes the entire antigen binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by co-expressed polynucleotides can associate, e.g., via disulfide bonds or other means, to form a functional antigen binding molecule. For example, a light chain portion of an immunoglobulin can be encoded by a separate polynucleotide from a heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an immunoglobulin.

[0522] In some aspects, an isolated polynucleotide encodes a polypeptide as described herein included in a bispecific antibody according to the application.

[0523] In one aspect, an isolated polynucleotide encoding an anti-PD1 / anti-LAG3 bispecific antibody is provided, wherein the first antigen binding domain that binds specifically to PD1 comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0524] Preparation of bispecific antibodies for use in the application

[0525] Antibodies can be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0526] In the case of a natural antibody or a natural antibody fragment, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acids encode the amino acid sequence making up the VL of the antibody and / or the amino acid sequence making up the VH of the antibody (e.g. the light chain and / or the heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors. In the case of certain bispecific antibodies having heterodimeric heavy chains, four nucleic acids are required, one for a first light chain, one for a first heavy chain comprising a first heteromonomeric Fc region polypeptide, one for a second light chain, and one for a second heavy chain comprising a second heteromonomeric Fc region polypeptide. The four nucleic acids can be comprised in one or more nucleic acid molecules or expression vectors. For example, such nucleic acids encode the amino acid sequence making up the first VL of the antibody and / or the amino acid sequence making up the first VH of the antibody comprising a first heteromonomeric Fc region and / or the amino acid sequence making up the second VL of the antibody and / or the amino acid sequence making up the second VH of the antibody comprising a second heteromonomeric Fc region (e.g. the first light chain and / or the second light chain and / or the first heavy chain and / or the second heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors, typically these nucleic acids are on two or three expression vectors, i.e. one vector can comprise more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs and T cell bispecifics (see e.g. Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric heavy chains comprises a so-called “knob mutation” (T366W, and optionally one of S354C or Y349C) and the other of the heteromonomeric heavy chains comprises a so-called “hole mutation” (T366S, L368A and Y407V, and optionally Y349C or S354C) (see e.g. Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0527] In one aspect, an isolated nucleic acid encoding a bispecific antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence that makes up a VL of an antigen binding domain that specifically binds PD1 and LAG3, respectively, and / or an amino acid sequence that makes up a VH of the antigen binding domain (e.g., in a light chain and / or a heavy chain of an antibody). In another aspect, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In another aspect, a host cell comprising such a nucleic acid is provided. In one such aspect, a host cell comprises (e.g., has been transformed with) (1) a first vector comprising a first pair of nucleic acids encoding amino acid sequences, one of which comprises a first VL of an antibody, and the other of which comprises a first VH of the antibody; and a second vector comprising a second pair of nucleic acids encoding amino acid sequences, one of which comprises a second VL of an antibody, and the other of which comprises a second VH of the antibody, or (2) a first vector comprising a first nucleic acid encoding an amino acid sequence, a second vector comprising a pair of nucleic acids encoding amino acid sequences, and a third vector comprising a pair of nucleic acids encoding amino acid sequences, the first vector comprising a first nucleic acid encoding an amino acid sequence comprises one of the variable domains (preferably a light chain variable domain); one of the pair of nucleic acids encoding amino acid sequences comprised by the second vector comprises a light chain variable domain, and the other of the pair of nucleic acids comprises a first heavy chain variable domain; one of the pair of nucleic acids encoding amino acid sequences comprised by the third vector comprises the respective other light chain variable domain as in the second vector, and the other of the pair of nucleic acids comprises a second heavy chain variable domain, or (3) a first vector comprising a nucleic acid encoding an amino acid sequence, a second vector comprising a nucleic acid encoding an amino acid sequence, a third vector comprising a nucleic acid encoding an amino acid sequence, and a fourth vector comprising a nucleic acid encoding an amino acid sequence, the first vector comprising a nucleic acid encoding an amino acid sequence comprises a first VL of an antibody, the second vector comprising a nucleic acid encoding an amino acid sequence comprises a first VH of an antibody, the third vector comprising a nucleic acid encoding an amino acid sequence comprises a second VL of an antibody, and the fourth vector comprising a nucleic acid encoding an amino acid sequence comprises a second VH of an antibody. In one aspect, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one aspect, a method of making a bispecific antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0528] For recombinant production of the anti-HLA-G / anti-CD3 bispecific antibodies and / or anti-PD1 / anti-LAG3 bispecific antibodies described herein, a nucleic acid encoding the bispecific antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific to genes encoding the heavy and light chains of the antibody).

[0529] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, in particular when glycosylation and Fc effector functions are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., in: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254 describing expression of antibody fragments in E. coli.) Antibodies can be isolated from bacterial cell paste in soluble fraction after expression and can be further purified.

[0530] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable clones or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathway has been “humanized” to produce antibodies with a partial or fully human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0531] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculoviral strains have been identified which can be used in conjunction with insect cells, particularly Spodoptera frugiperda cells.

[0532] Plant cell cultures can also be utilized as hosts. See e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0533] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0534] Assays

[0535] The physical / chemical properties and / or biological activities of the bispecific antibodies provided herein can be identified, screened for, or characterized by a variety of assays known in the art.

[0536] 1. Affinity determination

[0537] The affinity of the bispecific antigen-binding molecules, antibodies and antibody fragments provided herein for the corresponding antigens can be determined by surface plasmon resonance (SPR) using standard test equipment, e.g., a Biacore® instrument (GE Healthcare) and the receptor or target protein, e.g., obtainable by recombinant expression, according to the methods set forth in the Examples. Specific illustrative and exemplary embodiments for measuring binding affinity have been described in Examples 2, 8 or 11 in WO 2018 / 185043. According to one aspect, Kd is measured by surface plasmon resonance using a BIACORE® T100 instrument (GE Healthcare) at 25°C. D .

[0538] 2. Binding and other assays

[0539] In one aspect, the antigen binding activity of the bispecific antibodies of the application is tested, e.g., by known methods such as ELISA, Western blotting, etc. The binding of the anti-PD1 / anti-LAG3 bispecific antibodies provided herein to the corresponding recombinant antigens or antigen-expressing cells can be assessed by ELISA as described in Examples 8 or 11 of WO 2018 / 185043. In a further aspect, fresh peripheral blood mononuclear cells (PBMCs) can be used in the binding assay to show binding to different peripheral blood mononuclear cells (PBMCs), e.g., monocytes, NK cells and T cells.

[0540] In another aspect, a cell dimerization assay is used to demonstrate dimerization or at least binding / interaction of the two different receptors PD1 and LAG3, which fuse in the cytosol with two fragments of an enzyme when connected or cross-linked with the bispecific antibody against both targets. Thus, only one receptor alone is shown to be without enzymatic activity. For this specific interaction, the cytoplasmic C-terminus of both receptors is individually fused to a heterologous subunit of a reporter enzyme. The individual enzyme subunit alone does not show reporter activity. However, binding to both receptors simultaneously is expected to lead to local cytosolic aggregation of both receptors, complementation of both heterologous enzyme subunits, and finally formation of a specific and functional enzyme, which will hydrolyze a substrate, thereby generating a chemiluminescent signal (Example 11 of WO 2018 / 185043).

[0541] 3. Activity assays

[0542] In one aspect, assays are provided for identifying anti-PD1 / anti-LAG3 bispecific antibodies having biological activity. Biological activity can include, for example, enhancing activation and / or proliferation of different immune cells, especially T cells, secretion of immunomodulatory cytokines such as IFNy or TNF-a, blocking of the PD1 pathway, blocking of the LAG3 pathway, ability to kill tumor cells. Antibodies having such biological activity in vivo and / or in vitro are also provided. In certain aspects, the antibodies of the application are tested for such biological activity. In one aspect, an immune cell assay is provided that measures activation of lymphocytes from one individual (donor X) against lymphocytes from another individual (donor Y). Mixed lymphocyte reaction (MLR) can demonstrate the effect of blocking the PD1 pathway on lymphocyte effector cells. T cells in the assay are tested for activation and their IFN-g secretion in the presence or absence of the bispecific antibodies of the application. The assay is described in more detail in Example 9 of WO 2018 / 185043.

[0543] Pharmaceutical compositions, formulations, and routes of administration

[0544] In further aspects, the present application provides pharmaceutical compositions comprising the anti-HLA-G / anti-CD3 antibodies and the anti-PD1 / anti-LAG3 antibodies provided herein, for example, for use in any of the methods of treatment described below. In one embodiment, the pharmaceutical composition comprises the anti-HLA-G / anti-CD3 antibodies and the anti-PD1 / anti-LAG3 antibodies provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises the antibodies provided herein and at least one additional therapeutic agent, for example, as described below.

[0545] The pharmaceutical compositions of the present application comprise a therapeutically effective amount of one or more bispecific antibodies dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. According to the present disclosure, a pharmaceutically or pharmacologically acceptable carrier is one that is biologically or pharmacologically compatible for in vivo use, for example, in humans or animals. According to the present disclosure, the preparation of pharmaceutical compositions comprising at least one antibody and optionally an additional active ingredient will be known to those of skill in the art in light of the disclosure herein, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. In particular, the compositions are in a form suitable for lyophilization or solution in aqueous solution. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers and combinations thereof, as would be known to one of ordinary skill in the art.

[0546] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the antigen binding molecules of the application can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the fusion protein can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the fusion protein of the application in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility can be readily accomplished by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and / or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions, or emulsions, the preferred methods of preparation are vacuum-drying or freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level. Suitable pharmaceutical excipients include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty vehicles, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can be prepared from these

[0547] The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

[0548] Exemplary pharmaceutical excipients herein also include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

[0549] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter of which contains formulations comprising histidine-acetate buffers.

[0550] In addition to the previously described compositions, the bispecific antibodies can be formulated as long-acting formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. Thus, for example, the fusion protein can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as a sparingly soluble derivative (for example, as a sparingly soluble salt).

[0551] Pharmaceutical compositions comprising the bispecific antigen binding molecules of the application can be manufactured by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0552] The bispecific antibodies disclosed herein can be formulated into compositions in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that retain the biological effectiveness of the free acids or bases and are not biologically undesirable. These salts include acid addition salts, such as those formed with the free amino groups of a proteinaceous composition, for example, with inorganic acids such as for example, hydrochloric or phosphoric acids or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides or such organic bases as isopropylamine, trimethylamine, histidine or procaine. The pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0553] The compositions herein can also contain more than one active ingredient necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in conjunction with the compounds of this application in amounts that are effective for the purpose intended.

[0554] In one aspect, a pharmaceutical composition comprising an anti-HLA-G / anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier, and a second medicament comprising an anti-PD1 / anti-LAG3 antibody described herein is provided. In one aspect, the pharmaceutical composition is used to treat a cancer expressing HLA-G. In a particular aspect, the pharmaceutical composition is used to treat a cancer expressing HLA-G, in particular a disease selected from the group consisting of lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the naso-pharynx, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancers, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, lymphomas, lymphocytic leukemias, including any of the foregoing in a refractory form, or a combination of one or more of the foregoing cancers.

[0555] Formulations to be used for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0556] Administration of anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies

[0557] Both the anti-HLA-G / anti-CD3 bispecific antibodies and the anti-PD1 / anti-LAG3 antibodies (both of which are referred to herein as agents) can be administered by any suitable means, including parenterally, intrapulmonarily, and intranasally, and, if desired for local treatment, intralesionally. However, the methods described herein are particularly useful for therapeutic agents administered by parenteral, particularly intravenous, infusion.

[0558] Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various administration schedules are contemplated herein, including but not limited to single or multiple administrations, bolus administrations, and pulse infusions at various time points. In one aspect, the therapeutic agent is administered parenterally, particularly via intravenous administration. In a specific aspect, the substance is administered by intravenous infusion. On the other hand, the substance is administered subcutaneously.

[0559] Both the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to practitioners. Both the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody need not be, but are optionally, formulated with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other agents depends on the amount of therapeutic agent present in the formulation, the type of condition or treatment, and the other factors discussed above. These are generally used in the same dosages and by administration routes as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0560] For the prevention or treatment of disease, the appropriate dosage of anti-HLA-G / anti-CD3 bispecific antibody and anti-PD1 / anti-LAG3 antibody, used in combination with each other or with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of anti-HLA-G / anti-CD3 bispecific antibody, the severity and course of the disease, whether the antibody is for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the treating physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) of the agent can be an initial candidate dosage, e.g., for a patient weighing 70 kg, for example. Depending on the results of this initial dosage, it may be necessary to increase or decrease the dosage of the agent. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the bispecific antibody would be in the range from about 0.005 mg / kg to about 10 mg / kg. In other examples, dosages can also include about 1 pg / kg body weight, about 5 pg / kg body weight, about 10 pg / kg body weight, about 50 pg / kg body weight, about 100 pg / kg body weight, about 200 pg / kg body weight, about 350 pg / kg body weight, about 500 pg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight to about 1000 mg / kg body weight or more, and any range derivable therein. In an example derivable from the numbers listed herein, a dosage from about 5 mg / kg body weight to about 100 mg / kg body weight, from about 5 pg / kg body weight to about 500 mg / kg body weight, etc., can be used, e.g., based on the numbers above. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, e.g. every week or every three weeks (e.g., so that the patient receives from about two to about twenty, or, e.g., about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses, can be administered. However, other dosage regimens can be useful. The progress of the therapy is easily monitored by conventional techniques and assays.However, other dosage regimens may be useful.The progress of this therapy can be easily monitored by conventional techniques and assays.

[0561] In one aspect, both the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody are administered as a single administration. In certain aspects, the therapeutic agents are administered as two or more administrations. In one such aspect, the agents are administered weekly, biweekly, or every three weeks, particularly every two weeks. In one aspect, the agents are administered in a therapeutically effective amount. In one aspect, the agents are administered at a dose of about 10 μg / kg, about 100 μg / kg, about 200 μg / kg, about 300 μg / kg, about 400 μg / kg, about 500 μg / kg, about 600 μg / kg, about 700 μg / kg, about 800 μg / kg, about 900 μg / kg, or about 1000 μg / kg. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody is administered at a dose that is higher than the dose of the anti-HLA-G / anti-CD3 bispecific antibody in a corresponding treatment regimen without the anti-PD1 / anti-LAG3 antibody. In one aspect, administration of the anti-HLA-G / anti-CD3 bispecific antibody comprises an initial administration of a first dose of the anti-HLA-G / anti-CD3 bispecific antibody and one or more subsequent administrations of a second dose of the anti-HLA-G / anti-CD3 bispecific antibody, wherein the second dose is higher than the first dose. In one aspect, administration of the anti-HLA-G / anti-CD3 bispecific antibody comprises an initial administration of a first dose of the anti-HLA-G / anti-CD3 bispecific antibody and one or more subsequent administrations of a second dose of the anti-HLA-G / anti-CD3 bispecific antibody, wherein the first dose is no less than the second dose.

[0562] In one aspect, in a treatment regimen according to the present invention, administration of the anti-HLA-G / anti-CD3 bispecific antibody is the first administration of the anti-HLA-G / anti-CD3 bispecific antibody to a subject (at least within the same treatment course). In one aspect, the subject is not administered an anti-PD1 / anti-LAG3 antibody prior to administration of the anti-HLA-G / anti-CD3 bispecific antibody. In another aspect, the subject is not administered an anti-PD1 / anti-LAG3 antibody prior to administration of the anti-HLA-G / anti-CD3 bispecific antibody.

[0563] In the present invention, the combination of an anti-HLA-G / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 antibody can be used in combination with one or more other agents for therapy. For example, at least one additional therapeutic agent can be co-administered. In certain aspects, the additional therapeutic agent is an immunotherapeutic agent.

[0564] Such combination therapies encompass combined administration (where two or more therapeutic agents are administered within the same pharmaceutical composition or separately) and separate administration, in which case, administration of the therapeutic agents can occur simultaneously or sequentially, with or without a lapse of time between the administration of the individual therapeutic agents. In one embodiment, administration of the therapeutic agents and administration of the additional therapeutic agent occur within about one month of each other, or within about one, two or three weeks, or within about one, two, three, four, five or six days.

[0565] Therapeutic methods and compositions

[0566] HLA-G is primarily expressed on cytotrophoblasts in the placenta, but various tumors, including pancreatic, breast, skin, colorectal, gastric and ovarian cancers, have been reported to express HLA-G.

[0567] In one aspect, a method of treating or delaying progression of a cancer expressing HLA-G in a subject is provided, the method comprising administering to the subject an effective amount of an anti-HLA-G / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody.

[0568] In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In some embodiments, a method for increasing cytokine secretion, including IFNy, and / or T cell activation is provided, the method comprising administering to the subject an effective amount of an anti-HLA-G / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody. In particular embodiments, the method comprises T cell-mediated tumor cell killing. The "individual" or "subject" according to any of the above aspects is preferably a human.

[0569] In further aspects, a composition for use in cancer immunotherapy comprising an anti-HLA-G / anti-CD3 antibody and an anti-PD1 / anti-LAG3 antibody is provided. In certain embodiments, a composition comprising an anti-HLA-G / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody is provided for use in a method of cancer immunotherapy.

[0570] In a further aspect, provided herein is the use of a composition comprising an anti-HLA-G / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating a cancer expressing HLA-G. In one aspect, the medicament is for treating a cancer expressing HLA-G. In a further aspect, the medicament is for use in a method of treating a cancer expressing HLA-G, the method comprising administering to an individual having a cancer expressing HLA-G an effective amount of the medicament. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In a further aspect, the medicament is for use in activating T cells, particularly against HLA-G expressing tumor cells. The cancer expressing HLA-G is selected from lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the naso-pharynx, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancers, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. In particular, the cancer expressing HLA-G is selected from renal cell carcinoma, colorectal cancer, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0571] In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below. The "individual" according to any of the above embodiments can be a human.

[0572] The combination therapies described above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations); and separate administration, in which case the anti-PD1 / anti-LAG3 bispecific antibody as reported herein can be administered prior to, simultaneously with, and / or following the administration of one or more additional therapeutic agents. In one aspect, administration of an effective amount of the anti-HLA-G / anti-CD3 bispecific antibody, administration of an effective amount of the anti-PD1 / anti-LAG3 antibody, and administration of the additional therapeutic agent occur within about one month of one another, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of one another.

[0573] The anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody as reported herein (and any additional therapeutic agents) can each be administered by any appropriate means that anhygienic, including parenterally, intrapulmonarily, and intranasally, and, if desired for local treatment, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be effected by any appropriate route, e.g., by injection, such as intravenous or subcutaneous injection, depending partly on whether short-term or long-term administration is involved. Various administration schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0574] The anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody as reported herein will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the part of the body to be treated, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibodies need not, but may, be formulated with one or more agents currently used in preventing or treating the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages are used, or any dosage and route of administration is also envisioned, as appropriate.

[0575] Those of skill in the art will readily recognize that in many cases, the bispecific molecule can not provide a cure, but can only provide partial benefit. In some embodiments, a physiological change that has some benefit is also considered therapeutically beneficial. Thus, in some aspects, the amount of bispecific antibody that provides a physiological change is considered an "effective amount" or "therapeutically effective amount."

[0576] The anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies defined herein are suitably administered to a patient at one time or over a series of treatments. From about 1 pg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) of bispecific antibody can be an initial candidate dosage for administration to the patient, depending on the type and severity of the disease. A typical daily dose can range from about 1 pg / kg to 10 mg / kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, the treatment is typically continued until a desired suppression of disease symptoms has occurred. One exemplary dosage of an anti-HLA-G / anti-CD3 bispecific antibody would be in the range from about 0.05 pg / kg to about 1000 pg / kg. For anti-PD1 / anti-LAG3 antibodies, dosages can also include about 0.01 mg / kg body weight, about 0.05 mg / kg body weight, about 2 mg / kg body weight, about 4 mg / kg body weight, about 10 mg / kg body weight, about 20 mg / kg body weight, about 30 mg / kg body weight, about 40 mg / kg body weight, about 45 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 300 mg / kg body weight, about 400 mg / kg body weight, about 500 mg / kg body weight, about 600 mg / kg body weight, about 800 mg / kg body weight, about 1000 mg / kg body weight, to about 1200 mg / kg body weight or more, and any range derivable therein. In an example of a range derivable from the numbers listed herein, a range of about 5 mg / kg body weight to about 100 mg / kg body weight, about 0.05 pg / kg body weight to about 500 mg / kg body weight, etc., can be used. In one aspect, an anti-HLA-G / anti-CD3 bispecific antibody can be administered to a patient at a dose of about 0.01 mg, from 2.5 mg to about 10 mg, or about 20 mg, or about 30 mg. Such doses can be administered intermittently, e.g. every week or every three weeks (e.g. so that the patient receives from about two to about twenty, or e.g. about six doses of the fusion protein). An initial higher loading dose, followed by one or more lower doses, can be administered. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and assays.In one aspect, the anti-PD1 / anti-LAG3 antibody can be administered to a patient at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg.

[0577] A bispecific antibody comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3 as defined herein will typically be used in an amount effective to achieve the intended purpose. For use in therapy or prevention of a disease condition, a bispecific antibody of the application, or a pharmaceutical composition thereof, is administered in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0578] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. The dose can be formulated in an animal model to achieve a circulating concentration range that includes the IC50as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. 50 In vivo data, e.g., animal model data, can also be used to estimate initial dosages. A practitioner of ordinary skill in the art could readily optimize dosages for administration to humans based on animal data.

[0579] Initial dosages can also be estimated from in vivo data, e.g., animal model data. A practitioner of ordinary skill in the art could readily optimize dosages for administration to humans based on animal data.

[0580] Dosages and intervals between dosages can be adjusted to provide plasma levels of the bispecific antibody that are sufficient to maintain therapeutic effect. Common patient dosages for injection range from about 0.1 to 50 mg / kg / day, typically from about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.

[0581] In cases of local administration or selective uptake, effective local concentrations of the bispecific antibody can not be related to plasma concentrations. A person of ordinary skill in the art would be able to optimize therapeutically effective local dosages without undue experimentation.

[0582] A therapeutically effective dose of a bispecific antibody described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of the fusion protein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine LD 50LD50 (the dose lethal to 50% of the population) and ED50 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 50 / ED50 50 Bispecific antibodies exhibiting a large therapeutic index are preferred. In one embodiment, the bispecific antibodies according to the application exhibit a high therapeutic index. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage that are suitable for use in humans. The dosage is preferably within a range with little or no toxicity. The dosage can vary within this range depending upon a variety of factors, e.g., the dosing regimen, the route of administration, the severity of the subject's condition, etc. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl, et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety).

[0583] The physician in charge of the patient treated with the bispecific antibodies of the present application will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, etc. Conversely, the physician will also know to adjust the treatment to higher levels if the clinical response is insufficient (excluding toxicity). The size of the dose administered in the management of the target condition will vary with the severity of the condition to be treated, the route of administration, etc. For example, the severity of the condition can be assessed, in part, by standard prognostic assessment methods. Further, the dose and possibly the dose frequency will also vary according to the age, body weight, and response of the individual patient.

[0584] Such other agents are present in combination with the fusion protein in amounts that are effective for the intended purpose. The effective amount of such other agents depends on the amount of fusion protein used, the type of condition or treatment, and other factors discussed above. These bispecific antibodies are generally used in the same dosages and

[0585] Such combination therapies as described above encompass combined administration (where two or more therapeutic agents are administered within the same combination composition or in separate composition) and separate administration, in which case administration of the bispecific antibody can occur prior to, simultaneously with, and / or following administration of the additional therapeutic agent and / or adjuvant.

[0586] H. Articles of Manufacture

[0587] In another aspect of the application, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-PD1 / anti-LAG3 bispecific antibody as defined above.

[0588] The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can further comprise (a) a first container comprising a composition comprising an anti-HLA-G / anti-CD3 bispecific antibody described herein; and (b) a second container comprising a composition comprising an anti-PD1 / anti-LAG3 antibody. The article of manufacture in this embodiment of the application can further include a package insert indicating that the compositions can be used to treat the particular condition.

[0589] Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and

[0590] Table B (sequences):

[0591]

[0592] General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). The numbering and referencing of the amino acids of the antibody chains is according to the numbering system according to Kabat (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) as defined above.

[0593] Aspects of the application

[0594] Some aspects of the application are listed below.

[0595] 1. An anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating a cancer expressing HLA-G, wherein the anti-HLA-G / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody.

[0596] 2. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to para 1, wherein the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

[0597] 3. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to para 1 or 2, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain, which is an IgG Fc domain, in particular an IgGl Fc domain or an IgG4 Fc domain, and wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular to an Fcy receptor.

[0598] 4. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paras 1 to 3, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain of human IgGl subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

[0599] 5. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paras 1 to 4, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen binding domain that specifically binds to lymphocyte-activation gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1 comprises a VH domain comprising

[0600] (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1,

[0601] (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and

[0602] (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0603] a VL domain comprising

[0604] (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4,

[0605] (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO:5, and

[0606] (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO:6.

[0607] 6. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 5, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising

[0608] (a) a VH domain comprising

[0609] (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 11 ;

[0610] (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 12; and

[0611] (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 13; and

[0612] a VL domain comprising

[0613] (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 14;

[0614] (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 15; and

[0615] (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 16; or

[0616] (b) a VH domain comprising

[0617] (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 19;

[0618] (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 20; and

[0619] (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 21; and

[0620] a VL domain comprising

[0621] (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22;

[0622] (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and

[0623] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0624] 7. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 6, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to PD1, the first antigen binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10.

[0625] 8. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 7, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising

[0626] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or

[0627] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0628] 9. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 5 or 7, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising

[0629] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising the amino acid sequence of SEQ ID NO: 28, or

[0630] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29; and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, or

[0631] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31 ; and a VL domain comprising the amino acid sequence of SEQ ID NO: 32, or

[0632] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 33; and a VL domain comprising the amino acid sequence of SEQ ID NO: 34, or

[0633] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 64; and a VL domain comprising the amino acid sequence of SEQ ID NO: 65.

[0634] 10. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 8, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises

[0635] a first antigen binding domain that specifically binds to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10;

[0636] and a second antigen binding domain that specifically binds to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

[0637] 11. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 10, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1 and a Fab fragment that specifically binds to LAG3.

[0638] 12. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 11, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1, wherein the variable domains VL and VH are replaced with each other such that VL is part of the heavy chain and VH is part of the light chain.

[0639] 13. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 12, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises monovalent binding to PD1 and monovalent binding to LAG3.

[0640] 14. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 8 and 10 to 13, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises

[0641] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37; and a second light chain comprising the amino acid sequence of SEQ ID NO: 38; or

[0642] (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 39; and a second light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0643] 15. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 8 and 10 to 14, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37; and a second light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0644] 16. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 15, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to HLA-G, the first antigen binding domain comprising a heavy chain variable region (V H CD3) and a light chain variable region (V L CD3), and the second antigen binding domain comprising a heavy chain variable region (V H HLA-G) and a light chain variable region (V LHLA-G).

[0645] 17. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 15, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to HLA-G, wherein the first antigen binding domain that specifically binds to CD3 comprises a VH domain comprising

[0646] (i) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 41,

[0647] (ii) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and

[0648] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and

[0649] a VL domain comprising

[0650] (i) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 44,

[0651] (ii) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and

[0652] (iii) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 46.

[0653] 18. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 17, wherein the first antigen binding domain that specifically binds to CD3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 47; and a VL domain comprising the amino acid sequence of SEQ ID NO: 48.

[0654] 19. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 18, wherein the second antigen binding domain that specifically binds to HLA-G comprises

[0655] a) a VH domain comprising

[0656] i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49,

[0657] ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 50, and

[0658] iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 51, and

[0659] a) a VL domain, said VL domain comprising

[0660] i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 52,

[0661] ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 53, and

[0662] iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 54, or

[0663] b) a VH domain, said VH domain comprising

[0664] i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 91,

[0665] ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 92, and

[0666] iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 93, and

[0667] a) a VL domain, said VL domain comprising

[0668] i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 94,

[0669] ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 95, and

[0670] iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 95.

[0671] 20. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 19, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen binding domain that specifically binds to HLA-G, the second antigen binding domain comprising

[0672] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 55; and a VL domain comprising the amino acid sequence of SEQ ID NO: 56, or

[0673] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 97; and a VL domain comprising the amino acid sequence of SEQ ID NO: 98.

[0674] 21. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 20, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen binding domain that specifically binds to HLA-G.

[0675] 22. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 21, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises an Fc domain comprising one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

[0676] 23. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 22, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 57; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 58; a first light chain comprising the amino acid sequence of SEQ ID NO: 59; and a second light chain comprising the amino acid sequence of SEQ ID NO: 60.

[0677] 24. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of paragraphs 1 to 23, wherein the anti-HLA-G / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, and wherein the combination is administered at intervals of about one to three weeks.

[0678] 25. A pharmaceutical composition comprising a combination of an anti-HLA-G / anti- CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody for use in treating a disease, in particular a cancer, in particular a cancer expressing HLA-G, in a combined, sequential or simultaneous manner.

[0679] 26. A pharmaceutical composition comprising an anti-HLA-G / anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier, and a second medicament comprising an anti-PD1 / anti-LAG3 bispecific antibody and optionally a pharmaceutically acceptable carrier.

[0680] 27. The pharmaceutical composition according to paragraph 26 for use in the treatment of a cancer expressing HLA-G, in particular a cancer selected from the group consisting of lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the naso-pharynx, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of the soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancers, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including any refractory version of any of the above cancers, or a combination of one or more of the above cancers.

[0681] 28. Use of an anti-HLA-G / anti-CD3 bispecific antibody in combination with an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for the treatment of a cancer, in particular a cancer expressing HLA-G.

[0682] 29. A method of treating a cancer, in particular a cancer expressing HLA-G, in a subject, the method comprising administering to the subject an effective amount of an anti-HLA-G / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 bispecific antibody.

[0683] 30. The method according to paragraph 29, wherein the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition, or separately in two or more different compositions.

[0684] 31. The method according to paragraph 29 or 30, wherein the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously.

[0685] 32. The method of any one of paragraphs 29 to 31, wherein the anti-HLA-G / anti-CD3 bispecific antibody is administered simultaneously with, before, or after administration of the anti-PD1 / anti-LAG3 bispecific antibody.

[0686] 33. An anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating an HLA-G-expressing cancer, wherein the anti-HLA-G / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3,

[0687] wherein the first antigen-binding domain that specifically binds to PD1 comprises: a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and

[0688] wherein the second antigen-binding domain th...

Claims

1. An anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating a cancer expressing HLA-G, wherein the anti-HLA-G / anti-CD3 bispecific antibody is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen binding domain that specifically binds to lymphocyte-activation gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1 comprises: a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

2. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to claim 1, wherein the anti-HLA-G / anti-CD3 bispecific antibody is administered together with the anti-PD1 / anti-LAG3 bispecific antibody in a single composition or separately in two or more different compositions.

3. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to claim 1 or 2, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain, in particular an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain, and wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular to an Fcy receptor.

4. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of claims 1 to 3, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, which comprises (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

16. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or (b) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21 ; and a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

24.

5. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of claims 1 to 4, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to PD1, the first antigen binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO:

10.

6. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of claims 1 to 5, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

7. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of claims 1 to 3 or 5, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 27; and a VL domain comprising the amino acid sequence of SEQ ID NO: 28, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 29; and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 31 ; and a VL domain comprising the amino acid sequence of SEQ ID NO: 32, or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 33; and a VL domain comprising the amino acid sequence of SEQ ID NO: 34, or (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 64; and a VL domain comprising the amino acid sequence of SEQ ID NO:

65.

8. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of claims 1 to 6, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and a second antigen binding domain that specifically binds to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO:

18.

9. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of claims 1 to 8, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1 and a Fab fragment that specifically binds to LAG3.

10. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method according to any one of claims 1 to 6 or 8 or 9, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 35; a first light chain comprising the amino acid sequence of SEQ ID NO: 36; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 37; and a second light chain comprising the amino acid sequence of SEQ ID NO:

38.

11. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method of any one of claims 1 to 10, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen binding domain that specifically binds to CD3, the first antigen binding domain comprising: a heavy chain variable region (V H CD3) comprising a CDR-H1 sequence of SEQ ID NO: 41, a CDR-H2 sequence of SEQ ID NO: 42, and a CDR-H3 sequence of SEQ ID NO: 43; and / or a light chain variable region (V L CD3) comprising a CDR-L1 sequence of SEQ ID NO: 44, a CDR-L2 sequence of SEQ ID NO: 45, and a CDR-L3 sequence of SEQ ID NO:

46.

12. The anti-HLA-G / anti-CD3 bispecific antibody for use in a method of any one of claims 1 to 11, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen binding domain that specifically binds to HLA-G, the second antigen binding domain comprising: a heavy chain variable region (V H HLA-G) comprising the CDR-H1 sequence of SEQ ID NO: 49, the CDR-H2 sequence of SEQ ID NO: 50, and the CDR-H3 sequence of SEQ ID NO: 51; and / or a light chain variable region (V L HLA-G) comprising the CDR-L1 sequence of SEQ ID NO: 52, the CDR-L2 sequence of SEQ ID NO: 53, and the CDR-L3 sequence of SEQ ID NO:

54.

13. A composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in treating cancer, in particular an HLA-G expressing cancer, wherein the treatment comprises administering the composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with a composition comprising an anti-HLA-G / anti-CD3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to Programmed Cell Death Protein 1 (PD1) and a second antigen binding domain that specifically binds to Lymphocyte-Activating Gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1 comprises: a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 ; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

14. The composition of claim 13, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to PD1 comprising: a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO:

10.

15. The composition of claim 13 or 14, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3 comprising (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11 ; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or (b) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

22. (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21 ; and a VL domain comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

24.

16. The composition of claims 13 to 15, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, said second antigen binding domain comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO: 18, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

17. The composition of claims 13 to 16, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first Fab fragment that specifically binds to PD1, said first Fab fragment comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and a second Fab fragment that specifically binds to LAG3, said second Fab fragment comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO:

18.

18. A pharmaceutical composition comprising an anti-HLA-G / anti-CD3 bispecific antibody in combination with an anti-PD1 / anti-LAG3 bispecific antibody for the treatment of a disease, in particular a cancer, in particular an HLA-G expressing cancer, in a combined, sequential or simultaneous manner.

19. The pharmaceutical composition according to claim 18 for use in the treatment of cancer, in particular HLA-G expressing cancer, in particular a disease selected from the group consisting of lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, nasopharyngeal cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethra cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, bile duct cancer, central nervous system (CNS) cancer tumors, spinal cord axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, lymphomas, lymphocytic leukemias, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers.

20. Use of a combination of an anti-HLA-G / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a medicament for treating cancer, particularly HLA-G-expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene-3 (LAG3), wherein the first antigen-binding domain that specifically binds to PD1 comprises: a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and The VL domain comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

21. The use according to claim 20, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain comprises (a) VH domain, which contains (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11; (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 12; and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 13; and a VL domain comprising (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 14; (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 15; and (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 16; or (b) a VH domain comprising (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 19; (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 20; and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 21; and a VL domain comprising (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 22; (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 23; and (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO:

24.

22. The use of claim 20 or 21, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first Fab fragment that specifically binds to PD1, the first Fab fragment comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and a second Fab fragment that specifically binds to LAG3, the second Fab fragment comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO:

18.

23. A method for treating cancer, particularly cancer expressing HLA-G, in a subject, the method comprising administering to the subject an effective amount of an anti-HLA-G / anti-CD3 antibody and an effective amount of an anti-PD1 / anti-LAG3 bispecific antibody, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first antigen binding domain that specifically binds to programmed cell death protein 1 (PD1) and a second antigen binding domain that specifically binds to lymphocyte-activation gene-3 (LAG3), wherein the first antigen binding domain that specifically binds to PD1 comprises a VH domain comprising (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 1; (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:

3. (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:3; and a VL domain comprising (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO:4; (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO:5; and (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO:

6.

24. The method of claim 23, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen binding domain that specifically binds to LAG3, the second antigen binding domain comprising (a) a VH domain comprising (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 11 ; (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 12; and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 13; and a VL domain comprising (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 14; (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 15; and (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 16; or (b) a VH domain comprising (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 19; (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 20; and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 21 ; and a VL domain comprising (i) CDR-L1, comprising the amino acid sequence of SEQ ID NO: 22; (ii) CDR-L2, comprising the amino acid sequence of SEQ ID NO: 23; and (iii) CDR-L3, comprising the amino acid sequence of SEQ ID NO:

24.

25. The method of claim 23 or 24, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first Fab fragment that specifically binds to PD1, the first Fab fragment comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 9; and a VL domain comprising the amino acid sequence of SEQ ID NO: 10; and a second Fab fragment that specifically binds to LAG3, the second Fab fragment comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 17; and a VL domain comprising the amino acid sequence of SEQ ID NO:

18.

26. The method of any one of claims 23-25, wherein the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

27. The method of any one of claims 23-26, wherein the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously.

28. The method of any one of claims 23-27, wherein the anti-HLA-G / anti-CD3 bispecific antibody is administered concurrently with, prior to, or subsequent to administration of the anti-PD1 / anti-LAG3 bispecific antibody.

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