Combination therapy using PD1-LAG3 bispecific antibody and HLA-G T cell bispecific antibody

Abstract

The present invention relates to a combination therapy using an anti-PD1 / anti-LAG3 bispecific antibody and an HLA-G T cell activating bispecific antibody, the use of this combination therapy for the treatment of cancer, and a method for using this combination therapy.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a combination therapy using a PD1-LAG3 bispecific antibody and an HLA-G T cell activating bispecific antibody, the use of this combination therapy for the treatment of cancer, and a method for using this combination therapy. [Background technology]

[0002] background T-cell activating bispecific antibodies are a promising cancer therapy designed to utilize cytotoxic T cells against tumor cells. When such antibodies simultaneously bind CD3 on T cells and antigens expressed on tumor cells, it leads to T-cell activation and subsequent lysis of 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 non-classical HLA class I heavy chain paralog. This class I molecule is a heterodimer (beta-2 microglobulin) consisting of a heavy chain and a light chain. The heavy chain is fixed to the membrane but can also be excreted / secreted. HLA-G is mainly expressed on the trophoblast cell layer of the placenta. Several tumors (including pancreas, breast, skin, colorectal, stomach, and ovary) express HLA-G (Lin, A. et al., Mol Med. 21 (2015) 782-791; Amiot, L., et al., Cell Mol Life Sci. 68 (2011) 417-431). Expression has also been reported to be associated with inflammatory diseases, GvHD, and pathological conditions such as cancer. HLA-G expression has been reported to be associated with poor prognosis in cancer. Tumor cells evade host immune surveillance by inducing immune tolerance / suppression through HLA-G expression.

[0004] Because HLA-G shares high homology (over 98%) with other MHC I molecules, it is difficult to produce truly HLA-G specific antibodies that do not cross-react to other MHC I molecules. Due to the high polymorphism and high homology of the HLA family, many antibodies lack truly specific HLA-G binding properties and often bind to or cross-react with other HLA family members (as an MHC complex with β2M, or in a form without β2M), or simply do not inhibit the binding of the HLA-G β2M MHC complex to its receptors ILT2 and / or ILT4 (considered non-antagonist antibodies). Antibodies that specifically bind to HLA-G are described in International Publications 2019 / 202040, 2019 / 202041, and 2022129120. These publications also describe T cell bispecific antibodies that contain a binding moiety that specifically binds to HLA-G.

[0005] The antitumor activity of HLA-G-TCBs has been demonstrated in vitro using different HLA-G-positive tumor cell lines and in vivo using mouse models. T cell activation, IFNγ secretion, and cytotoxicity induced by HLA-G-TCBs were shown to be dose-dependent and correlate with HLA-G density on the cell surface and the percentage of HLA-G-positive cells. Consistent with the mode of action of TCBs, tumor growth inhibition was accompanied by tumor T cell infiltration and activation, reflected by increased cytokine secretion (including IFNγ) and increased expression of T cell activation markers including CD69, CD25, and granzyme B. Evaluation of HLA-G expression on tumor cell lines upon IFNγ stimulation in vitro and in PDX (patient-derived xenograft) tumors treated with HLA-G TCBs revealed that HLA-G expression can be upregulated upon IFNγ stimulation and TCB treatment, respectively, and may positively influence the antitumor activity of HLA-G-TCBs in vivo.

[0006] T cell exhaustion can pose a challenge when redirected T cells are unable to effectively recognize and destroy target tumor cells, reducing the effectiveness of T cell-targeted immunotherapy. Exhausted T cells are characterized by persistent expression of the inhibitory molecule PD1 (programmed cell death protein 1), and it has been found that blocking the PD1-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 result in a reversal of T cell exhaustion, possibly due to resistance mechanisms, the immunosuppressive activity of MDSCs, and / or regulatory T cells.

[0007] Lymphocyte-activating gene-3 (LAG3 or CD223) was first discovered in experiments designed to selectively isolate molecules expressed in IL-2-dependent NK cell lines (Triebel F et al., Cancer Lett. 235 (2006), 147-153). LAG3 is a unique transmembrane protein with structural homology to CD4 and four extracellular immunoglobulin superfamily-like domains (D1-D4). The distal IgG domain of this membrane 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) required for LAG3 to negatively affect T cell function. LAG3 can be cleaved at the connective peptide (CP) by metalloproteinases to produce a soluble form, which is detectable in serum. Similar to 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 (pDCs) and is upregulated after T cell activation. LAG3 regulates T cell function and T cell homeostasis. A subset of conventional T cells that are anerious or dysfunctional express LAG3. +T cells are enriched in 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 can antagonist LAG3 activity and 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 an effective antitumor immune response and provide survival benefits to more cancer patients than currently available checkpoint inhibitors. By preferentially targeting PD1 / LAG3 co-expressing dysfunctional T cells and potentially reducing the targeting of LAG3-expressing Tregs in the tumor microenvironment, PD1-LAG3 BsAbs may be able to restore an antitumor immune response while avoiding the reactivation of Treg-mediated immunosuppressive effects.

[0009] To overcome effects caused by a lack of co-stimulation during T-cell bispecific antibody-mediated cancer immunotherapy, such as poor sustained T-cell response, novel combination therapies using two or more bispecific antibodies are needed to improve immunotherapy through additive stimulating effects that cannot be achieved with a single molecule. [Overview of the project]

[0010] Summary of the Invention This invention relates to a combination therapy using 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). The anti-PD1 / anti-LAG3 bispecific antibodies described herein have been found to offer better selectivity and efficacy, and are therefore more advantageous than anti-PD1 antibodies, particularly when combined with anti-HLA-G / anti-CD3 bispecific antibodies. These anti-PD1 / anti-LAG3 bispecific antibodies are further characterized by exhibiting reduced sync effect (indicated by reduced internalization by T cells), and these bispecific antibodies preferentially bind to conventional T cells rather than to Tregs, protecting T cell effector function from Treg suppression, resulting in increased tumor-specific T cell effector function and increased in vivo tumor eradication. Based on these properties, they are advantageous for use in combination with T cell bispecific antibodies, particularly anti-HLA-G / anti-CD3 bispecific antibodies.

[0011] This specification describes an anti-HLA-G / anti-CD3 bispecific antibody for use in methods of treating cancer, particularly HLA-G expressing cancer, which is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody.

[0012] The present invention relates to an anti-HLA-G / anti-CD3 bispecific antibody for use in the methods defined above herein, 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 is VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 3, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 6 This invention provides an anti-HLA-G / anti-CD3 bispecific antibody, which includes [specific antibody name].

[0013] In one embodiment, an anti-HLA-G / anti-CD3 bispecific antibody is provided for use in a method of treating cancer, particularly HLA-G expressing cancer, 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.

[0014] Furthermore, the present invention provides an anti-HLA-G / anti-CD3 bispecific antibody for use in methods of treating cancer, particularly 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, and the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain which is an IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor. More specifically, the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A and P329G (numbered by the Kabat EU index).

[0015] In one embodiment, an anti-HLA-G / anti-CD3 bispecific antibody for use in the method described above herein, 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 (a) A VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) containing a VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 16; or (b) VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 21, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 A bispecific anti-HLA-G / anti-CD3 antibody is provided, which includes the above.

[0016] In another embodiment, an anti-HLA-G / anti-CD3 bispecific antibody for use in the methods disclosed herein is provided, 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.

[0017] In a further embodiment, 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, and the second antigen-binding domain (a) A VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, or (b) VH domain containing the amino acid sequence of SEQ ID NO: 25 and VL domain containing the amino acid sequence of SEQ ID NO: 26 A bispecific anti-HLA-G / anti-CD3 antibody is provided, which includes the above.

[0018] In an additional embodiment, an anti-HLA-G / anti-CD3 bispecific antibody for use in the method described herein, 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 (a) A VH domain containing the amino acid sequence of SEQ ID NO: 27 and a VL domain containing the amino acid sequence of SEQ ID NO: 28, or (b) A VH domain containing the amino acid sequence of SEQ ID NO: 29 and a VL domain containing the amino acid sequence of SEQ ID NO: 30, or (c) A VH domain containing the amino acid sequence of SEQ ID NO: 31 and a VL domain containing the amino acid sequence of SEQ ID NO: 32, or (d) A VH domain containing the amino acid sequence of SEQ ID NO: 33 and a VL domain containing the amino acid sequence of SEQ ID NO: 34, or (e) A bispecific anti-HLA-G / anti-CD3 antibody is provided, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 64 and a VL domain containing the amino acid sequence of SEQ ID NO: 65.

[0019] Furthermore, an anti-HLA-G / anti-CD3 bispecific antibody for use in the methods disclosed herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody is A first antigen-binding domain that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, An anti-HLA-G / anti-CD3 bispecific antibody is provided, comprising a second antigen-binding domain that specifically binds to LAG3, including a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18.

[0020] In a further embodiment, an anti-HLA-G / anti-CD3 bispecific antibody is provided for use in methods of treating cancer, particularly HLA-G expressing cancer, 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 embodiment, the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1, and the variable domains VL and VH are substituted for each other such that VL is part of the heavy chain and VH is part of the light chain.

[0021] In another embodiment, an anti-HLA-G / anti-CD3 bispecific antibody is provided for use in the methods disclosed herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a monovalent conjugation to PD-1 and a monovalent conjugation to LAG3.

[0022] In a further embodiment, an anti-HLA-G / anti-CD3 bispecific antibody for use in the methods disclosed above herein 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. Furthermore, an anti-PD1 / anti-LAG3 bispecific antibody is provided herein, wherein the anti-PD1 / anti-LAG3 bispecific antibody described above comprises an Fc domain comprising a modification that facilitates the association of the first and second subunits of the Fc domain. In one embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided, wherein, according to the knob-into-hole method, the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole. In particular, the first subunit of the Fc domain contains amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S and Y407V (Kabat EU index numbering).

[0023] In a particular embodiment, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, particularly HLA-G expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody is (a) A first heavy chain containing the amino acid sequence of SEQ ID NO: 35, a first light chain containing the amino acid sequence of SEQ ID NO: 36, a second heavy chain containing the amino acid sequence of SEQ ID NO: 37, and a second light chain containing the amino acid sequence of SEQ ID NO: 38, or (b) An anti-HLA-G / anti-CD3 bispecific antibody is provided, comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 35, a first light chain containing the amino acid sequence of SEQ ID NO: 36, a second heavy chain containing the amino acid sequence of SEQ ID NO: 39, and a second light chain containing the amino acid sequence of SEQ ID NO: 40.

[0024] More specifically, the anti-PD1 / anti-LAG3 bispecific antibody comprises a first heavy chain containing the amino acid sequence of SEQ ID NO: 35, a first light chain containing the amino acid sequence of SEQ ID NO: 36, a second heavy chain containing the amino acid sequence of SEQ ID NO: 37, and a second light chain containing the amino acid sequence of SEQ ID NO: 38.

[0025] Furthermore, an anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, particularly HLA-G-expressing cancer, the anti-HLA-G / anti-CD3 bispecific antibody being for use in combination with an anti-PD1 / anti-LAG3 bispecific antibody, the anti-HLA-G / anti-CD3 bispecific antibody comprising a first antigen-binding domain that specifically binds to CD3 and comprising a heavy-chain variable region (V H CD3) and a light-chain variable region (V L CD3), and a second antigen-binding domain that specifically binds to HLA-G and comprising a heavy-chain variable region (V H HLA-G) and a light-chain variable region (V L HLA-G), is provided.

[0026] 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 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-L@ sequence of SEQ ID NO: 46.

[0027] More specifically, the anti-HLA-G / anti-CD3 bispecific antibody comprises a first antigen-binding domain that specifically binds to CD3 and comprising a heavy-chain variable region (V H CD@) 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.

[0028] In one aspect, the anti-HLA-G / anti-CD3 bispecific antibody for use in a method of treating cancer, particularly HLA-G-expressing cancer, comprises a second antigen-binding domain that specifically binds to HLA-G, the second antigen-binding domain comprising The heavy chain variable region (V) includes the CDR-H1 sequence of sequence number 49, the CDR-H2 sequence of sequence number 50, and the CDR-H3 sequence of sequence number 51. H HLA-G); and / or The light chain variable region (V) includes the CDR-L1 sequence of sequence number 52, the CDR-L2 sequence of sequence number 53, and the CDR-L3 sequence of sequence number 54. L Includes HLA-G.

[0029] In particular, the second antigen-binding domain is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 55. H HLA-G), and / or light chain variable region containing the amino acid sequence of SEQ ID NO: 56 (V L The antibody includes HLA-G. In a further embodiment, an anti-HLA-G / anti-CD3 bispecific antibody for use in methods treating cancer, particularly HLA-G expressing cancer, includes a third antigen-binding domain that binds to HLA-G. In another embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes an Fc domain comprising one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0030] In one embodiment, a method is provided for producing an anti-HLA-G / anti-CD3 bispecific antibody comprising a first Fab fragment that specifically binds to CD3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 47 and a VL domain containing the amino acid sequence of SEQ ID NO: 48, and a second and optionally third Fab fragment that specifically binds to HLA-G, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 55 and a VL domain containing the amino acid sequence of SEQ ID NO: 56.

[0031] In a further embodiment, an anti-HLA-G / anti-CD3 bispecific antibody is provided for use in a method of treating cancer, particularly 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, the combination of which is administered at intervals of 1 to 3 weeks.

[0032] In one further embodiment, a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in the treatment of cancer, particularly HLA-G expressing cancer, wherein the treatment comprises administration of the composition comprising the 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 is a VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 3, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) A composition is provided comprising a VL domain, which comprises CDR-L3 containing the amino acid sequence of SEQ ID NO: 6.

[0033] In one embodiment, 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.

[0034] In one further embodiment, the composition comprises an anti-PD1 / anti-LAG3 bispecific antibody having a second antigen-binding domain that specifically binds to LAG3, (a) A VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) containing a VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 16; or (b) VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 21, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 Includes.

[0035] In one embodiment, the composition is an anti-PD1 / anti-LAG3 bispecific antibody comprising an antigen-binding domain that specifically binds to LAG3, (a) A VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, or (b) VH domain containing the amino acid sequence of SEQ ID NO: 25 and VL domain containing the amino acid sequence of SEQ ID NO: 26 It contains anti-PD1 / anti-LAG3 bispecific antibodies.

[0036] In one particular embodiment, the composition is an anti-PD1 / anti-LAG3 bispecific antibody, A first Fab fragment that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, A second Fab fragment that specifically binds to LAG3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, and It contains anti-PD1 / anti-LAG3 bispecific antibodies.

[0037] Furthermore, a composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in the treatment of cancer, particularly HLA-G expressing cancer, wherein the treatment comprises the administration of the composition comprising an anti-PD1 / anti-LAG3 bispecific antibody in combination with the composition comprising an anti-HLA-G / anti-CD3 bispecific antibody, wherein the anti-HLA-G / anti-CD3 bispecific antibody has a heavy chain variable region (V H CD3) and light chain variable region (V L The first antigen-binding domain includes CD3, and the heavy chain variable region (V H HLA-G) and light chain variable region (V L A composition is provided that includes a second antigen-binding domain containing HLA-G.

[0038] In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody has a heavy chain variable region (V) containing 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. H CD3), and / or the light chain variable region (V) including 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. L It contains a first antigen-binding domain including CD3. More specifically, anti-HLA-G / anti-CD3 bispecific antibodies contain a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 47. H CD3), and / or the light chain variable region containing the amino acid sequence of SEQ ID NO: 48 (V L It includes a first antigen-binding domain containing CD3. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody has a heavy chain variable region (V) containing 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. H HLA-G), and / or the light chain variable region (V) including 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. LIt contains a second antigen-binding domain including HLA-G. In particular, the second antigen-binding domain contains a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 55. H HLA-G), and / or light chain variable region containing the amino acid sequence of SEQ ID NO: 56 (V L The antibody includes HLA-G. In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes a third antigen-binding domain that binds to HLA-G. In particular, the first antigen-binding domain that specifically binds to CD3 includes a VH domain containing the amino acid sequence of SEQ ID NO: 47 and a VL domain containing the amino acid sequence of SEQ ID NO: 48, and the second (and optionally third) antigen-binding domain that specifically binds to HLA-G includes a VH domain containing the amino acid sequence of SEQ ID NO: 55 and a VL domain containing the amino acid sequence of SEQ ID NO: 56. In another embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes an Fc domain containing one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0039] In a further aspect, a pharmaceutical product, (A) A first composition comprising an anti-HLA-G / anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier as active ingredients; and (B) A second composition comprising an anti-PD1 / anti-LAG3 bispecific antibody and a pharmaceutically acceptable carrier as active ingredients, Pharmaceutical products are provided for use in combination, sequential, or concurrent treatment of diseases, particularly cancer, especially HLA-G expressing cancers.

[0040] In another embodiment, a combination of anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 bispecific antibodies is used to treat diseases, particularly cancers, particularly HLA-G expressing cancers, particularly lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer. Pharmaceutical compositions are provided for use in combination, sequential, or simultaneous treatment of one or more of the following cancers: cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, nasopharyngeal carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumor, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia (including refractory versions of any of the above cancers), or one or more of the above cancers. In particular, the pharmaceutical compositions are intended for use in the treatment of solid tumors, including, but not limited to, renal cell carcinoma, colorectal carcinoma, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0041] In another embodiment, the use of a combination of an anti-HLA-G / CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody in the manufacture of a pharmaceutical for treating or delaying the progression of proliferative disorders, particularly 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 is a VH domain. (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 3, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 6 Uses including the following are provided.

[0042] In one further embodiment, the anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) A VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) containing a VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 16; or (b) VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 21, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 Includes.

[0043] In another embodiment, the 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 pharmaceutical for treating or delaying the progression of proliferative disorders, particularly cancer, particularly HLA-G expressing cancer, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first Fab fragment 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 Fab fragment 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.

[0044] In a further embodiment, a method for treating cancer, particularly HLA-G expressing cancer, comprising administering an effective amount of anti-HLA-G / anti-CD3 antibody and an effective amount of anti-PD1 / anti-LAG3 bispecific antibody to the subject, 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 VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 3, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) A method is provided which includes a VL domain comprising CDR-L3 containing the amino acid sequence of SEQ ID NO: 6.

[0045] In one embodiment, the anti-PD1 / anti-LAG3 bispecific antibody includes a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) A VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) containing a VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 16; or (b) VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 21, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 A method is provided that includes this.

[0046] In another embodiment, a method for treating cancer is provided, comprising an anti-PD1 / anti-LAG3 bispecific antibody comprising a first Fab fragment 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 Fab fragment 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.

[0047] In one embodiment, an anti-HLA-G / anti-CD3 bispecific antibody has a heavy chain variable region (V H CD3) and light chain variable region (V L A first antigen-binding domain that specifically binds to CD3 (including CD3), and a heavy chain variable region (V H HLA-G) and light chain variable region (V L A method for treating cancer is provided, comprising a second antigen-binding domain that specifically binds to HLA-G, including HLA-G. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) including 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. H CD3), and / or the light chain variable region (V) including 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. L It contains a first antigen-binding domain including CD3. More specifically, anti-HLA-G / anti-CD3 bispecific antibodies contain a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 47. H CD3), and / or the light chain variable region containing the amino acid sequence of SEQ ID NO: 48 (V L It includes a first antigen-binding domain containing CD3. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody has a heavy chain variable region (V) containing 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. H HLA-G), and / or the light chain variable region (V) including 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. L It contains a second antigen-binding domain including HLA-G. In particular, the second antigen-binding domain contains a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 55. H HLA-G), and / or light chain variable region containing the amino acid sequence of SEQ ID NO: 56 (V LThe antibody includes HLA-G. In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes a third antigen-binding domain that binds to HLA-G. In particular, the first antigen-binding domain that specifically binds to CD3 includes a VH domain containing the amino acid sequence of SEQ ID NO: 47 and a VL domain containing the amino acid sequence of SEQ ID NO: 48, and the second (and optionally third) antigen-binding domain that specifically binds to HLA-G includes a VH domain containing the amino acid sequence of SEQ ID NO: 55 and a VL domain containing the amino acid sequence of SEQ ID NO: 56. In another embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes an Fc domain containing one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0048] In one embodiment, 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. In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously. In another embodiment, the anti-HLA-G / anti-CD3 bispecific antibody is administered simultaneously with, before, or after, the anti-PD1 / anti-LAG3 bispecific antibody.

[0049] In any of the above embodiments, the subject is preferably a mammal, particularly a human. [Brief explanation of the drawing]

[0050] [Figure 1A-1B]Schematic diagrams of specific anti-HLA-G / anti-CD3 bispecific antibodies used in the examples (Figure 1A) and specific anti-PD1 / anti-LAG3 bispecific antibodies (Figure 1B). These molecules are described in more detail in Example 1. Figure 1A shows an exemplary bispecific anti-HLA-G / anti-CD3 antibody in 2+1 format (referred to as HLA-G TCB). The molecule contains a single antigen-binding moiety for CD3, two antigen-binding moieties for HLA-G, and an Fc domain. Figure 1B shows an anti-PD1 / anti-LAG3 bispecific antibody in 1+1 CrossMab format, where the PD1-binding domain contains crossFab (VH / VL domain exchange) and the LAG3-binding domain contains CH1 and CK domains with amino acid mutations to assist in correct pairing ("charged variant"). The Fc portions of both antibodies shown here contain knob-in-hole mutations (indicated by black circles) as well as amino acid mutations L234A, L235A, and P329G, which nearly completely eliminate Fcγ receptor binding of the human IgG1 Fc domain. [Figure 2]This protocol describes the in vivo efficacy study #1173 of HLA-G TCB (P1AD7977) as monotherapy against PD-L1 antibody alone and in combination with PD1-LAG3 bispecific antibody (P1AA0927) or PD-L1 antibody (P1AE0828) in fully humanized NSG mice carrying BC004 PDX (patient-derived xenograft). Humanized NSG mice were injected with 2 × 10⁶ BC004 cells in a total volume of 20 μL of PBS into the mammary fat pad (imfp). After the tumors reached an average volume of approximately 200 mm³, mice were randomized to one of five groups receiving the following: A) histidine buffer (vehicle) as a control; B) HLA-G-TCB (0.5 mg / kg intravenously once weekly); C) anti-PD-L1 antibody (10 mg / kg intravenously once weekly); D) HLA-G-TCB (0.5 mg / kg intravenously once weekly) + anti-PD-L1 antibody (10 mg / kg intravenously once weekly); E) HLA-G-TCB (0.5 mg / kg intravenously once weekly) + PD1-LAG3 bispecific Ab (3 mg / kg intravenously once weekly). The table below the protocol defines the subgroups of mice that received different treatment combinations. The experiment is described in Example 2. [Figure 3] The results of Study #1173 are presented. Tumor volume was measured twice weekly using a caliper. Figure 3A shows a direct comparison of results for all subgroups over the period from day 34 to day 65. Data are presented as tumor volume (median + / - IQR). Figure 3B shows the measured tumor volume (mm3) for each individual animal over the period from day 34 to day 65, demonstrating the homogeneity of the antitumor response across various subgroups. [Figure 4]This document presents the protocol for in vivo efficacy study #1655 of HLAG TCB (P1AD7977) in fully humanized NSG mice carrying BC004 PDX (patient-derived xenograft) at two different concentrations (1.5 mg / kg or 3 mg / kg) as monotherapy and in combination with a PD1-LAG3 bispecific antibody (P1AA0927), or in combination with a PD1 antibody (P1AA6975) + anti-LAG3 antibody (P1AD8676). Humanized NSG mice were injected with 2 × 10⁶ BC004 cells in a total volume of 20 μL of PBS into the mammary fat pad (imfp). After the tumors reached an average volume of approximately 200 mm³, mice were randomized to one of five groups receiving the following: A) histidine buffer (vehicle) as a control; B) HLA-G-TCB (0.5 mg / kg intravenously once weekly); C) HLA-G-TCB (0.5 mg / kg intravenously once weekly) and anti-PD1-LAG3 bispecific antibody (3 mg / kg intravenously once weekly); D) HLA-G-TCB (0.5 mg / kg intravenously once weekly) and anti-PD1-LAG3 bispecific antibody (1.5 mg / kg intravenously once weekly); E) HLA-G-TCB (0.5 mg / kg intravenously once weekly) + anti-PD1 Ab (1.5 mg / kg intravenously once weekly) + anti-LAG3 antibody (1.5 mg / kg intravenously once weekly). The table below the protocol defines the subgroups of mice that received different combinations of treatment. The experiment is described in Example 2. [Figure 5] The results of Study #1655 are presented. Tumor volume was measured twice weekly using a caliper. Figure 5A shows a direct comparison of results for all subgroups over the period from day 35 to day 60. Data are presented as tumor volume (median + / - IQR). Figure 5B shows the measured tumor volume (mm3) for each individual animal over the period from day 35 to day 60, demonstrating the homogeneity of the antitumor response across various subgroups. [Figure 6]The results of the immunopharmacodynamic analysis performed in study #1655 are shown. For this purpose, single-cell suspensions obtained by collecting and processing tumors and staining them with a cocktail of fluorescently labeled antibodies to detect immune cell markers were subjected to flow cytometry analysis after the end of treatment days 17 and 32. Figure 6A shows the level of immune cell infiltration (indicated by the number of CD45+ cells) for different treatment groups on days 17 and 32. Figure 6B shows the level of T cell infiltration (indicated by the number of CD3+ cells) and T cell activation (indicated by granzyme B expression as a percentage of CD8+ T cells) for different treatment groups on days 17 and 32. [Modes for carrying out the invention]

[0051] Detailed description of the invention definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly used in the art to which this invention pertains. In interpreting this specification, the following definitions apply, wherever singular terms are used, including their plural forms and vice versa.

[0052] The term "antibody" as used herein is used in its broadest sense and encompasses, but is not limited to, a wide range of antibody structures, including monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0053] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous collection of antibodies, i.e., the individual antibodies in the collection are identical and / or bind to the same epitope, except for possible variant antibodies, such as naturally occurring mutations or mutations that occur during the production of monoclonal antibody preparations, where such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on an antigen.

[0054] As used herein, the term “monospecific” antibody means an antibody having one or more binding sites, each binding to the same epitope of the same antigen. The term “bispecific” means that an antibody can specifically bind to two binding sites, each formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), which bind to at least two distinct antigenic determinants, for example, to different antigens or different epitopes on the same antigen. Such a bispecific antibody is in a 1+1 format. Other bispecific antibody formats include a 2+1 format (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, a bispecific antibody contains two antigen-binding sites, each of which is specific to a different antigenic determinant.

[0055] As used in this application, the term "valency" refers to the presence of a specific number of binding domains within an antigen-binding molecule. In this case, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding domains, respectively, within the antigen-binding molecule. The bispecific antibodies of the present invention are at least "bivalent" and may be "trivalent" or "polyvalent" (e.g., "tetravalent" or "hexavalent"). In certain embodiments, the antibodies of the present invention are bispecific and have two or more binding sites. That is, even if there are more than two binding sites (i.e., the antibody is trivalent or polyvalent), the antibody may be bispecific.

[0056] In this specification, the terms “full-length antibody,” “complete antibody,” and “whole antibody” are used interchangeably to refer to antibodies having a structure substantially similar to that of a native antibody. “Native antibody” refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, a native IgG class antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, CH3), also called heavy chain constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a light chain constant domain (CL), also called light chain constant domains. Antibody heavy chains are classified into one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), and μ(IgM), some of which may be further classified into subtypes such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). Antibody light chains can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domain.

[0057] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an 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), multispecific antibodies made from antibody fragments, and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For an overview of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994). See also International Publication No. 93 / 16185 and U.S. Patents No. 5,571,894 and 5,587,458. For Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues with increased in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments containing two antigen-binding domains, which may be bivalent or bispecific; see, for example, European Patent No. 404,097; International Publication No. 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). A single-domain antibody is an antibody fragment that contains all or part of the heavy chain variable domains or all or part of the light chain variable domains of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).Furthermore, the antibody fragments are composed of single-chain polypeptides characterized by the ability of the VH domain to associate with the VL domain, or by the ability of the VL domain to associate with the VH domain and bind to a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody. The antibody fragments can be produced by a variety of techniques, including but not limited to production by recombinant host cells (e.g., E. coli and phages) as described herein, as well as proteolytic digestion of complete antibodies.

[0058] When a complete antibody is digested with papain, two identical antigen-binding fragments called "Fab" fragments are obtained, each containing the variable domains of the heavy and light chains, the constant domain of the light chain, and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to a light chain fragment containing the VL domain and constant domain of the light chain (CL), and an antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues containing one or more cysteines from the antibody hinge region added to the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH is a Fab' fragment in which the cysteine ​​residue of the constant domain has a free thiol group. Pepsin treatment yields an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0059] The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which either the variable or constant regions of the heavy and light chains are exchanged. Two possible chain compositions of the crossover Fab molecule are possible and are included in the bispecific antibodies of the present invention. In one case, the variable regions of the Fab heavy and light chains are exchanged, i.e., the crossover Fab molecule includes a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This crossover Fab molecule is called CrossFab. (VLVH)It is also called [another name]. On the other hand, when the constant regions of the Fab heavy chain and light chain are exchanged, the crossover Fab molecule includes a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1). This crossover Fab molecule is called CrossFab. (CLCH1) It is also called [another name].

[0060] A "single-stranded Fab fragment" or "scFab" is a polypeptide comprising 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 domain and the linker have one of the following sequences from the N-terminus to the C-terminus: (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 the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-stranded Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules will be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (for example, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0061] A "crossover single-strand Fab fragment" or "x-scFab" is a polypeptide comprising 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 domain and the linker have one of the following sequences in the N-terminus to C-terminus direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL. Here, VH and VL together form an antigen-binding domain that specifically binds to a given antigen, and the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules will be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0062] A "single-chain variable fragment (scFv)" is a linkage of the antibody's heavy chain (V) using a short linker peptide of 10 to approximately 25 amino acids. H ) and light chain (V L It is a fusion protein of the variable region of ). The linker usually contains a lot of glycine to improve flexibility and serine or threonine to improve solubility. H The N-terminus and V L It is possible to link the C-terminuses, and vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Furthermore, the antibody fragment is composed of a single-chain polypeptide characterized by the ability to associate with the VH domain, i.e., with the VL domain, or to associate with the VL domain, i.e., with the VH domain and bind to a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0063] "Scaffold antigen-binding proteins" are known in the art, and for example, fibronectin and engineered ankyrin repeat proteins (DARPin) 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 present invention, the scaffold antigen-binding protein is CTLA-4 (epibody), lipocalin (anticalin), protein A-derived molecules, e.g., the Z-domain (afibody), A-domain (avimer / maxibody) of protein A, serum transferrin (transbody); designed ankyrin repeat protein (DARPin), variable domain of antibody light or heavy chain (single-domain antibody, sdAb), variable domain of antibody heavy chain (nanobody, aVH), V NAR Fragment, fibronectin (adonectin), C-type lectin domain (tetranectin); variable domain (V) of novel antigen receptor beta-lactamase NARThe group consists of fragments, human gamma-crystallin or ubiquitin (affilin molecules); Knitz-type domains of human protease inhibitors; microbodies, e.g., Notchin family proteins, peptide aptamers, and fibronectin (adnectin). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a CD28 family receptor primarily expressed on CD4+ T cells. Its extracellular domain has a variable domain-like Ig folding. The loop corresponding to the CDR of the antibody may be replaced with heterologous sequences to give different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as organisms (e.g., U.S. Patent No. 7166697B1). Organisms are approximately the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipokalin is a family of extracellular proteins that carry small hydrophobic molecules such as steroids, bilines, retinoids, and lipids. Lipokalin has a rigid beta-sheet secondary structure with many loops at the open end of a conical structure, which can be manipulated to bind to different target antigens. Antikarin is 160-180 amino acids in size and is derived from lipokalin. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250,297, B1 and U.S. Patent Application Publication No. 2007,022,4633. Affibodies are scaffolds derived from protein A of Staphylococcus aureus that can be manipulated to bind to antigens. The domain consists of three helical bundles of approximately 58 amino acids. Libraries are generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and European Patent No. 1641818A1. Abimer is a multi-domain protein derived from the A-domain scaffold family. The native domain, approximately 35 amino acids long, adopts a defined disulfide-bonded structure.Diversity is generated by the shuffling of innate variations exhibited by the 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). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be manipulated to bind to different target antigens by inserting peptide sequences into a permissible surface loop. An example of a manipulated transferrin scaffold is a transbody. For further details, see J. Biol. Chem 274, 24066-24073 (1999). Engineered ankyrin repeat proteins (DARPin) are derived from ankyrin, a family of proteins that mediate the adhesion of intrinsic membrane proteins of the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha helices and a beta turn. A single ankyrin repeat can be manipulated to bind to different target antigens by randomizing the residues in the first alpha helix and beta turn of each repeat. Its binding interface can be increased by increasing the number of modules (affinity maturation method). 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 U.S. Patent Application Publication No. 20040132028A1.

[0064] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of a camel-derived antibody heavy chain (nanobody or V). H (H fragment). Furthermore, the term single-domain antibody refers to autonomous human heavy chain variable domains (aVH) or shark-derived V. NARThis includes fragments. Fibronectin is a scaffold that can be manipulated to bind to antigens. Adnectin consists of a scaffold with the native amino acid sequence of the 10th domain of 15 repeat units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be manipulated so that adnectin can specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent No. 20080139791, International Publication No. 2005056764, and U.S. Patent No. 6818418B1. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). The microbodies are derived from naturally occurring microproteins with a length of 25–50 amino acids and containing 3–4 cysteine ​​crosslinks, examples of which include KalataBI, conotoxin, and Nottin. The microproteins have loops that can be manipulated to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on the manipulated Nottin domain, see International Publication No. 2008098796.

[0065] An antigen-binding molecule that binds to the same epitope as the reference molecule refers to an antigen-binding molecule that blocks 50% or more of the binding of the reference molecule to that antigen in a competitive assay, and conversely, the reference molecule blocks 50% or more of the binding of the antigen-binding molecule to that antigen in a competitive assay.

[0066] As used herein, the terms “antigen-binding domain” or “antigen-binding site” refer to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term “antigen-binding domain” refers to a portion of an antibody that specifically binds to some or all of a given antigen and is complementary to some or all of that antigen. If the antigen is large, the antigen-binding molecule may bind to only a specific portion of the antigen, which is called an epitope. An antigen-binding domain may be provided, for example, by one or more variable domains (also called variable regions). In particular, an antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one embodiment, an antigen-binding domain may bind to its antigen and block or partially block its function. Antigen-binding domains that specifically bind to PD1 and LAG3 include antibodies and fragments thereof, as further defined herein. In addition, an antigen-binding domain may include a binding domain based on a scaffold antigen-binding protein, e.g., a designed repeat protein or a designed repeat domain (see, for example, International Publication 2002 / 020565).

[0067] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope” and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-antigen complex (e.g., a contiguous extension of amino acids or a conformational structure composed of discontinuous amino acids from different regions). Useful antigenic determinants can be found, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, in serum, and / or in the extracellular matrix (ECM). Proteins useful as antigens herein may be any native form of protein derived from vertebrates, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. In certain embodiments, the antigen is a human protein. When referring to a particular protein of the present invention, the term encompasses “full-length” untreated proteins and any form of protein obtained from cell treatment. The term also encompasses naturally occurring protein variants, such as splice variants or allele variants.

[0068] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesirable or nonspecific 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) techniques (analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of an antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen as measured, for example, by SPR. In certain embodiments, the molecule that binds to the antigen 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 less, for example, 10 -7 M~10 -13 M, for example 10-9 M~10 -13 The dissociation constant (K) of M D ) has.

[0069] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding pair (e.g., an antigen). Unless otherwise stated herein, "binding affinity" refers to the endogenous binding affinity that reflects the 1:1 interaction between members of the binding pair (e.g., antibody and antigen). The affinity of molecule X for its binding pair Y is typically expressed by the dissociation constant (K). D It can be expressed by the dissociation rate constant and the binding rate constant (k, respectively). off and k on This is the ratio of the rate constants. Therefore, as long as the ratio of the rate constants remains the same, equivalent affinity can include different rate constants. Affinity can be measured by methods common in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0070] As used herein, the term “high affinity” of an antibody refers to its affinity for a target antigen. D 10 -9 M or less, and more specifically 10 -10 This refers to antibodies with an affinity of M or less. "Low affinity" antibodies are K D 10 -8 This refers to the antibodies mentioned above.

[0071] An "affinity-mature" antibody is an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to an unmodified parent antibody, and such modifications result in an improved affinity of the antibody for the antigen.

[0072] As used herein, the terms “HLA-G,” “Human HLA-G,” and “HLAG” refer to the 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 an MHC class I complex with β2-microglobulin (B2M or β2m). In one embodiment, HLA-G refers to the MHC class I complex of HLA-G and β2-microglobulin. In a preferred embodiment, HLA-G refers to the cell surface-bound MHC class I complex of HLA-G and β2-microglobulin, also known as HLA-G1 (see, for example, Figure 1 in International Publication No. 2022 / 129120A1, and Blaschitz et al., Molecular Human Reproduction, 11(2005)699-710, in particular Figure 1).

[0073] As used herein, a “human HLA-G binding”, “(human) HLA-G specific binding”, “(human) HLA-G binding”, or “anti-HLA-G” antibody (single-specific, multi-specific, or bi-specific) or antigen-binding domain is 5.0 × 10⁻¹⁴ -8 K below mol / L D In one embodiment, the K value is 1.0 x 10⁹ mol / l or less. D Value, in one embodiment, 5.0x10 -8 mol / l ~ 1.0 x 10 -13 K in mol / l D The binding affinity value refers to the antibody / antigen binding portion that specifically binds to the (human) HLA-G antigen or its extracellular domain (ECD). In one embodiment, the antibody binds to the HLA-G β2M MHC I complex containing SEQ ID NO: 106.

[0074] Binding affinity is determined by a standard binding assay, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), using a construct containing, for example, the extracellular domain of HLA-G (e.g., its naturally occurring three-dimensional structure). In one embodiment, binding affinity is determined by a standard binding assay using an exemplary soluble HLA-G containing an MHC class I complex including SEQ ID NO: 106.

[0075] HLA-G has a typical MHC I fold and consists of two chains. Chain 1 consists of three domains: alpha-1, alpha-2, and alpha-3. The alpha-1 and alpha-2 domains have two alpha helices that form adjacent peptide binding grooves. Small peptides (approximately 9mer) can bind to these grooves, as with other MHC proteins. Chain 2 is beta-2 microglobulin (β2M), which is shared with various other MHC proteins.

[0076] HLA-G molecules can form functionally active complex oligomeric structures (Kuroki, K et al. Eur J Immunol. 37(2007) 1727-1729). Disulfide-bonded dimers are formed between the Cys42 groups of two HLA-G molecules (Shiroishi M et al., J Biol Chem 281(2006) 10439-10447). Trimeric and tetrameric complexes are, for example, Kuroki, K et al. Eur J Immunol. 37(2007) 1727-1729, Allan DS, et al. J Immunol Methods. 268(2002) 43-50, and T Gonen-Gross et al., J Immunol 171(2003) 1343-1351). Unlike most other MHC class I molecules, HLA-G possesses several free cysteine ​​residues. Boyson et al., Proc Nat Acad Sci USA, 99:16180 (2002) reported that recombinant soluble HLA-G5 can form a disulfide dimer with an intermolecular Cys42-Cys42 disulfide bond. Furthermore, the membrane-bound form of HLA-G1 can also form a disulfide dimer on the cell surface of JEG3 cell lines that endogenously express HLA-G. Disulfide dimer 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)).

[0077] HLA-G is primarily expressed on the trophoblast cell layer of the placenta. Several tumors (including those of the pancreas, breast, skin, colorectal, stomach, and ovary) express HLA-G (Lin, A. et al., Mol Med. 21 (2015) 782-791; Amiot, L., et al., Cell Mol Life Sci. 68 (2011) 417-431). Expression has also been reported to be associated with pathological conditions such as inflammatory diseases, GvHD, and cancer. HLA-G expression has been reported to be associated with poor prognosis in cancer. Tumor cells evade host immune surveillance by inducing immune tolerance / suppression via HLA-G expression.

[0078] In the case of HLA-G, there are seven isoforms, three of which are secretory and four are membrane-bound. The most important functional isoforms of HLA-G include beta-2-microglobulin (β2M)-related HLA-G1 and HLA-G5. However, the immunotolerogenic effects of these isoforms differ and depend on the ligand morphology (monomer, dimer) and the affinity of the ligand-receptor interaction.

[0079] HLA-G proteins can be produced using standard molecular biology techniques. The nucleic acid sequences of HLA-G isoforms are publicly known in the art. See, for example, GenBank accession number AY359818.

[0080] HLA-G isomerism promotes signal transduction via ILTs (Ig-like transcripts), particularly ILT2, ILT4, or combinations thereof.

[0081] ILTs are involved in regulating immune cell activation and represent the Ig type of activating and inhibitory receptors that control the function of immune cells (Borges, L., et al., Curr Top Microbial Immunol, 244:123-136 (1999)). ILTs are classified into three groups: (i) inhibitory, i.e., those containing a cytoplasmic immunoreceptor tyrosine-dependent inhibitory motif (ITIM) and transmitting inhibitory signals (ILT2, ILT3, ILT4, ILT5, and LIR8); (ii) activating, i.e., those containing a short cytoplasmic tail and charged amino acid residues in a transmembrane domain (ILT1, ILT7, ILT8, and LIR6 alpha) and delivering activation signals via the cytoplasmic immunoreceptor tyrosine-dependent activation motif (ITAM) of the associated common gamma chain of the Fc receptor; and (iii) soluble molecules lacking a transmembrane domain, such as ILT6. Several recent studies have highlighted the immunomodulatory role of ILTs on the surface of antigen-presenting cells (APCs). The most characterized immunosuppressive receptors, ILT2, ILT3, and ILT4 receptors, are expressed on a broad range of immune cells, including monocytes, B cells, dendritic cells, plasmacytoid dendritic cells, and subsets of NK cells and T cells. ILT2 is expressed on a subset of T cells and has been shown to inhibit the activation and proliferation of these cells during 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, CC, et al., Nat Immunol, 3:237-243 (2002)). ILT expression on DCs is strictly regulated by inflammatory stimuli, cytokines, and growth factors, and is downregulated after DC activation (Ju, XS, et al., Gene, 331:159-164 (2004)).The expression of ILT2 and ILT4 receptors is highly regulated by histone acetylation and contributes to strictly controlled gene expression only in the myeloid cells (Nakajima, H., J Immunol, 171:6611-6620 (2003)).

[0082] The involvement of inhibitory receptors ILT2 and ILT4 can alter the cytokine and chemokine secretion / release profiles of monocytes and inhibit Fc receptor signaling (Colonna, M., et al. J Leukoc Biol, 66:375-381 (1999)). The role and function of ILT3 on DCs have been accurately described by the Suciu-Foca group (Suciu-Foca, N., Int Immunopharmacol, 5:7-11 (2005)). The ligand for ILT3 is unknown, but ILT4 is known to bind to the third domain of HLA class I molecules (HLA-A, HLA-B, HLA-C, and HLA-G) and compete with CD8 for MHC class I binding (Shiroishi, M., Proc Natl Acad Sci USA, 100:8856-8861 (2003)). HLA-G is the preferred ligand for some inhibitory ILT receptors. HLA-G plays a potential role in maternal-fetal tolerance and in mechanisms by which tumor cells evade immune recognition and destruction (Hunt, JS, et al., Faceb J, 19:681-693 (2005)). Regulation of DC function by HLA-G-ILT interactions is most likely a crucial pathway in DC biology. Human monocyte-derived DCs highly expressing ILT2 and ILT4 receptors have been shown to still maintain a stable tolerance-like phenotype (CD80low, CD86low, HLA-DRlow) that can induce T cell anergy when treated with HLA-G and stimulated with allogeneic T cells (Ristich, V., et al., Eur J Immunol, 35:1133-1142 (2005)). Furthermore, HLA-G interactions with DCs highly expressing ILT2 and ILT4 receptors have resulted in downregulation of several genes involved in the MHC class II presentation pathway. IFN-gamma-inducible lysosomal thiol reductase (GILT), a lysosomal thiol reductase abundantly expressed by professional APCs, was significantly reduced in HLA-G modified DCs.Since the in vivo T cell response for antigen selection was reduced in animals lacking GILT after target gene disruption, the repertoire of primed CD4+ T cells may be influenced by GILT DC expression (Marie, M., et al., Science, 294:1361-1365 (2001)). HLA-G / ILT interactions on DCs interfere with the association and transport of MHC class II molecules to the cell surface, potentially reducing the efficiency of presentation or expression of structurally abnormal MHC class II molecules. It has been determined that HLA-G significantly reduces the transcription of the invariant chain (CD74), HLA-DMA, and HLA-DMB genes on human monocyte-derived DCs that highly express the ILT inhibitory receptor (Ristich, V., et al; Eur J Immunol 35:1133-1142 (2005)).

[0083] Another receptor for HLA-G is KIR2DL4, because KIR2DL4 binds to cells that express HLA-G (US Patent No. 2003232051; Cantoni, C. et al. Eur J Immunol 28(1998)1980; Rajagopalan, S. and EOLong. [A published errata can be found in J Exp Med 191(2000)2027] J Exp Med 189(1999)1093; Ponte, M. et al. PNAS USA 96(1999)5674). KIR2DL4 (also called 2DL4) is a member of the KIR family (also called CD158d) that shares structural features with both activating and inhibitory receptors (Selvakumar, A. et al. Tissue Antigens 48(1996)285). 2DL4 possesses cytoplasmic ITIMs suggesting inhibitory function and positively charged amino acids in its transmembrane region, a characteristic feature of KIR activation. Unlike other clonally distributed KIRs, 2DL4 is transcribed by all NK cells (Valiante, NM et al. Immunity 7 (1997) 739; Cantoni, C. et al. Eur J Immunol 28 (1998) 1980; Rajagopalan, S. and EOLong. [Published errata can be found in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093).

[0084] The term "inhibition of ILT2 binding to HLA-G on JEG-3 cells (ATCC HTB36)" refers to the inhibition of the binding interaction of (recombinant) ILT2 in the assay described, for example, in Example 5 of International Publication No. 2022 / 129120.

[0085] The term "reduction" (and its grammatical variations such as "reduce" or "reducing") refers to a decrease in any quantity, measured by appropriate methods known in the art, such as a reduction in the number of B cells or a reduction in cytokine release. For clarity, this term also includes reduction to zero (or below the detection limit of the analytical method), i.e., complete elimination or removal. Conversely, "increased" refers to an increase in any quantity.

[0086] As used herein, “T cell antigen” refers to the antigenic determinants presented on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0087] As used herein, “T cell activating therapeutic agent” refers to a therapeutic agent capable of inducing T cell activation in a target, and more particularly a therapeutic agent designed to induce T cell activation in a target. Examples of T cell activating therapeutic agents include bispecific antibodies (also called “T cell bispecific antibodies” or “TCBs”) that specifically bind to activated T cell antigens such as CD3, and target cell antigens such as HLA-G. Further examples include chimeric antigen receptors (CARs) that include a T cell activation domain and an antigen-binding portion that specifically binds to a target cell antigen such as HLA-G.

[0088] As used herein, “activated T cell antigen” refers to an antigenic determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, that can induce or enhance T cell activation upon interaction with antigen-binding molecules. Specifically, the interaction of an antigen-binding molecule with a T cell-activating antigen can induce T cell activation by triggering a cascade of signaling in the T cell receptor complex. An exemplary activated T cell antigen is CD3.

[0089] Unless otherwise specified, the term “CD3” refers to any native CD3 derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The term encompasses “full-length,” untreated CD3, and any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allele variants. In one embodiment, CD3 is human CD3, in particular the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also Sequence ID 66. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1. See also SEQ ID NO: 67.

[0090] 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 antigen-binding molecule specific to CD3 and HLA-G," "anti-HLA-G / anti-CD3 bispecific antibody," "HLA-G CD3 TCB," or "HLA-G TCB" are used interchangeably herein and refer to bispecific antibodies that can bind to CD3 and HLA-G with sufficient affinity to be useful as diagnostic and / or therapeutic agents when the antibody targets CD3 and HLA-G.

[0091] The term "PD1," also known as programmed cell death protein 1, is a 288-amino acid type I membrane protein 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 possesses two ligands: PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The structure of this protein includes an extracellular IgV domain, followed by a transmembrane domain and an intracellular tail. The intracellular tail contains two phosphorylation sites located within an immunoreceptor tyrosine system inhibitory motif and an immunoreceptor tyrosine system switch motif, suggesting 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 during ligand binding. PD1 is not expressed in naive T cells, but is upregulated in accordance with T cell receptor (TCR)-mediated activation and is observed in 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 properly. Although PD1 has a relatively broad expression pattern, its most important role is likely that of a co-inhibitory receptor for T cells (Chinai et al, Trends in Pharmacological Sciences 36 (2015), 587-595). Therefore, current therapeutic approaches focus on blocking the interaction between PD1 and its ligands to improve T cell response. The terms “programmed death 1,” “programmed cell death 1,” “protein PD-1,” “PD-1,” “PD1,” “PDCD1,” “hPD-1,” and “hPD-I” may be used interchangeably and include variants, isoforms, species homologs, and analogs of human PD1 that have at least one epitope in common with PD-1. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org) deposit number Q15116 (SEQ ID NO: 68).

[0092] The terms “anti-PD1 antibody” and “antibody containing an antigen-binding domain that binds to PD1” refer to an antibody capable of binding to PD1 (particularly the PD1 polypeptide expressed on the cell surface) with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when targeting PD1. In one embodiment, the degree of binding of an anti-PD1 antibody to an unrelated non-PD1 protein is less than 10% of the binding of the antibody to PD1, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance assay using a biosensor system such as the Biacore® system. In certain embodiments, an antigen-binding protein that binds to human PD1 has a binding affinity of K for binding to human PD1 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM. D It has a value (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~

[0093] 10 -13 M). In a preferred embodiment, each K of the binding affinity D The value is determined by a surface plasmon resonance assay using the extracellular domain (ECD) (PD1-ECD) of human PD1 for PD1 binding affinity. The term "anti-PD1 antibody" also includes bispecific antibodies capable of binding to PD1 and a second antigen.

[0094] In a specific embodiment, the anti-PD1 antibody is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pizilizumab), PDR001 (spartalizumab), SHR1210 (canrelizumab), MEDI-0680 (AMP-514), REGN2810, and BGB-108. In a particular embodiment, the anti-PD1 antibody is pembrolizumab, or an antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 75 and a light chain containing the amino acid sequence of SEQ ID NO: 76. Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD1 antibody described in International Publication No. 2009 / 114335 (CAS Registry No. 1374853-91-4). In a particular embodiment, the anti-PD1 antibody is nivolumab, or an antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 77 and a light chain containing the amino acid sequence of SEQ ID NO: 78. Nivolumab (CAS Registry No. 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 International Publication No. 2006 / 121168 (CAS Reg. No. 946414-94-4). In another specific embodiment, the anti-PD1 antibody comprises a heavy chain variable domain VH containing the amino acid sequence of SEQ ID NO. 7 and a light chain variable domain VL containing the amino acid sequence of SEQ ID NO. 8, or a humanized variant thereof. In a specific embodiment, the anti-PD1 antibody comprises a heavy chain variable domain VH containing the amino acid sequence of SEQ ID NO. 9 and a light chain variable domain VL containing the amino acid sequence of SEQ ID NO. 10.

[0095] The terms “LAG3,” “Lag-3,” “Lymphocyte Activator Gene-3,” or “CD223,” as used herein, refer to any native LAG3 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. 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, e.g., splice variants or allele variants. In a preferred embodiment, the term “LAG3” refers to human LAG3. The amino acid sequence of an exemplary processed (signal sequence-free) LAG3 is shown in SEQ ID NO: 69. The amino acid sequence of an exemplary extracellular domain (ECD) LAG3 is shown in SEQ ID NO: 70.

[0096] The terms “anti-LAG3 antibody” and “antibody that binds to LAG3” refer to an antibody capable of binding to LAG3 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when targeting LAG3. In one embodiment, the degree of binding of an anti-LAG3 antibody to an unrelated non-LAG3 protein is less than about 10% of the binding of the antibody to LAG3 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to LAG3 has a degree of ≤1 μM, ≤100 nM, ≤0 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The dissociation constant (K) of M D ) has. In certain embodiments, the anti-LAG3 antibody binds to an epitope of LAG3 that is conserved in LAG3 from different species. In a preferred embodiment, the “anti-LAG3 antibody,” the “antibody that specifically binds to human LAG3,” and the “antibody that binds to human LAG3” are 1.0 × 10 -8 K below mol / L D Value, in one embodiment, 1.0 x 10 -9K below mol / L D Value, in one embodiment, 1.0 x 10 -9 mol / l ~ 1.0 x 10 -13 K in mol / l D This refers to an antibody that specifically binds to the human LAG3 antigen or its extracellular domain (ECD) based on its binding affinity. In this regard, binding affinity is determined using a standard binding assay, e.g., surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), for example, using the LAG3 extracellular domain. The term "anti-LAG3 antibody" also includes bispecific antibodies capable of binding to LAG3 and a second antigen. In one embodiment, an anti-LAG3 antibody is relatrimab or BMS-986016, or an antibody comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 27 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the anti-LAG3 antibody comprises a heavy chain variable domain VH containing the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain VL containing the amino acid sequence of SEQ ID NO: 18, or a heavy chain variable domain VH containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable domain VL containing the amino acid sequence of SEQ ID NO: 26, or a heavy chain variable domain VH containing the amino acid sequence of SEQ ID NO: 64 and a light chain variable domain VL containing the amino acid sequence of SEQ ID NO: 65.

[0097] "Bispecific antibody comprising 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 antigen-binding molecule specific to PD1 and LAG3," or "anti-PD1 / anti-LAG3 (bispecific) antibody" are used interchangeably herein and refer to a bispecific antibody capable of binding to PD1 and LAG3 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent when targeting PD1 and LAG3.

[0098] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, the blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, the bispecific antibodies of the present invention block PD1 and LAG3 signaling to restore a functional response by T cells (e.g., proliferation, cytokine production, target cell death) from a dysfunctional state to antigen stimulation.

[0099] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain involved in the binding of antigen-binding molecules to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0100] As used herein, the terms “hypervariable region” or “HVR” mean each of the regions of the antibody variable domain, e.g., “complementarity-determining regions” (CDRs), that are hypervariable within the sequence and determine antigen-binding specificity. Generally, an antibody contains six CDRs, three located in the VH (CDR-H1, CDR-H2, CDR-H3) and three located in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs as used herein include: (a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs produced at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

[0101] Unless otherwise specified, CDR is determined in accordance with Kabat et al. above. Those skilled in the art will understand that the designation of CDR may also be determined in accordance with Chothia, McCallum, or any other scientifically recognized nomenclature.

[0102] The terms “Kabat-like variable domain residue numbering” or “Kabat-like amino acid position numbering” and their variations refer to the numbering system used for the heavy-chain or light-chain variable domains of antibody formulations by Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions into the FR or HVR of the variable domain. For example, a heavy-chain variable domain may contain a single amino acid insertion after H2 residue 52 (Kabat-like residue 52a) and a residue inserted after heavy-chain FR residue 82 (e.g., Kabat-like residues 82a, 82b, and 82c). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with the homologous region of the sequence numbered by “standard” Kabat. Generally, native quadruple-chain antibodies contain six HVRs, with three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0103] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0104] For the purposes of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework, as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0105] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species.

[0106] The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0107] A “humanized” antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the HVR (e.g., CDR) corresponds to those of the non-human antibody, and all or substantially all of the FR corresponds to those of the human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. “Humanized” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” as encompassed in the present invention are those in which the constant region has been further modified or altered from the constant region of the original antibody to produce the properties according to the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0108] A “human” antibody is one that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0109] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical and / or bind to the same epitope, with the exception of possible variant antibodies, such as those containing naturally occurring mutations or arising during the production of a monoclonal antibody preparation, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such and other exemplary methods for producing monoclonal antibodies are described herein.

[0110] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an antibody heavy chain, which includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In particular, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain always contains C-terminal lysine, but variants without C-terminal lysine are included in this invention.

[0111] The IgG Fc region includes an IgG CH2 domain and an IgG CH3 domain. The “CH2 domain” of the human IgG Fc region typically extends from an amino acid residue at approximately amino acid position 231 to an amino acid residue at approximately amino acid position 340. In one embodiment, a carbohydrate chain is bound to the CH2 domain. The CH2 domain as used herein may be either a native sequence CH2 domain or a variant CH2 domain. The “CH3 domain” includes an extension of residues at the C-terminus of the CH2 domain in the Fc region (i.e., from an amino acid residue at approximately position 341 of IgG to an amino acid residue at approximately position 447). The CH3 region as used herein may be either a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced “projection” (“knob”) on one of its chains and an introduced “cavity” (“hole”) corresponding to 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, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0112] The “knob-into-hole” technique is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the projection can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the projection is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In specific embodiments, the knob modification includes the amino acid substitution T366W in one of the two subunits of the Fc domain, and the whole modification includes the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In more specific embodiments, the subunit of the Fc domain constituting the knob modification further includes the amino acid substitution S354C, and the subunit of the Fc domain constituting the whole modification further includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues forms a disulfide bridge between the two subunits of the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0113] The “region corresponding to the Fc region of immunoglobulins” is intended to include naturally occurring allelic variants of the Fc region of immunoglobulins, as well as variants that produce substitutions, additions, or deletions but have modifications that do not substantially reduce the effector function of the immunoglobulin (e.g., antibody-dependent cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulins without substantially losing biological function. Such variants can be selected according to general rules known in the art to minimize the impact on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0114] The term "effector function" refers to the function of biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immunoconjugate-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0115] An "activated Fc receptor" is an Fc receptor that, following involvement by the Fc region of an antibody, triggers a signaling event that stimulates receptor-hosting cells to perform effector functions. Examples of activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activated Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

[0116] The term "peptide linker" refers to a peptide containing 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 include, for example, (G4S)n and (SG4) n or G4(SG4) n is a peptide linker, where "n" is generally a number between 1 and 10, typically a number between 2 and 4, and particularly 2. Particularly interesting peptide linkers 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 specifically (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72).

[0117] "Fused to" or "connected to" means that the components (e.g., an antigen-binding domain and an Fc domain) are linked either directly by a peptide bond or via one or more peptide linkers.

[0118] As used herein, the term "amino acid" refers to a group of naturally occurring carboxy α-amino acids including alanine (three-letter notation: ala, one-letter notation: 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).

[0119] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences, introducing gaps as necessary to achieve maximum percentage sequence identity, and disregarding conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art of a person skilled in the art using commonly available computer software such as BLAST, BLAST-2, ALIGN.SAWI, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. However, as used herein, the amino acid sequence identity percentage value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office (Washington DC, 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) and can also be compiled from source code. The ALIGN-2 program should be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity % of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or, can be described as a given amino acid sequence A having or containing a certain amino acid sequence identity % to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y

[0120] Here, X is the number of amino acid residues scored as identity matches by the sequence alignment program ALIGN-2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B is not equal to the amino acid sequence identity % of B to A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein are obtained as described in the paragraph immediately preceding the use of the ALIGN-2 computer program.

[0121] In certain embodiments, amino acid sequence variants of the bispecific antibodies of the present invention provided herein are envisioned. 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 bispecific antibodies may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the molecule, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding). Notable sites for substitutional mutagenesis include HVR and framework (FR). Conservative substitutions are listed in Table A under the heading "Preferred Substitutions" and are further described below with reference to amino acid side chain classes (1) to (6). Amino acid substitutions can be introduced into the target molecule, and the product can be screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC.

[0122] Amino acids can be classified according to their general side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0123] Non-conservative substitution involves swapping one member of one of these classes with one of another. [Table A]

[0124] The term “amino acid sequence variant” includes substantial variants in which amino acid substitutions exist in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized antibody or human antibody). Generally, a variant(s) selected for further study and obtained will have alterations (e.g., improvements) (e.g., increased affinity, decreased immunogenicity) of specific biological properties (e.g., increased affinity, decreased immunogenicity) and / or substantially retain the specific biological properties of the parent antigen-binding molecule compared to the parent antigen-binding molecule. Exemplary substitution variants are affinity-matured antibodies and can be readily generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, a variant antigen-binding molecule has one or more HVR residues that have been mutated, phage-displayed, and screened for specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such alterations do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative alterations (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within an HVR. A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at the positions of amino acids that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition to this, the crystal structure of the antigen-antigen binding molecule complex is used to identify contact sites between the antibody and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they contain the desired properties.

[0125] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is a bispecific antibody with an N-terminal methionyl residue. Other insertion variants of molecules include N-terminal or C-terminal fusions to polypeptides that increase the serum half-life of bispecific antibodies.

[0126] In certain embodiments, the bispecific antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Glycosylated variants of a molecule can be easily obtained by altering the amino acid sequence so that one or more glycosylation sites are created or removed. For example, the carbohydrate that binds to the Fc domain may be modified. Native antibodies produced by mammalian cells typically contain branched bibranched oligosaccharides that are commonly bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the bibranched oligosaccharide structure. In some embodiments, oligosaccharide modifications in the bispecific antibodies of the present invention may be made to create variants having specific improved properties. In one embodiment, a variant of a bispecific antibody is provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. Such a fucosylated variant may have improved ADCC function. See, for example, U.S. Patent Publication 2003 / 0157108 (Presta, L.) or U.S. Patent 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Further variants of the bispecific antibody of the present invention include those having a bifid oligosaccharide, for example, a bifid oligosaccharide attached to the Fc region being bifid by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. See, for example, International Publication No. 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6602684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide bound to the Fc domain are also provided.Such antibody variants may possess improved CDC functionality and are described, for example, in International Publication No. 1997 / 30087 (Patel et al.), International Publication No. 1998 / 58964 (Raju, S.), and International Publication No. 1999 / 22764 (Raju, S.).

[0127] In certain embodiments, it may be desirable to create cysteine-modified variants of the bispecific antibody of the present invention, for example, “thioMAb” in which one or more residues of the molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the molecule. By substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site of the antibody, and this reactive thiol group can be used to conjugate the antibody to other parts, such as the drug part or the linker-drug part, to create an immunoconjugate. In certain embodiments, one or more of the following residues may 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. The cysteine-modified antigen-binding molecule may be generated, for example, as described in U.S. Patent No. 7,521,541.

[0128] In certain embodiments, the bispecific antibodies provided herein may be modified to include further non-proteinoid moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, the polymers may be the same molecule or different molecules. Generally, the number and / or types of polymers used in derivatization are not limited, but can be determined based on limitations including specific properties or functions of the antibody being improved, and whether the bispecific antibody derivative is used under specified conditions.

[0129] In another embodiment, an antibody-nonprotein portion conjugate is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonprotein portion is a carbon nanotube (Kam, NWet al., Proc. Natl. Acad. Sci. USA 102(2005) 11600-11605). The radiation may have any wavelength, but is not limited, and is not harmful to normal cells, but includes wavelengths that heat the nonprotein portion to a temperature that kills cells proximal to the antibody-nonprotein portion.

[0130] An "immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic drugs.

[0131] The term "polynucleotide" refers to an isolated nucleic acid molecule or structure, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or other types of bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments.

[0132] The term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA that has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of this invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that originally contained polynucleotide molecules, but the polynucleotide molecules are extrachromosomal or located at chromosomal locations different from their native chromosomal locations. Isolated RNA molecules include the in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and double-stranded forms. Furthermore, isolated polynucleotides or nucleic acids of this invention include such molecules produced by synthesis. In addition, polynucleotides or nucleic acids may be regulatory elements such as promoters, ribosome-binding sites, or transcription terminators, or may contain regulatory elements.

[0133] A nucleic acid or polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to the reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference nucleotide sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These modifications to the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or somewhere between these terminal positions, and may be individually scattered among residues in the reference sequence, or scattered among one or more consecutive groups in the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0134] The term "expression cassette" refers to a polynucleotide, generated by recombination or synthesis, comprising a set of specific nucleic acid elements that enable the transcription of a particular nucleic acid in target cells. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, in particular, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence or fragment thereof encoding the bispecific antigen-binding molecule of the present invention.

[0135] The terms “vector” or “expression vector” are synonymous with “expression construct” and refer to a DNA molecule used to introduce and induce the expression of a specific gene to which it operably binds within a target cell. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors that have been incorporated into the genome of the host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector enables the transcription of large amounts of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation mechanism. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence or fragment thereof encoding the bispecific antigen-binding molecule of the present invention.

[0136] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. Offspring may contain mutations, but may not be completely identical to the parent cells in terms of nucleic acid content. Hereinafter, mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included. Host cells are any type of cell line that can be used to produce the bispecific antigen-binding molecules of the present invention. In particular, host cells are prokaryotic or eukaryotic host cells. Host cells include cultured cells, such as mammalian cultured cells, to name just a few: 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, but also cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.

[0137] The "effective dose" of a drug refers to the amount of that drug required to cause a certain physiological change in the cells or tissues to which it is administered.

[0138] The "therapeutic effective dose" of a drug, such as a pharmaceutical composition, refers to the effective amount in the dosage and duration required to obtain the desired therapeutic or preventive outcome. For example, a therapeutic effective dose of a drug might eliminate, reduce, delay, minimize, or prevent the side effects of a disease.

[0139] The "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, 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.

[0140] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained herein is effective, and which does not contain additional components that are unacceptably toxic to the subject to which the preparation is administered.

[0141] "Pharmacologically acceptable excipients" refer to components in a pharmaceutical composition other than the active ingredient that are non-toxic to the target substance. Examples of pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and preservatives.

[0142] The term "package insert" is used to refer to the instructions that are typically included in the product packaging of a therapeutic product, and include information on indications, usage, dosage, administration, combination therapies, contraindications and / or precautions regarding the use of the therapeutic product.

[0143] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of the treated individual, and may be performed for preventive purposes or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological outcomes of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating symptoms, and achieving remission or improving prognosis. In some embodiments, the molecules of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0144] As used herein, the term "cancer" refers to lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer. This includes cancers such as colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia (including refractory versions of any of the above cancers), or combinations of one or more of the above cancers. In one embodiment, the term cancer refers to HLA-G expressing cancers. In particular, HLA-G expressing cancers include selected renal cell carcinoma, colorectal carcinoma, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0145] The term "HLA-G expression" is intended to indicate a significant level of HLA-G expression within cells, preferably on the cell surface of cells from tumors or cancers, preferably solid tumors. Patients with "HLA-G expressing cancer" can be determined by standard assays known in the art. For example, HLA-G antigen expression can be measured via immunohistochemistry (IHC) detection, using FACS, or via PCR-based detection of the corresponding mRNA.

[0146] As used herein, the terms “HLA-G expressing cancer” or “HLA-G positive cancer” refer to all cancers in which cancer cells express the HLA-G antigen. Preferably, as used herein, HLA-G expressing cancers include lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, and gastric cancer. This refers to cancer, including colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, nasopharyngeal carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumor, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, 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, HLA-G expressing cancers include selected renal cell carcinoma, colorectal carcinoma, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0147] The terms “treatment method,” “treatment method,” or their equivalents, when applied to cancer, for example, refer to a procedure or set of actions designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of cancer. “Treatment method” of cancer or another proliferative disorder does not necessarily mean that cancer cells or other disorders are actually eliminated, that the number of cells or disorders is actually reduced, or that the symptoms of cancer or other disorders are actually alleviated. Often, treatment methods for cancer are performed even when the probability of success is low, but are still considered to induce an overall beneficial course of action given the patient’s medical history and estimated survival.

[0148] The terms “combination,” “simultaneous administration,” or “administering simultaneously” refer to administering anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 bispecific antibodies as two separate formulations (or as a single formulation). Simultaneous administration may be simultaneous or sequential in either order, and it is desirable that there be a period during which both (or all) activators exert biological activity simultaneously. Anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 bispecific antibodies are administered simultaneously or sequentially (e.g., intravenously (iv) by serial infusion (one being an anti-HLA-G / anti-CD3 bispecific antibody and the other an anti-PD1 / anti-LAG3 bispecific antibody)). When both therapeutic agents are administered sequentially, the doses are either administered on the same day in two separate doses, or one agent is administered on day 1 and the second agent is administered between days 2 and 7, preferably between days 2 and 4. Thus, the term "sequential" refers to the first component (anti-HLA-G / anti-CD3 bispecific antibody or anti-PD1 / The term "simultaneously" means within 7 days after administration of the anti-LAG3 bispecific antibody, preferably within 4 days after administration of the first component. With respect to the maintenance doses of the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 bispecific antibody, the term "simultaneous administration" means that the maintenance doses can be administered simultaneously, for example, weekly, if the treatment cycle is appropriate for both drugs. Alternatively, the anti-PD1 / anti-LAG3 bispecific antibody may be administered every 2 weeks, for example, and the anti-HLA-G / anti-CD3 bispecific antibody every 3 weeks. Or, the maintenance doses may be administered sequentially and simultaneously within one day or several days.

[0149] Exemplary anti-HLA-G / anti-CD3 bispecific antibody used in the present invention The combination of anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 bispecific antibodies has been found to exhibit higher efficacy in humanized mouse tumor models than anti-HLAG / anti-CD3 bispecific antibody monotherapy or combinations of anti-HLAG / anti-CD3 bispecific antibodies with anti-PD1 and anti-LAG3 antibodies. This data demonstrates that the combination with anti-PD1 / anti-LAG3 bispecific antibodies is advantageous over combinations with anti-PD1 and / or anti-LAG3 monospecific antibodies because it provides better efficacy, as indicated by stronger inhibition of tumor growth and better activation of T cells.

[0150] This invention relates to anti-HLA-G / anti-CD3 bispecific antibodies and their use in combination with anti-PD1 / anti-LAG3 bispecific antibodies, particularly their use in methods for treating or delaying the progression of HLA-G expressing cancers. The anti-HLA-G / anti-CD3 bispecific antibodies used herein are bispecific antibodies comprising a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to HLA-G. Therefore, they target HLA-G expressing tumor cells.

[0151] Therefore, the anti-HLA-G / anti-CD3 bispecific antibodies used herein have a heavy chain variable region (V) that specifically binds to CD3. H CD3) and light chain variable region (V L The first antigen-binding domain includes CD3, and the heavy chain variable region (V) specifically binds to HLA-G. H HLA-G) and light chain variable region (V L It includes a second antigen-binding domain containing HLA-G.

[0152] In certain embodiments, the anti-HLA-G / anti-CD3 bispecific antibody for use in combination contains a heavy chain variable region (V) including 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. HCD3), and / or a light chain variable region (V) 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 L A first antigen-binding domain comprising an anti-HLA-G / anti-CD3 bispecificity and comprising the heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 47 H CD3), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 48 L A first antigen-binding domain comprising an anti-HLA-G / anti-CD3 bispecificity. In a further aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD3), and / or a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 48 L CD3).

[0153] In another aspect, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen-binding domain that specifically binds to PD1 and comprises a heavy chain variable region (V) 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 H HLA-G), and / or a light chain variable region (V) 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 L HLA-G). More specifically, the anti-HLA-G / anti-CD3 bispecific antibody comprises a second antigen-binding domain comprising a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 55 H HLA-G), and / or a light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 56 L HLA-G). In a further aspect, the anti-HLA-G / anti-CD3 bispecificity comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 55 HHLA-G), and / or light chain variable region containing the amino acid sequence of SEQ ID NO: 56 (V L The antibody comprises a second antigen-binding domain containing HLA-G. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody comprises an antigen-binding domain that specifically binds to CD3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 47 and a VL domain containing the amino acid sequence of SEQ ID NO: 48, and a second antigen-binding domain that specifically binds to HLA-G, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 55 and a VL domain containing the amino acid sequence of SEQ ID NO: 56.

[0154] In another specific embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes a third antigen-binding domain that binds to HLA-G. Specifically, the anti-HLA-G / anti-CD3 bispecific antibody includes a heavy chain variable region (V) 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. H HLA-G); and / or a light chain variable region (V) including the CDR-L1 sequence of sequence number 52, the CDR-L2 sequence of sequence number 53, and the CDR-L3 sequence of sequence number 54. L It contains a third antigen-binding domain including HLA-G. More specifically, anti-HLA-G / anti-CD3 bispecific antibodies contain a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 55. H Light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 56 (HLA-G), and / or the amino acid sequence of SEQ ID NO: 56. L It includes a third antigen-binding domain containing HLA-G. In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 55. H HLA-G), and / or light chain variable region containing the amino acid sequence of SEQ ID NO: 56 (V LThe antibody comprises a third antigen-binding domain containing HLA-G. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody comprises an antigen-binding domain that specifically binds to CD3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 47 and a VL domain containing the amino acid sequence of SEQ ID NO: 48, and a second and optionally third antigen-binding domain that specifically binds to HLA-G, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 55 and a VL domain containing the amino acid sequence of SEQ ID NO: 56.

[0155] In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody used herein is a full-length antibody. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody used herein is a human IgG class antibody, particularly a human IgG1 class antibody.

[0156] In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody is a bispecific antibody in which the first antigen-binding domain is a cross-Fab molecule in which the variable or constant domains of the Fab heavy chain and Fab light chain are exchanged, and the second and third antigen-binding domains, if present, are conventional Fab molecules.

[0157] Fab molecules can be fused to each other via the Fc domain, or via peptide linkers containing one or more amino acids, typically about 2 to 20 amino acids, either directly or with each other. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (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 specifically (G4S)2 or GGGGSGGGGS (SEQ ID NO: 72). A peptide linker particularly suitable for fusing the Fab light chains of first and second Fab molecules to each other is (G4S)2. Another suitable linker includes sequence (G4S)4 (SEQ ID NO: 74). Furthermore, the linker may include (part of) the immunoglobulin hinge region. In particular, when a Fab molecule fuses to the N-terminus of an Fc domain subunit, it may fuse with or without an additional peptide linker via the immunoglobulin hinge region or a portion thereof.

[0158] In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody is a bispecific antibody in which (i) a second antigen-binding domain is fused to the N-terminus of the first antigen-binding domain at the C-terminus of the Fab heavy chain, the first antigen-binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and a third antigen-binding domain is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain (as shown in Figure 1A), or (ii) a first antigen-binding domain is fused to the N-terminus of the second antigen-binding domain at the C-terminus of the Fab heavy chain, the second antigen-binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and a third antigen-binding domain is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0159] In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody comprises an Fc domain containing one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor. In particular, the anti-HLA-G / anti-CD3 bispecific antibody comprises an IgG1 Fc domain containing amino acid substitutions L234A, L235A, and P329G (according to EU numbering).

[0160] In certain embodiments, 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 of SEQ ID NO: 57; a second polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 58; a third polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 59; and a fourth polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 60. In even more specific embodiments, 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).

[0161] In another embodiment, the anti-HLA-G / anti-CD3 bispecific antibody for use in combination includes a heavy chain variable region (V) containing 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. H CD3), and / or the light chain variable region (V) including 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. L It contains a first antigen-binding domain including CD3. More specifically, anti-HLA-G / anti-CD3 bispecificity is due to the heavy chain variable region (V) which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 89. H CD3), and / or the light chain variable region (V) which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 90.L The antibody comprises a first antigen-binding domain including CD3. In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 89. H CD3), and / or the light chain variable region containing the amino acid sequence of SEQ ID NO: 90 (V L Includes CD3).

[0162] In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody has a heavy chain variable region (V) including 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. H HLA-G), and / or the light chain variable region (V) including 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. L It contains a second antigen-binding domain including HLA-G. More specifically, anti-HLA-G / anti-CD3 bispecific antibodies contain a heavy chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 97. H Light chain variable region (V) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of sequence number 98 (HLA-G) and / or the amino acid sequence of sequence number 98. L It includes a second antigen-binding domain containing HLA-G. In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 97. H HLA-G), and / or light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 98 L It contains a second antigen-binding domain, which includes HLA-G.

[0163] In certain embodiments, 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 of SEQ ID NO: 99, a second polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 100, a third polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 101, and a fourth polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 102. In even more specific embodiments, 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.

[0164] Specific anti-HLA-G / anti-CD3 bispecific antibodies are listed in PCT Publication No. 2022 / 24024 or International Publication No. 2022 / 129120.

[0165] In a further embodiment, the anti-HLA-G / anti-CD3 bispecific antibody may also include a bispecific T cell engager (BiTE®).

[0166] Exemplary bispecific anti-PD1 / anti-LAG3 antibodies for use in the present invention The combinations provided herein utilize novel bispecific antibodies that possess particularly advantageous properties, such as productivity, stability, binding affinity, biological activity, specific targeting of specific T cells, targeting efficiency, and reduced toxicity, 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). Specific bispecific anti-PD1 / anti-LAG3 antibodies for use herein are described in International Publication No. 2018 / 185043A1.

[0167] In certain embodiments, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, exhibiting reduced internalization upon binding to the T cell surface. Internalization represents a critical sink for molecules that can be rapidly reexpressed on the cell surface ready for the target receptor to inhibit TCR signaling, while being degraded within hours. In further embodiments, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, preferentially binding to conventional T cells rather than Tregs. This is advantageous because targeting LAG3 on Tregs using blocking antibodies can worsen by increasing its repressive function and ultimately masking the positive blocking effect on other T cells. In further embodiments, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, capable of protecting T cell effector function from Treg suppression. In another embodiment, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, which, when cultured with the tumor cell line ARH77 as shown in the assay presented herein, can induce granzyme B secretion by CD4 T cells. In a further embodiment, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, which exhibits increased tumor-specific T cell effector function and / or enhances the cytotoxic effect of T cells. In yet another embodiment, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, which exhibits increased tumor eradication in vivo.

[0168] In one embodiment, the present invention relates to 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 is VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 3, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) Provides an anti-PD1 / anti-LAG3 bispecific antibody comprising a VL domain and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 6.

[0169] In one embodiment, an anti-PD1 / anti-LAG3 bispecific antibody comprises an IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, the Fc domain reducing or even disabling effector function. In particular, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors.

[0170] In a further embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided 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 IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor.

[0171] In another embodiment, an anti-PD1 / anti-LAG3 bispecific antibody for use in combination comprises 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 is (a) A VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) containing a VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 16; or (b) VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 21, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 A bispecific anti-PD1 / anti-LAG3 antibody is provided, which includes the above.

[0172] In a further embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided 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 containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10.

[0173] In another embodiment, an anti-PD1 / anti-LAG3 bispecific antibody for use in combination comprises 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 is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, or (b) VH domain containing the amino acid sequence of SEQ ID NO: 25 and VL domain containing the amino acid sequence of SEQ ID NO: 26 A bispecific anti-PD1 / anti-LAG3 antibody is provided, which includes the above.

[0174] In a further embodiment, an anti-PD1 / anti-LAG3 bispecific antibody for use in combination comprises 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 is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 27 and a VL domain containing the amino acid sequence of SEQ ID NO: 28, or (b) A VH domain containing the amino acid sequence of SEQ ID NO: 29 and a VL domain containing the amino acid sequence of SEQ ID NO: 30, or (c) A VH domain containing the amino acid sequence of SEQ ID NO: 31 and a VL domain containing the amino acid sequence of SEQ ID NO: 32, or (d) A VH domain containing the amino acid sequence of SEQ ID NO: 33 and a VL domain containing the amino acid sequence of SEQ ID NO: 34, or (e) A bispecific anti-PD1 / anti-LAG3 antibody is provided, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 64 and a VL domain containing the amino acid sequence of SEQ ID NO: 65.

[0175] In another embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided 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 containing the amino acid sequence of SEQ ID NO: 81 and a VL domain containing the amino acid sequence of SEQ ID NO: 82.

[0176] In a particular embodiment, an anti-PD1 / anti-LAG3 bispecific antibody for use in combination comprises a first antigen-binding domain that specifically binds to PD1 and a second antigen-binding domain that specifically binds to LAG3, The first antigen-binding domain that specifically binds to PD1 includes a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10. A bispecific anti-PD1 / anti-LAG3 antibody is provided, wherein the second antigen-binding domain that specifically binds to LAG3 comprises a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, or a VH domain containing the amino acid sequence of SEQ ID NO: 25 and a VL domain containing the amino acid sequence of SEQ ID NO: 26.

[0177] In one embodiment, the bispecific antibody of the present invention comprises a first antigen-binding domain that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, and a second antigen-binding domain that specifically binds to LAG3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18.

[0178] In a further embodiment, the anti-PD1 / anti-LAG3 bispecific antibody of the present invention 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: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0179] In a further embodiment, 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 antibody, a humanized antibody, or a chimeric antibody. In particular, the bispecific antibody is a humanized antibody or a chimeric antibody.

[0180] In one embodiment, an 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).

[0181] In one embodiment, an anti-PD1 / anti-LAG3 bispecific antibody is provided 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 and a first Fab fragment containing an antigen-binding domain that specifically binds to PD1, and a second Fab fragment containing an antigen-binding domain that specifically binds to LAG3. In a particular embodiment, in one of the Fab fragments, the variable domains VL and VH are substituted for 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 embodiment, in the first Fab fragment containing an antigen-binding domain that specifically binds to PD1, the variable domains VL and VH are substituted for each other.

[0182] In certain embodiments, 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 (a) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35, and a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36, 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 (b) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 35, and a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 36, 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, is provided.

[0183] ​​​​​​​​​​​In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies presented herein, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0186] The following sections describe preferred embodiments of the bispecific antigen-binding molecules of the present invention, including Fc domain modifications that reduce Fc receptor binding and / or effector function. In one embodiment, the present invention relates to anti-PD1 / anti-LAG3 bispecific antibodies and / or anti-HLA-G / anti-CD3 bispecific antibodies in which the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. In particular, the Fc domain is an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0187] The Fc domain confers desirable pharmacokinetic properties to the bispecific antibody of the present invention, including a long serum half-life and a desirable tissue-to-blood distribution ratio, which contribute to good accumulation in target tissues. However, at the same time, this can lead to the undesirable situation where the bispecific antibody of the present invention targets cells expressing the Fc receptor rather than cells that possess the preferred antigen. Therefore, in certain embodiments, the Fc domain of the bispecific antibody of the present invention exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG Fc domain, particularly the IgG1 Fc domain or IgG4 Fc domain. More specifically, the Fc domain is the IgG1 FC domain.

[0188] In one such embodiment, the Fc domain (or the bispecific antigen-binding molecule of the present invention containing said Fc domain) exhibits a binding affinity to the Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%, compared to the native IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing said IgG1 Fc domain), and / or exhibits effector function of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%, compared to the native IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing said IgG1 Fc domain). In one embodiment, the Fc domain (or the bispecific antigen-binding molecule of the present invention containing said Fc domain) does not substantially bind to the Fc receptor and / or induce effector function. In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In specific embodiments, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the domain of the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the bispecific antigen-binding molecule of the present invention containing said Fc domain) exhibits a binding affinity to FcRn that is greater than about 70%, particularly greater than about 80%, and more specifically greater than about 90%, compared to the native IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing said IgG1 Fc domain).

[0189] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to an unengineered Fc domain. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule of the present invention contains one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In another embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least half, at least one-fifth, or at least one-tenth. In one embodiment, the bispecific antigen-binding molecule of the present invention containing an engineered Fc domain exhibits a binding affinity to the Fc receptor of less than 20%, particularly less than 10%, and more specifically less than 5%, compared to the bispecific antibody of the present invention containing an unengineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a specific embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more specifically, a human FcγRIIB. In some embodiments, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically, a human FcγRIIIa, FcγRI, or FcγRIIa, most specifically a human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly to C1q, is also reduced. In one embodiment, binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn (i.e., retention of the binding affinity of the Fc domain to the receptor) is achieved when the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) exhibits a binding affinity greater than approximately 70% of the binding affinity of the unmodified form of the Fc domain (or the bispecific antigen-binding molecule of the present invention containing this unmodified form of Fc) to FcRn.An Fc domain, or a bispecific antigen-binding molecule of the present invention containing such Fc domain, may exhibit an affinity greater than approximately 80% and even greater than approximately 90% of such affinity. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule of the present invention is engineered to reduce effector function compared to an unengineered Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cell-mediated cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0190] Antibodies with reduced effector function include those having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant, which has substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, as well as Fc variants with substitutions at alanine residues 265 and 297 (U.S. Patent No. 7,332581). Specific antibody variants with improved or reduced binding to FcR have been described. (e.g., U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields, R. Let al., J. Biol. Chem. 276(2001) 6591-6604.)

[0191] In one embodiment of the present invention, the Fc domain includes amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some embodiments, the Fc domain includes amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain includes an amino acid substitution at position P329 and includes further amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. Furthermore, in certain embodiments, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions almost completely eliminates the binding of the human IgG1 Fc domain to the Fcγ receptor, as described in PCT application publication number International Publication 2012 / 130831A1. The document also describes methods for preparing such mutant Fc domains and for determining their properties, such as Fc receptor binding or effector function. Such antibodies are IgG1 having mutations L234A and L235A, or mutations L234A, L235A, and P329G (numbering follows the EU index of Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0192] In one embodiment, the anti-PD1 / anti-LAG3 bispecific antibody and / or anti-HLA-G / anti-CD3 bispecific antibody may have (i) a homodimer Fc region of a human IgG1 subclass having any mutations P329G, L234A and L235A, or (ii) a homodimer Fc region of a human IgG4 subclass having any mutations P329G, S228P and L235E, or (iii) any mutations P329G, L234A, L235A, I253A, H310A and H435A. (iv) a homodimer Fc region of a human IgG1 subclass having mutations P329G, L234A, L235A, H310A, H433A and Y436A, or (iv) one Fc region polypeptide containing mutation T366W and the other Fc region polypeptide containing mutations T366S, L368A and Y407V, or one Fc region polypeptide containing mutations T366W and Y349C and the other Fc region polypeptide containing mutations T366S, L368A and Y407V (v) A heterodimer Fc region containing S354C, or one Fc region polypeptide containing mutants T366W and S354C, and the other Fc region polypeptide containing mutants T366S, L368A, Y407V and Y349C, or (v) both Fc region polypeptides containing mutants P329G, L234A and L235A, one Fc region polypeptide containing mutant T366W, and the other Fc region polypeptide containing mutants T366S, L368A and Y407V, Alternatively, one Fc region polypeptide contains mutants T366W and Y349C, and the other Fc region polypeptide contains mutants T366S, L368A, Y407V and S354C, or one Fc region polypeptide contains mutants T366W and S354C, and the other Fc region polypeptide contains mutants T366S, L368A, Y407V and Y349C, containing heterodimer Fc regions of a human IgG1 subclass (all positions according to Kabat's EU index).

[0193] In one embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228, particularly the amino acid substitution S228P (Kabat numbering). In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing amino acid substitutions L235E, S228P, and P329G. This amino acid substitution reduces Fab arm exchange of the IgG4 antibody in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). Accordingly, in one embodiment, a bispecific antibody is provided that comprises a heterodimer Fc region of a human IgG4 subclass, wherein both Fc region polypeptides (all at their respective positions according to the Kabat EU index) contain mutants P329G, S228P, and L235E, one Fc region polypeptide contains mutant T366W and the other Fc region polypeptide contains mutants T366S, L368A, and Y407V, or one Fc region polypeptide contains mutants T366W and Y349C and the other Fc region polypeptide contains mutants T366S, L368A, Y407V, and S354C, or one Fc region polypeptide contains mutants T366W and S354C and the other Fc region polypeptide contains mutants T366S, L368A, Y407V, and Y349C.

[0194] Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn) are responsible for the transfer of mature IgG to the fetus (Guyer, R. Let al., J. Immunol. 117 (1976) 587-593 and Kim, J. K et al., J. Immunol. 24 (1994) 2429-2434), as described in U.S. Patent No. 2005 / 0014934. These antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include substitutions in one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, those with a substitution in Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, AR and Winter, G., Nature 322(1988)738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and International Publication No. 94 / 29351.

[0195] Binding to the Fc receptor can be easily measured, for example, by ELISA or surface plasmon resonance (SPR) using standard instruments such as the BIAcore instrument (GE Healthcare), and the Fc receptor can be obtained by recombinant expression. Such appropriate binding assays are described herein. Alternatively, the binding affinity of the Fc domain or a cell-activating bispecific antigen-binding molecule containing the Fc domain to the Fc receptor may be evaluated using cell lines known to express a specific Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor). The effector function of the Fc domain or the bispecific antibody of the present invention containing the Fc domain can be measured by methods known in the art. An appropriate assay for measuring ADCC is described herein. Other examples of in vitro assays for evaluating the ADCC activity of a target molecule 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) and U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96). (登録商標) Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, for example, as disclosed by Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0196] The following sections describe preferred embodiments of the bispecific antibodies of the present invention, including Fc domain modifications that reduce Fc receptor binding and / or effector function. In one embodiment, 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 the Fcγ receptor, particularly to the Fc receptor. In another embodiment, 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 embodiment, the Fc domain is an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0197] Modification of the Fc domain to promote heterodimerization The bispecific antigen-binding molecules described herein include different antigen-binding domains fused to one or the other of two subunits of the Fc domain, and therefore the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Several combinations of two polypeptides are possible through recombinant co-expression of these polypeptides and subsequent dimerization. To increase the yield and purity of the bispecific antibodies of the present invention in recombinant production, it is advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecules disclosed herein that promote the association of the desired polypeptides.

[0198] Accordingly, in certain embodiments, anti-PD1 / anti-LAG3 bispecific antibodies and / or anti-HLA-G / anti-CD3 antibodies are provided, which include a modification in which the Fc domain facilitates the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located within the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located within the CH3 domain of the Fc domain.

[0199] In specific embodiments, the modification is a so-called "knob-into-hole" modification, comprising 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. Accordingly, the present invention 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 the first subunit of the Fc domain comprises a knob by the knob-in-hole method and the second subunit of the Fc domain comprises a hole. In specific embodiments, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (Kabat EU index numbering).

[0200] The knob-into-hole technique is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996); and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the projection can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the projection is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0201] Accordingly, in one embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen-binding molecule of the present invention, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a repositionable protrusion within the CH3 domain of the first subunit within a cavity in 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 generating a cavity within the CH3 domain of the second subunit, within which the protrusion in the CH3 domain of the first subunit is repositionable. The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In certain embodiments, the threonine residue at position 366 in the CH3 domain of the first subunit of the Fc domain is replaced with a tryptophan residue (T366W), and the tyrosine residue at position 407 in the CH3 domain of the second subunit of the Fc domain is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0202] In a further embodiment, the serine residue at position 354 in the first subunit of the Fc domain is further replaced with a cysteine ​​residue (S354C), and the tyrosine residue at position 349 in the second subunit of the Fc domain is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues leads to the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a specific embodiment, the first subunit of the Fc domain contains amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S, and Y407V (Kabat EU index numbering).

[0203] However, other knob-in-hole technologies described in European Patent No. 1870459 may also be used alternatively or additionally. In one embodiment, the multispecific antibody contains 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" (numbered according to the Kabat EU index).

[0204] In one embodiment, the bispecific antibody contains the T366W mutation in the CH3 domain of the "knob chain," the T366S, L368A, and Y407V mutations in the CH3 domain of the "hole" chain, and further contains the R409D and K370E mutations in the CH3 domain of the "knob chain," and the D399K and E357K mutations in the CH3 domain of the "hole chain" (numbered according to the Kabat EU index).

[0205] 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 further comprises 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 follows the Kabat EU index).

[0206] In an alternative aspect, modifications that promote the association of the first and second subunits of the Fc domain include, for example, modifications that intervene in the electrostatic steering effect, as described in PCT Publication No. WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues with charged amino acid residues at the interface of the two Fc domain subunits such that homodimer formation is electrostatically unfavorable but heterodimerization is electrostatically favorable.

[0207] In addition to the "knob-into-hole technology", other techniques for modifying the CH3 domains of the heavy chains of multispecific antibodies to force heterodimerization are also known in the art. These techniques, in particular those described 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 contemplated herein as alternatives to "knob-into-hole" in combination with bispecific antibodies.

[0208] In one embodiment, in a bispecific antibody, the method described in European Patent No. 1870459 is used to assist in the heterodimerization of the first and second heavy chains of a multispecific antibody. This method is based on the introduction of oppositely charged amino acids to specific amino acid positions at the CH3 / CH3 domain interface between the first and second heavy chains.

[0209] Accordingly, in this embodiment, in 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 their respective antibody CH3 domains, and the amino acid sequences of the CH3 domains of the first and second heavy chains each contain a series of amino acids located within the interface in the tertiary structure of the antibody, wherein, from the series of amino acids located at the interface, the first amino acid in one heavy chain's CH3 domain is substituted with a positively charged amino acid, and from the series of amino acids located at the interface, the second amino acid in the other heavy chain's CH3 domain is substituted with a negatively charged amino acid. This embodiment of the bispecific antibody is also referred to herein as a "CH3(+ / -) manipulated bispecific antibody" (where the abbreviation "+ / -" represents an amino acid charged to the opposite charge to that introduced into each CH3 domain).

[0210] In one embodiment, in a CH3(+ / -) manipulated 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.

[0211] In one embodiment, in a CH3(+ / -) manipulated 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.

[0212] In one embodiment, in a CH3(+ / -) manipulated bispecific antibody, the positively charged amino acid is K, and the negatively charged amino acid is E.

[0213] In one embodiment, in a CH3(+ / -) manipulated bispecific antibody, in the CH3 domain of one heavy chain, amino acid R at position 409 is substituted with D, and amino acid K at position 409 is substituted with E; in the CH3 domain of the other heavy chain, amino acid D at position 399 is substituted with K, and amino acid E at position 357 is substituted with K (numbering according to the Kabat EU index).

[0214] In one embodiment, the method described in International Publication No. 2013 / 157953 is used to assist in the heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with K, and in the CH3 domain of the other heavy chain, amino acid L at position 351 is substituted with D (numbered according to the Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with K, and amino acid L at position 351 is substituted with K, and in the CH3 domain of the other heavy chain, amino acid L at position 351 is substituted with D (numbered according to the Kabat EU index).

[0215] In another embodiment, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with K, and amino acid L at position 351 is substituted with K, and in the CH3 domain of another heavy chain, amino acid L at position 351 is substituted with D (Kabat EU index numbering). Furthermore, at least one of the following substitutions is included in the CH3 domain of the other heavy chain: amino acid Y at position 349 is substituted with E, amino acid Y at position 349 is substituted with D, and amino acid L at position 368 is substituted with E (Kabat EU index numbering). In one embodiment, amino acid L at position 368 is substituted with E (Kabat EU index numbering).

[0216] In one embodiment, the method described in International Publication No. 2012 / 058768 is used to assist in the heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, amino acid L at position 351 is substituted with Y, and amino acid Y at position 407 is substituted with A, and in the CH3 domain of the other heavy chain, amino acid T at position 366 is substituted with A, and amino acid K at position 409 is substituted with F (numbered according to the 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 at positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N), and 392 (originally K) is substituted (numbered according to the Kabat EU index). Preferred substitutions are as follows: Replace amino acid T at position 411 with an amino acid selected from N, R, Q, K, D, E, and W (numbered according to the Kabat EU index). Replace amino acid D at position 399 with an amino acid selected from R, W, Y, and K (numbered according to the Kabat EU index). Replace amino acid S at position 400 with an amino acid selected from E, D, R, and K (numbered according to the Kabat EU index). Replace amino acid F at position 405 with an amino acid selected from I, M, T, S, V, and W (numbered according to the Kabat EU index). Replace amino acid N at position 390 with an amino acid selected from R, K, and D (numbered according to the Kabat EU index). The amino acid K at position 392 is replaced with an amino acid selected from V, M, R, L, F, and E (numbered according to the Kabat EU index).

[0217] In another embodiment, the bispecific antibody is manipulated according to International Publication No. 2012 / 058768), namely, in the CH3 domain of one heavy chain, amino acid L at position 351 is substituted with Y, and amino acid Y at position 407 is substituted with A, and in the CH3 domain of the other heavy chain, amino acid T at position 366 is substituted with V, and amino acid K at position 409 is substituted with F (numbered according to the Kabat EU index). In yet another embodiment of the multispecific antibody, in the CH3 domain of one heavy chain, amino acid Y at position 407 is substituted with A, and in the CH3 domain of the other heavy chain, amino acid T at position 366 is substituted with A, and amino acid K at position 409 is substituted with F (numbered according to the Kabat EU index). In the latter embodiment described above, in the CH3 domain of the other heavy chain, amino acid K at position 392 is substituted with E, amino acid T at position 411 is substituted with E, amino acid D at position 399 is substituted with R, and amino acid S at position 400 is substituted with R (numbering according to the Kabat EU index).

[0218] In one embodiment, the method described in International Publication No. 2011 / 143545 is used to assist in the heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, amino acid modifications in the CH3 domains of both heavy chains are introduced at positions 368 and / or 409 (numbered according to the Kabat EU index).

[0219] In one embodiment, the method described in International Publication No. 2011 / 090762 is used to assist in the heterodimerization of the first and second heavy chains of a bispecific antibody. International Publication No. 2011 / 090762 relates to amino acid modification by the "knob-into-hole" (KiH) technique. In one embodiment, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with W, and in the CH3 domain of the other heavy chain, amino acid Y at position 407 is substituted with A (numbered according to the Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with Y, and in the CH3 domain of the other heavy chain, amino acid Y at position 407 is substituted with T (numbered according to the Kabat EU index).

[0220] In one embodiment, the method described in International Publication No. 2009 / 089004 is used to assist in the heterodimerization of the first and second heavy chains of a bispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, amino acid K or N at position 392 is substituted with a negatively charged amino acid (in one embodiment, by E or D, in one preferred embodiment, by D), and in the CH3 domain of the other heavy chain, amino acid D at position 399, amino acid E or D at position 356, or amino acid E at position 357 is substituted with a positively charged amino acid (in one embodiment, by K or R, in one preferred embodiment, by K, and in one preferred embodiment, by K at position 399 or 356) (numbering by Kabat EU index). In a further embodiment, in addition to the above substitutions, in one heavy chain's CH3 domain, amino acid K or R at position 409 is substituted with a negatively charged amino acid (in one embodiment, by E or D, and in a preferred embodiment, by D) (numbered by the Kabat EU index). In one even further embodiment, in addition to, or instead of, the above substitutions, in one heavy chain's CH3 domain, amino acid K at position 439 and / or amino acid K at position 370 are independently substituted with negatively charged amino acids (in one embodiment, by E or D, and in a preferred embodiment, by D) (numbered by the Kabat EU index).

[0221] In one embodiment, the method described in International Publication No. 2007 / 147901 is used to assist in the heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, amino acid K at position 253 is substituted with E, amino acid D at position 282 is substituted with K, and amino acid K at position 322 is substituted with D. In the CH3 domain of the other heavy chain, amino acid D at position 239 is substituted with K, amino acid E at position 240 is substituted with K, and amino acid K at position 292 is substituted with D (numbered according to the Kabat EU index).

[0222] The C-terminus of the heavy chain of a bispecific antibody, as reported herein, may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus, in which one or two of the C-terminal amino acid residues are removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG.

[0223] In one embodiment of all the embodiments reported herein, the bispecific antibody comprising a heavy chain containing the C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of all the embodiments reported herein, the bispecific antibody comprising a heavy chain containing the C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbered according to the Kabat EU index).

[0224] Modifications in the Fab domain In one embodiment, an anti-PD1 / anti-LAG3 bispecific antibody and / or anti-HLA-G / anti-CD3 antibody is provided, in which either the variable domains VH and VL or the constant domains CH1 and CL are exchanged in one of the Fab fragments. The bispecific antibody is prepared according to Crossmab technology.

[0225] Multispecific antibodies with domain substitution / exchange in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in International Publication 2009 / 080252, International Publication 2009 / 080253 and Schaefer, W. et al, PNAS, 108(2011)11187-1191. These multispecific antibodies significantly reduce byproducts resulting from mismatches between the light chain for the primary antigen and the incorrect heavy chain for the secondary antigen (compared to approaches without such domain exchange).

[0226] In certain embodiments, an anti-PD1 / anti-LAG3 bispecific antibody and / or anti-HLA-G / anti-CD3 antibody is provided, wherein in one of the Fab fragments, the variable domains VL and VH are substituted for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In certain embodiments, the bispecific antibody is a bispecific antibody in which the variable domains VL and VH are substituted for each other in a first Fab fragment containing an antigen-binding domain that specifically binds to PD1.

[0227] In another embodiment, to further improve correct pairing, anti-PD1 / anti-LAG3 bispecific antibodies and / or anti-HLA-G / anti-CD3 antibodies may contain different charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the cross- or non-cross-linked CH1 and CL domains. These modifications are described, for example, in International Publication 2015 / 150447, International Publication 2016 / 020309 and PCT / European Patent 2016 / 073408.

[0228] In a particular embodiment, an anti-PD1 / anti-LAG3 bispecific antibody and / or anti-HLA-G / anti-CD3 antibody is provided, wherein in one of the Fab fragments in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered according to the Kabat EU index), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index). In a particular embodiment, the bispecific antibody is a bispecific antibody in which, in the constant domain CL of a second Fab fragment containing an antigen-binding domain that specifically binds to TIM3, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered according to the Kabat EU index), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

[0229] In a particular embodiment, an anti-PD1 / anti-LAG3 bispecific antibody and / or anti-HLA-G / anti-CD3 antibody is provided, wherein in one of the CL domains, the amino acid at position 123 (EU numbering) is replaced by arginine (R), the amino acid at position 124 (EU numbering) is replaced by lysine (K), and in one of the CH1 domains, the amino acid at position 147 (EU numbering) and the amino acid at position 213 (EU numbering) are replaced by glutamic acid (E). In a particular embodiment, the bispecific antibody is a bispecific antibody in which, in a Fab fragment containing an antigen-binding domain that specifically binds to LAG3, the amino acid at position 123 (EU numbering) is replaced by arginine (R), the amino acid at position 124 (EU numbering) is replaced by lysine (K), and in one of the CH1 domains, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced by glutamic acid (E).

[0230] In a further embodiment, a bispecific antibody is a bivalent antibody, a) The first light chain and first heavy chain of an antibody that specifically binds to the first antigen, b) comprising 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 interchangeable.

[0231] The antibody in a) does not contain the modification reported in b), and the heavy and light chains in a) are isolated chains.

[0232] In antibody b), within the light chain, the variable light chain domain VL is replaced by the variable heavy chain domain VH of the antibody, and within the heavy chain, the variable heavy chain domain VH is replaced by the variable light chain domain VL of the antibody.

[0233] In one embodiment, in the constant domain CL of the first light chain of (i)(a), the amino acid at position 124 (Kabat numbering) is substituted with a positively charged amino acid, and in the constant domain CH1 of the first heavy chain of (a), the amino acid at position 147 or the amino acid at position 213 (Kabat EU index numbering) is substituted with a negatively charged amino acid, or (ii)(b), in the constant domain CL of the second light chain of (b), the amino acid at position 124 (Kabat numbering) is substituted with a positively charged amino acid, and in the constant domain CH1 of the second heavy chain of (b), the amino acid at position 147 or the amino acid at position 213 (Kabat EU index numbering) is substituted with a negatively charged amino acid.

[0234] In another embodiment, in the constant domain CL of the first light chain of (i)(a), the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one preferred embodiment, independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first heavy chain of (a), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat numbering). (Numbering by EU Index), or in the constant domain CL of the second light chain of (ii)(b), the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering by Kabat) (in a preferred embodiment, independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the second heavy chain of (b), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering by Kabat EU Index).

[0235] In one embodiment, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted with K (numbered according to the Kabat EU index).

[0236] In one embodiment, in the constant domain CL of the second heavy chain, the amino acid at position 123 is substituted with R, and the amino acid at position 124 is substituted with K (numbered according to the Kabat EU index).

[0237] In one embodiment, in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted with E (numbered according to the Kabat EU index).

[0238] In one embodiment, in the constant domain CL of the first light chain, amino acids at positions 124 and 123 are substituted with K, and in the constant domain CH1 of the first heavy chain, amino acids at positions 147 and 213 are substituted with E (numbered according to the Kabat EU index).

[0239] In one embodiment, in the constant domain CL of the first light chain, the amino acid at position 123 is substituted with R, and the amino acid at position 124 is substituted with K, and in the constant domain CH1 of the first heavy chain, both amino acids at positions 147 and 213 are substituted with E (numbering according to the Kabat EU index).

[0240] In one embodiment, in the constant domain CL of the second heavy chain, amino acids at positions 124 and 123 are substituted with K; in the constant domain CH1 of the second light chain, amino acids at positions 147 and 213 are substituted with E; in the variable domain VL of the first light chain, amino acid at position 38 is substituted with K; in the variable domain VH of the first heavy chain, amino acid at position 39 is substituted with E; in the variable domain VL of the second heavy chain, amino acid at position 38 is substituted with K; and in the variable domain VH of the second light chain, amino acid at position 39 is substituted with E (numbering by Kabat EU index).

[0241] In one embodiment, a bispecific antibody is a bivalent antibody. a) The first light chain and first heavy chain of an antibody that specifically binds to the first antigen, b) comprising 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 substituted for each other, and the constant domains CL and CH1 of the second light chain and the second heavy chain are substituted for each other.

[0242] The antibody in a) does not contain the modifications reported in b), and the heavy and light chains in a) are isolated chains. In the antibody in b), within the light chain, the variable light chain domain VL is replaced by the variable heavy chain domain VH of the antibody, and the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody. Within the heavy chain, the variable heavy chain domain VH is replaced by the variable light chain domain VL of the antibody, and the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of the antibody.

[0243] In one embodiment, a bispecific antibody is a bivalent antibody. a) The first light chain and first heavy chain of an antibody that specifically binds to the first antigen, b) comprising 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 CH1 of the second light chain and the second heavy chain are substituted for each other.

[0244] The antibody in a) does not contain the modifications reported in b), and the heavy and light chains in a) are isolated chains. In the antibody in b), within the light chain, the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody, and within the heavy chain, the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of the antibody.

[0245] In one embodiment, a bispecific antibody is a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains, b) A bispecific antibody comprising one, two, three, or four single-chain Fab fragments that specifically bind to a second antigen, The single-stranded Fab fragment in b) is fused to the full-length antibody in a) via a peptide linker at the C-terminus or N-terminus of the heavy or light chain of the full-length antibody.

[0246] In one embodiment, one or two identical single-chain Fab fragments bound to a second antigen are fused to a full-length antibody via a peptide linker at the C-terminus of the heavy or light chain of the full-length antibody.

[0247] In one embodiment, one or two identical single-chain Fab (scFab) fragments bound to a second antigen are fused to a full-length antibody via a peptide linker at the C-terminus of the heavy chain of the full-length antibody.

[0248] In one embodiment, one or two identical single-chain Fab (scFab) fragments bound to a second antigen are fused to a full-length antibody via a peptide linker at the C-terminus of the light chain of the full-length antibody.

[0249] In one embodiment, two identical single-chain Fab(scFab) fragments bound to a second antigen are fused to a full-length antibody via a peptide linker at the respective C-terminuses of the heavy or light chain of the full-length antibody.

[0250] In one embodiment, two identical single-chain Fab(scFab) fragments that bind to a second antigen are fused to a full-length antibody via a peptide linker at the C-terminus of each heavy chain of the full-length antibody.

[0251] In one embodiment, two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to a full-length antibody via a peptide linker at the C-terminus of each light chain of the full-length antibody.

[0252] In one embodiment, a bispecific antibody is a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains, b) ba) Antibody heavy chain variable domain (VH), or bb) A first polypeptide comprising an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), The first polypeptide is fused to the N-terminus of the VH domain of one of the two heavy chains of the full-length antibody via a peptide linker. (c) ca) Antibody light chain variable domain (VL), or cb) A second polypeptide comprising an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), The second polypeptide is a trivalent antibody comprising a second polypeptide fused to the N-terminus of the VL domain via a peptide linker to the other C-terminus of the two heavy chains of the full-length antibody. 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.

[0253] In one embodiment, the antibody heavy chain variable domain (VH) of polypeptide (b) and the antibody light chain variable domain (VL) of polypeptide (c) are linked and stabilized via interchain disulfide crosslinks by introducing disulfide bonds between the following positions. (i) From position 44 of the heavy chain variable domain to position 100 of the light chain variable domain, (ii) From position 105 of the heavy chain variable domain to position 43 of the light chain variable domain, (iii) From position 101 of the heavy chain variable domain to position 100 of the light chain variable domain (always numbered according to the Kabat EU index).

[0254] Techniques for introducing unnatural disulfide crosslinks for stabilization are described in International Publication No. 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, any disulfide bond between the variable domains of polypeptides b) and c) is located between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, any disulfide bond between the variable domains of polypeptides b) and c) is located between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (always numbered by Kabat). In one embodiment, a trivalent bispecific antibody that does not have the arbitrary disulfide stabilization between the variable domains VH and VL of the single-stranded Fab fragment is preferred.

[0255] In one embodiment, the bispecific antibody is a tripspecific antibody or a quadruplespecific antibody. a) A first light chain and a first heavy chain of a full-length antibody that specifically binds to the first antigen, b) comprising a second (modified) light chain and a second (modified) heavy chain of a full-length antibody, which specifically bind to a second antigen and in which the variable domains VL and VH are substituted for each other, and / or the constant domains CL and CH1 are substituted for each other. c) One to four antigen-binding domains that specifically bind to one or two additional antigens (i.e., a third and / or fourth antigen) are fused to the C-terminus or N-terminus of the light or heavy chain of a) and / or b) via a peptide linker.

[0256] The antibody in a) does not contain the modifications reported in b), and the heavy and light chains in a) are isolated chains.

[0257] In one embodiment, a triplicate or quadruplicate antibody comprises one or two antigen-binding domains that specifically bind to one or two further antigens, as described in c).

[0258] In one embodiment, the antigen-binding domain is selected from the group consisting of scFv fragments and scFab fragments.

[0259] In one embodiment, the antigen-binding domain is an scFv fragment.

[0260] In one embodiment, the antigen-binding domain is an scFab fragment.

[0261] In one embodiment, the antigen-binding domain is fused to the C-terminus of the heavy chains a) and / or b).

[0262] In one embodiment, a triplicate or quadruplicate antibody comprises one or two antigen-binding domains that specifically bind to one further antigen, as described in c).

[0263] In one embodiment, a triplicate or quadruplicate antibody contains two identical antigen-binding domains that specifically bind to a third antigen under c). In a preferred embodiment, both of these identical antigen-binding domains are fused to the C-terminus of the heavy chains a) and b) via the same peptide linker. In a preferred embodiment, the two identical antigen-binding domains are scFv or scFab fragments.

[0264] In one embodiment, a triplicate or quadruplicate antibody includes two antigen-binding domains that specifically bind to a third and a fourth antigen under c). In one embodiment, both of these antigen-binding domains are fused to the C-terminus of the heavy chain under a) and b) via the same peptide linkage. In a preferred embodiment, the two antigen-binding domains are scFv or scFab fragments.

[0265] In one embodiment, the bispecific antibody is a bispecific tetravalent antibody. a) Two light chains and two heavy chains (and two Fab fragments) of an antibody that specifically binds to the first antigen, b) comprising two further Fab fragments of an antibody that specifically binds to a second antigen, both of which are fused to either the C-terminus or N-terminus of the heavy chain of (a) via a peptide linker. The following modifications were made to the Fab fragment. (i) In both Fab fragments of a) or in both 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, (ii) In both Fab fragments of a), the variable domains VL and VH are replaced by each other, and the steady domains CL and CH1 are replaced by each other, and in both Fab fragments of b), either the variable domains VL and VH are replaced by each other, or the steady domains CL and CH1 are replaced by each other, (iii) In both Fab fragments of (a), the variable domains VL and VH are substituted for each other, or the steady domains CL and CH1 are substituted for each other, and in both Fab fragments of (b), the variable domains VL and VH are substituted for each other, and the steady domains CL and CH1 are substituted for each other, or (iv) In both Fab fragments of (a), the variable domains VL and VH are substituted for each other, and in both Fab fragments of (b), the constant domains CL and CH1 are substituted for each other, or (v) In both Fab fragments of a), the constant domains CL and CH1 are replaced by each other, and in both Fab fragments of b), the variable domains VL and VH are replaced by each other.

[0266] In one embodiment, both of the further Fab fragments are fused via a peptide linker to either the C-terminus of the heavy chain of a) or the N-terminus of the heavy chain of a).

[0267] In one embodiment, both of the further Fab fragments are fused to either of the C-terminuses of the heavy chain of a) via a peptide linker.

[0268] In one embodiment, both of the further Fab fragments are fused to the N-terminus of the heavy chain of a) via a peptide linker.

[0269] In one embodiment, the following modifications are made to the Fab fragment: 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.

[0270] In one embodiment, the bispecific antibody is a tetravalent antibody. a) A (modified) heavy chain of the first antibody, which specifically binds to the first antigen and contains a first VH-CH1 domain pair, wherein the N-terminus of the second VH-CH1 domain pair of the first antibody is fused to the C-terminus of the heavy chain via a peptide linker, b) The two light chains of the first antibody in a), c) A (modified) heavy chain of a second antibody, which specifically binds to a second antigen, contains a first VH-CL domain pair, and has the N-terminus of the second VH-CL domain pair of the second antibody fused to the C-terminus of the heavy chain via a peptide linker, d) comprising two (modified) light chains of the second antibody in c), each containing a CL-CH1 domain pair.

[0271] In one embodiment, a bispecific antibody is a) A heavy chain and light chain of a first full-length antibody that specifically binds to the first antigen, b) comprising a heavy chain and a light chain of a second full-length antibody, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker.

[0272] The antibody in a) does not contain the modifications reported in b), and the heavy and light chains are isolated chains.

[0273] In one embodiment, a bispecific antibody is a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains, b) comprising an Fv fragment that specifically binds to a second antigen, which includes a VH2 domain and a VL2 domain, wherein both domains are connected to each other via disulfide crosslinks, Either the VH2 domain or the VL2 domain is fused via a peptide linker to the heavy or light chain of a full-length antibody that specifically binds to the first antigen.

[0274] In bispecific antibodies, the heavy and light chains in a) are isolated chains.

[0275] In one embodiment, the other of the VH2 domain or VL2 domain is not fused to the heavy or light chain of a full-length antibody that specifically binds to the first antigen via a peptide linker.

[0276] In all embodiments 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.

[0277] In one embodiment, a bispecific antibody is a) Two Fab fragments that specifically bind to the first antigen, b) A single CrossFab fragment that specifically binds to the second antigen and in which the CH1 domain and CL domain are exchanged with each other, c) A trivalent antibody comprising a heavy chain in the first Fc region and one Fc region containing a heavy chain in the second Fc region, The C-terminuses of the CH1 domains of the two Fab fragments are ligated to the N-terminuses of the heavy chain Fc region polypeptide, while the C-terminus of the CL domain of the CrossFab fragment is ligated to the N-terminus of one of the VH domains of the Fab fragment.

[0278] In one embodiment, a bispecific antibody is a) Two Fab fragments that specifically bind to the first antigen, b) A single CrossFab fragment that specifically binds to the second antigen and in which the CH1 domain and CL domain are exchanged with each other, c) A trivalent antibody comprising a heavy chain in the first Fc region and one Fc region containing a heavy chain in the second Fc region, The C-terminus of the CH1 domain of the first Fab fragment is ligated to the N-terminus of one heavy chain Fc-region polypeptide, the C-terminus of the CL domain of the CrossFab fragment is ligated to the N-terminus of another heavy chain Fc-region polypeptide, and the C-terminus of the CH1 domain of the second Fab fragment is ligated 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.

[0279] In one embodiment, a bispecific antibody is a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains, b) A Fab fragment that specifically binds to a second antigen, comprising a heavy chain fragment and a light chain fragment, wherein the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of the antibody within the light chain fragment, and the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of the antibody within the heavy chain fragment, The heavy chain Fab fragment is inserted between one CH1 domain of the heavy chain of the full-length antibody and the respective 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, which is paired with the heavy chain of the full-length antibody, with the heavy chain Fab fragment inserted into it.

[0280] In one embodiment, a bispecific antibody is a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains, b) A Fab fragment that specifically binds to a second antigen containing a VH2 domain and a VL2 domain, 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, the C-terminus of the heavy chain fragment of the Fab fragment is conjugated to one N-terminus of the heavy chain 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 which forms a pair with the heavy chain of the full-length antibody, and the heavy chain fragment of the Fab fragment is conjugated to the Fab fragment.

[0281] Polynucleotides Isolated polynucleotides encoding the bispecific antibodies or fragments thereof described herein are further provided.

[0282] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide is composed of a base, specifically 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. Often, nucleic acid molecules are described by a base sequence, which represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo in a host or patient, for example. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into a target in vivo to generate an antibody (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823B1).

[0283] "Isolated" polynucleotides refer to nucleic acid molecules that have been separated from their natural environment. Isolated polynucleotides include nucleic acid molecules that are originally contained in cells but are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0284] The isolated polynucleotide encoding the bispecific antibody of the present invention may be expressed as a single polynucleotide encoding a complete antigen-binding molecule, or as a plurality (e.g., two or more) of co-expressed polynucleotides. Polypeptides encoded by the co-expressed polynucleotides may associate, for example, via disulfide bonds or other means to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide derived from the heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form an immunoglobulin.

[0285] In some embodiments, the isolated polynucleotide encodes a polypeptide contained in the bispecific antibody according to the present invention as described herein.

[0286] In one embodiment, an isolated polynucleotide encoding an anti-PD1 / anti-LAG3 bispecific antibody is provided, the first antigen-binding domain which 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.

[0287] Preparation of bispecific antibodies used in the present invention Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding antibodies are provided.

[0288] For a native antibody or native antibody fragment requiring two nucleic acids, one is for the light chain or fragment thereof, and the other is for the heavy chain or fragment thereof. Such nucleic acids encode the amino acid sequence containing the VL and / or VH of the antibody (e.g., the light chain and / or heavy chain(s) of the antibody). These nucleic acids may be on the same expression vector or on different expression vectors. For a particular bispecific antibody having a heterodimeric heavy chain requiring four nucleic acids, one is for the first light chain, one is for the first heavy chain containing the first heteromonomer Fc region polypeptide, one is for the second light chain, and one is for the second heavy chain containing the second heteromonomer Fc region polypeptide. These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors. For example, such nucleic acids encode an amino acid sequence containing a first VL and / or an amino acid sequence containing a first VH containing a first heteromonomer Fc region and / or an amino acid sequence containing a second VL and / or an amino acid sequence containing a second VH containing a second heteromonomer Fc region of the antibody (e.g., the first and / or second light chains and / or first and / or second heavy chains of the antibody). These nucleic acids may reside on the same or different expression vectors, and typically these nucleic acids reside on two or three expression vectors, i.e., one vector may contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMab and T cell bispecific antibodies (see, e.g., Schaefer, W. et al, PNAS, 108(2011)11187-1191). For example, one heteromonomer heavy chain may contain a so-called "knob mutation" (T366W and optionally one of S354C or Y349C), while the other may contain a so-called "hole mutation" (T366S, L368A, and Y407V and optionally Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0289] In one embodiment, an isolated nucleic acid encoding a bispecific antibody as described herein is provided. Such nucleic acid encodes an amino acid sequence (e.g., the light chain and / or heavy chain of the antibody) comprising an amino acid sequence comprising an antigen-binding domain VL and / or VH that specifically binds to PD1 and LAG3. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a first vector comprising a first nucleic acid pair encoding an amino acid sequence, one of which comprises the first VL of the antibody and the other comprises the first VH; and a second vector comprising a second nucleic acid pair encoding an amino acid sequence, one of which comprises the second VL of the antibody and the other comprises the second VH; or (2) a first vector comprising a first nucleic acid encoding an amino acid sequence, one of which comprises a variable domain (preferably a light chain variable domain); and a nucleic acid pair comprising a light chain variable domain (3) A third vector comprising a nucleic acid pair encoding an amino acid sequence, wherein one of the amino acid sequences comprises a chain variable domain, the other comprising a first heavy chain variable domain, and (4) a nucleic acid encoding an amino acid sequence, wherein one of the amino acid sequences comprises the other light chain variable domain from the second vector, and the other comprising a second heavy chain variable domain, or (5) a nucleic acid encoding an amino acid sequence, wherein the amino acid sequence comprises a first VL of the antibody, a second vector comprising a nucleic acid encoding an amino acid sequence comprising a first VH of the antibody, a third vector comprising a nucleic acid encoding an amino acid sequence comprising a second VL of the antibody, and a fourth vector comprising a nucleic acid encoding an amino acid sequence comprising a second VH of the antibody. In one embodiment, the host cell is a eukaryote, for example, a Chinese hamster ovary (CHO) cell or lymphocyte (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for producing a bispecific antibody is provided, the method comprising culturing a host cell comprising the nucleic acid encoding the antibody under conditions suitable for antibody expression, as provided above, and optionally recovering the antibody from the host cell (or host cell medium).

[0290] For the recombinant production of anti-HLA-G / anti-CD3 bispecific antibodies and / or anti-PD1 / anti-LAG3 bispecific antibodies described herein, for example, nucleic acids encoding such bispecific antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids may be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

[0291] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254 (which describes the expression of antibody fragments in Escherichia coli)). After expression, antibodies may be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0292] In addition to prokaryotes, eukaryotes such as filamentous fungi and yeasts are suitable as cloning or expression hosts for antibody-encoding vectors, including strains of fungi and yeast with "humanized" glycosylation pathways, resulting in the production of antibodies with partially or completely human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24(2006) 210-215.

[0293] Furthermore, suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified, and these may be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0294] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (PLANTIBODIES® technology for antibody production in transgenic plants).

[0295] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7), human embryonic kidney cells (e.g., 293 cells or 293 cells as described in Graham et al., J Gen Virol 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical tumor cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather et al., Annals). These are MRC5 cells and FS4 cells, as described in NYAcad.Sci.383(1982)44-68. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlaub, G. et al., Proc. Natl. Acad.Sci. USA 77(1980)4216-4220), including DHFR-CHO cells, 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, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol.248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp.255-268.

[0296] Assay The bispecific antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0297] 1. Affinity assay The affinity of the bispecific antigen-binding molecules, antibodies, and antibody fragments provided herein for the corresponding antigen can be determined by surface plasmon resonance (SPR) using a standard instrument such as a Biacore® instrument (GE Healthcare), a receptor, or a target protein that can be obtained by recombinant expression, according to the method described in the examples. Specific exemplary and exemplary embodiments for measuring binding affinity are described in Examples 2, 8, or 11 of International Publication No. 2018 / 185043. According to one embodiment, K D This is measured using a BIACORE® T100 instrument (GE Healthcare) by surface plasmon resonance at 25°C.

[0298] 2. Binding assays and other assays In one embodiment, the bispecific antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA and Western blotting. The binding of the anti-PD1 / anti-LAG3 bispecific antibody provided herein to the corresponding recombinant antigen or antigen-expressing cells can be evaluated by ELISA as described in Example 8 or 11 of International Publication No. 2018 / 185043. In a further embodiment, fresh peripheral blood mononuclear cells (PBMCs) may be used in a binding assay to demonstrate binding to different peripheral blood mononuclear cells (PBMCs), such as monocytes, NK cells, and T cells.

[0299] In another embodiment, a cell dimerization assay was used to demonstrate the dimerization or at least binding / interaction of two distinct receptors, PD1 and LAG3, fused to the cytosol with two fragments of the enzyme upon ligation or crosslinking with bispecific antibodies against both targets. This resulted in only one receptor exhibiting no enzymatic activity. For this specific interaction, the cytosolic C-terminuses of both receptors fused individually to heterologous subunits of the reporter enzyme. Only one enzyme subunit exhibited no reporter activity. However, simultaneous binding to both receptors is expected to lead to local cellular accumulation of both receptors, complementarity of the two heterologous enzyme subunits, and ultimately, the formation of a specific, functional enzyme that hydrolyzes the substrate and thereby generates a chemiluminescent signal (Example 11 of International Publication No. 2018 / 185043).

[0300] 3. Activity assay In one embodiment, an assay is provided for identifying anti-PD1 / anti-LAG3 bispecific antibodies having biological activity. Biological activity may include, for example, activation and / or proliferation of different immune cells, particularly T cells; secretion of immunomodulatory cytokines, such as IFNγ or TNF-alpha; blocking of the PD1 pathway; blocking of the LAG3 pathway; and the ability to enhance tumor cell death. Antibodies having such biological activity in vivo and / or in vitro are also provided. In certain embodiments, the antibodies of the present invention are tested for such biological activity. In one embodiment, an immunocytological assay is provided to measure the activation of lymphocytes from one individual (donor X) to lymphocytes from another individual (donor Y). The use of a mixed lymphocyte reaction (MLR) can demonstrate the effect of blocking the PD1 pathway against lymphocyte effector cells. T cells in this assay were tested for activation and their IFN-gamma secretion in the presence or absence of the bispecific antibodies of the present invention. This assay is described in detail in Example 9 of International Publication No. 2018 / 185043.

[0301] Pharmaceutical compositions, formulations, and routes of administration In further embodiments, the present invention provides pharmaceutical compositions comprising anti-HLA-G / anti-CD3 antibodies and anti-PD1 / anti-LAG3 antibodies provided herein for use in, for example, any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises the anti-HLA-G / anti-CD3 antibodies and anti-PD1 / anti-LAG3 antibodies provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any of the bispecific antibodies provided herein and at least one additional therapeutic agent (e.g., those described below).

[0302] The pharmaceutical compositions of the present invention comprise one or more bispecific antibodies in a therapeutically effective amount dissolved or dispersed in a pharmaceutically acceptable excipient. The expression “pharmaceutically or pharmacologically acceptable” means molecular entities and compositions that are generally non-toxic to the recipient at the dose and concentration employed, i.e., that do not cause adverse reactions, allergic reactions, or other adverse reactions when administered to animals such as humans. The preparation of pharmaceutical compositions comprising at least one antibody and optionally additional active ingredients will be known to those skilled in the art in light of this disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference. In particular, compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutically acceptable excipients” include any solvent, buffer, dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antimicrobial, antifungal), isotonic agent, salt, stabilizer, and combinations thereof, as known to those skilled in the art.

[0303] 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 molecule of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the fusion protein may be in powder form for preparation with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the required amount of the fusion protein of the present invention, along with various other components listed below, if necessary, into a suitable solvent. Sterility can be easily achieved, for example, by filtration with a sterile filtration membrane. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and / or other components. For sterile powders used to prepare sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which yields powders of the active ingredient and any further desired ingredients from an already sterile filtered liquid medium. The liquid medium should be adequately buffered if necessary, and the liquid diluent should be first isotonicized with sufficient saline or glucose before injection. The composition must be stable under manufacturing and storage conditions and protected from microbial contamination such as bacteria and fungi. It will be understood that endotoxin contamination should be kept to a minimum, e.g., less than 0.5 ng / mg of protein.Suitable pharmaceutically acceptable excipients include, but are not limited to, buffers, e.g., phosphates, citrates and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens, e.g., methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than approximately 10 residues) polypeptides, and Examples of active ingredients include proteins, such as serum albumin, gelatin, or immunoglobulin; 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). The aqueous injection suspension may also contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain appropriate stabilizers or agents that increase the solubility of the compound to enable the preparation of a highly concentrated solution. Furthermore, the suspension of the active compound may be prepared as a suitable oil injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethylcrete or triglycerides, or liposomes.

[0304] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), or encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such technologies are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing a polypeptide, which may be in the form of a molded article such as a film or microcapsule. In certain embodiments, sustained absorption of the injectable composition may be achieved by using a slow-absorbing agent in the composition, such as aluminum monostearate, gelatin, or a combination thereof.

[0305] Exemplary pharmaceutically acceptable excipients of the present invention further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Applications Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycans (e.g., chondroitinases).

[0306] An example of a lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which includes histidine-acetate buffer.

[0307] In addition to the compositions already described, bispecific antibodies may be formulated as depot formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Therefore, for example, the fusion protein may be formulated using a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a slightly insoluble derivative, for example, as a slightly insoluble salt.

[0308] The pharmaceutical composition containing the bispecific antigen-binding molecule of the present invention can be manufactured by conventional mixing, dissolution, emulsification, encapsulation, encapsulation, or lyophilization processes. The pharmaceutical composition can be formulated by conventional methods using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries that facilitate the processing of the protein into a pharmaceutically usable formulation. The appropriate formulation depends on the selected route of administration.

[0309] The bispecific antibodies disclosed herein may be formulated into compositions in the form of free acids or free bases, neutral or salt forms. A pharmaceutically acceptable salt is one that substantially retains the biological activity of the free acid or free base. These include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than their corresponding free base forms.

[0310] The compositions herein may also contain multiple active ingredients necessary for the specific indication being treated, preferably having complementary activities that do not adversely affect one another. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0311] In one embodiment, a pharmaceutical composition comprising an anti-HLA-G / anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier is provided, as well as a second pharmaceutical comprising the anti-PD1 / anti-LAG3 antibody described herein. In one embodiment, the pharmaceutical composition is for use in the treatment of HLA-G expressing cancers. In a particular embodiment, the pharmaceutical composition is for HLA-G expressing cancers, in particular lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer It is intended for use in the treatment of diseases selected from one or more of the following cancers: colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, nasopharyngeal carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia (including refractory versions of any of the cancers listed above), or combinations of one or more of the cancers listed above.

[0312] Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0313] Administration of anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies Both anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies (both referred to herein as "substances") may be administered by any preferred means, including parenteral administration, intrapulmonary administration, intranasal administration, and, if desired for local treatment, intralesional administration. However, the methods described herein are particularly useful with respect to therapeutic agents administered parenterally, especially by intravenous infusion.

[0314] Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. The administration may be by any preferred route, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic. Various administration schedules are considered herein, including but not limited to single doses, multiple doses at various time points, bolus administration, and pulse infusion. In one embodiment, the therapeutic agent is administered parenterally, particularly intravenously. In certain embodiments, the substance is administered by intravenous infusion. In another embodiment, the substance is administered subcutaneously.

[0315] Both anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies will be formulated, administered, and given in a manner consistent with best practices. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical symptoms, the cause of the disorder, the site of drug delivery, the method of administration, the administration scheduling, and other factors known to the healthcare provider. Both anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies may, but are not necessarily, be formulated together with one or more drugs currently used to prevent or treat the disorder in question. The effective dose of such other drugs will depend on the amount of therapeutic agent present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These will generally be used by the same dosages and routes of administration as described herein, or at approximately 1–99% of the dosages described herein, or by any dosage and route as empirically / clinically deemed appropriate.

[0316] For the prevention or treatment of disease, the appropriate dosage of anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies (when used in combination, or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of anti-HLA-G / anti-CD3 bispecific antibody, the severity and course of the disease, whether both agents are administered for preventive or therapeutic purposes, previous treatments, the patient's medical history and response to therapeutic agents, and the discretion of the attending physician. Each substance is administered appropriately to the patient, either as a single dose or over a series of treatments. Depending on the type and severity of the disease, a dose of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the substance may be the initial candidate dose to be administered to the target, whether, for example, by one or more separate doses or by continuous infusions. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. In repeated administrations over several days or more, treatment is usually continued, depending on the symptoms, until the desired suppression of disease symptoms occurs. One exemplary dosage of bispecific antibodies ranges from about 0.005 mg / kg to about 10 mg / kg. In other examples, the dosage may also include, per administration, about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 500 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 inducible range in between. Examples of doses that can be derived from the numbers listed herein include approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight, etc., which may be administered based on the numbers mentioned above. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to a patient.Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives approximately 2 to approximately 20 doses of antibody, or for example, approximately 6 doses). An initial higher loading dose may be administered, followed by one or more lower doses. However, other drug regimens may also be useful. The progress of this treatment is readily monitored by conventional techniques and assays. However, other drug regimens may also be useful. The progress of this treatment is readily monitored by conventional techniques and assays.

[0317] In one embodiment, the administration of both the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody is a single dose. In certain embodiments, the therapeutic agent is administered in two or more doses. In such embodiments, the substance is administered weekly, bi-weekly, or tri-weekly, particularly every two weeks. In one embodiment, the substance is administered in a therapeutically effective dose. In one embodiment, the substance is administered in doses of approximately 10 μg / kg, approximately 100 μg / kg, approximately 200 μg / kg, approximately 300 μg / kg, approximately 400 μg / kg, approximately 500 μg / kg, approximately 600 μg / kg, approximately 700 μg / kg, approximately 800 μg / kg, approximately 900 μg / kg, or approximately 1000 μg / kg. In one embodiment, an anti-HLA-G / anti-CD3 bispecific antibody is administered at a higher dose than the dose of the anti-HLA-G / anti-CD3 bispecific antibody in the corresponding treatment regimen, without the administration of an anti-PD1 / anti-LAG3 antibody. In one embodiment, the administration of the anti-HLA-G / anti-CD3 bispecific antibody includes an initial dose of a first dose of the anti-HLA-G / anti-CD3 bispecific antibody and one or more subsequent doses of a second dose of the anti-HLA-G / anti-CD3 bispecific antibody, where the second dose is higher than the first dose. In one embodiment, the administration of the anti-HLA-G / anti-CD3 bispecific antibody includes an initial dose of a first dose of the anti-HLA-G / anti-CD3 bispecific antibody and one or more subsequent doses of a second dose of the anti-HLA-G / anti-CD3 bispecific antibody, where the first dose is not lower than the second dose.

[0318] In one embodiment, the administration of the anti-HLA-G / anti-CD3 bispecific antibody in the treatment regimen according to the present invention is the first administration of the anti-HLA-G / anti-CD3 bispecific antibody to the subject (at least within the scope of the same treatment process). In one embodiment, the administration of the anti-PD1 / anti-LAG3 antibody is not performed on the subject before the administration of the anti-HLA-G / anti-CD3 bispecific antibody. In another embodiment, the anti-PD1 / anti-LAG3 antibody is administered before the administration of the anti-HLA-G / anti-CD3 bispecific antibody.

[0319] In the present invention, a 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 additional agents in a therapeutic setting. For example, at least one additional therapeutic agent can be administered simultaneously. In certain embodiments, the additional therapeutic agent is an immunotherapy agent.

[0320] The combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case the administration of a therapeutic agent may be performed before, simultaneously with, and / or after the administration of an additional therapeutic agent or drug. In one embodiment, the administration of a therapeutic agent and the administration of an additional therapeutic agent may occur within approximately one month of each other, or within approximately one, two, or three weeks, or within approximately one, two, three, four, five, or six days.

[0321] Treatment method and composition HLA-G is primarily expressed on trophotrophic cells of the placenta, but it has also been reported that various tumors (including those of the pancreas, breast, skin, colon, rectum, stomach, and ovaries) express HLA-G.

[0322] One embodiment provides a method for treating or delaying the progression of HLA-G expressing cancer in a subject, comprising administering an effective amount of anti-HLA-G / anti-CD3 antibody and an effective amount of anti-PD1 / anti-LAG3 antibody to the subject.

[0323] In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the target. In some embodiments, methods are provided herein for increasing cytokine secretion (including IFNγ) and / or T cell activation, comprising administering an effective amount of anti-HLA-G / anti-CD3 antibody and an effective amount of anti-PD1 / anti-LAG3 antibody to the target. In certain embodiments, the method comprises T cell-mediated death of tumor cells. The “individual” or “target” in any of the above embodiments is preferably human.

[0324] In further embodiments, compositions for use in cancer immunotherapy are provided, comprising an anti-HLA-G / anti-CD3 antibody and an anti-PD1 / anti-LAG3 antibody. In specific embodiments, compositions comprising an anti-HLA-G / anti-CD3 antibody and an effective amount of anti-PD1 / anti-LAG3 antibody are provided for use in a method of cancer immunotherapy.

[0325] In further embodiments, the use of a composition comprising an anti-HLA-G / anti-CD3 antibody and an effective amount of anti-PD1 / anti-LAG3 antibody in the manufacture or preparation of a pharmaceutical is provided herein. In one embodiment, the pharmaceutical is for the treatment of HLA-G expressing cancer. In one embodiment, the pharmaceutical is for the treatment of HLA-G expressing cancer. In further embodiments, the pharmaceutical is for use in a method of treating HLA-G expressing cancer, comprising administering an effective amount of the pharmaceutical to an individual having HLA-G expressing cancer. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In further embodiments, the pharmaceutical is for activating T cells, in particular HLA-G expressing tumor cells. HLA-G expressing cancers include lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer. Cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, nasopharyngeal carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumor, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, 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, HLA-G expressing cancers include selected renal cell carcinoma, colorectal carcinoma, non-small cell lung cancer, and pancreatic ductal adenocarcinoma (PDAC).

[0326] In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, as described below. The “individual” in any of the above embodiments may be a human.

[0327] The combination therapies described above include combined administration (containing two or more therapeutic agents in the same or separate formulations) and separate administrations, in which case, the administration of the anti-PD1 / anti-LAG3 bispecific antibody reported herein may be performed before, concurrently with, and / or after the administration of the additional therapeutic agent (one or more). In one embodiment, the administration of an effective dose of the anti-HLA-G / anti-CD3 bispecific antibody, the administration of an effective dose of the anti-PD1 / anti-LAG3 antibody, and the administration of the additional therapeutic agent are performed within approximately one month, or within approximately one, two, or three weeks, or within approximately one, two, three, four, five, or six days.

[0328] Both the anti-HLA-G / anti-CD3 bispecific antibody and the anti-PD1 / anti-LAG3 antibody (and any additional therapeutic agents) described herein may be administered by any preferred means, including parenteral, intrapulmonary and intranasal, and, if desired for local treatment, intrafocal administration. Parenteral administration may include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic. Various dosing schedules are considered herein, including but not limited to single doses, multiple doses at various time points, bolus doses, and pulse infusions.

[0329] As reported herein, both anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies will be formulated, administered, and given in a manner consistent with best practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical symptoms, the cause of the disorder, the site of drug delivery, the method of administration, the administration scheduling, and other factors known to the healthcare provider. Antibodies will be formulated, optionally but not necessarily, with one or more drugs currently used to prevent or treat the disorder in question. The effective dose of such other drugs will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These will generally be used by the same dosages and routes of administration as described herein, or at approximately 1–99% of the dosages described herein, or by any dosage and route as empirically / clinically deemed appropriate.

[0330] Those skilled in the art will readily recognize that in many cases, bispecific antibodies may not provide a cure but may offer only partial benefits. In some embodiments, physiological changes that provide some benefit may also be considered therapeutically beneficial. Therefore, in some embodiments, the amount of bispecific antibody that induces a physiological change may be considered an "effective dose" or "therapeutic effective dose."

[0331] Both anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 antibodies as defined herein are administered appropriately to the patient either in a single dose or over a series of treatments. Depending on the type and severity of the disease, bispecific antibodies in doses of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to the patient, whether, for example, by one or more separate doses or by sequential infusions. A typical daily dose may range from approximately 1 μg / kg to 10 mg / kg, depending on the factors described above. In repeated administrations over several days or more, the treatment is usually continued, depending on the symptoms, until the desired suppression of disease symptoms occurs. An example of an exemplary dose of anti-HLA-G / anti-CD3 bispecific antibodies would range from approximately 0.05 μg / kg to approximately 1000 μg / kg. For anti-PD1 / anti-LAG3 antibodies, the dose may also include approximately 0.01 mg / kg body weight, approximately 0.05 mg / kg body weight, approximately 2 mg / kg body weight, approximately 4 mg / kg body weight, approximately 10 mg / kg body weight, approximately 20 mg / kg body weight, approximately 30 mg / kg body weight, approximately 40 mg / kg body weight, approximately 45 mg / kg body weight, approximately 50 mg / kg body weight, approximately 100 mg / kg body weight, approximately 200 mg / kg body weight, approximately 300 mg / kg body weight, approximately 400 mg / kg body weight, approximately 500 mg / kg body weight, approximately 600 mg / kg body weight, approximately 800 mg / kg body weight, approximately 1000 mg / kg body weight, up to approximately 1200 mg / kg body weight or more, and any inducible range within these. In examples of the range derivable from the numbers enumerated herein, doses of approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, approximately 0.05 μg / kg / body weight to approximately 500 mg / kg / body weight, etc., may be administered based on the aforementioned numbers. In one embodiment, the anti-HLA-G / anti-CD3 bispecific antibody may be administered to the patient in doses ranging from approximately 0.01 mg to 2.5 mg to approximately 10 mg, or approximately 20 mg, or approximately 30 mg. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives approximately 2 to approximately 20 doses of the fusion protein, or for example, approximately 6 doses). An initial lower loading dose may be administered, followed by one or more higher doses. However, other drug regimens may also be useful. The progress of this treatment is readily monitored by conventional techniques and assays.In one embodiment, anti-PD1 / anti-LAG3 can be administered to a patient in doses ranging from approximately 100 mg to approximately 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, or 1500 mg.

[0332] A bispecific antibody comprising a first antigen-binding domain that specifically binds to PD1 as defined herein and a second antigen-binding domain that specifically binds to LAG3 would be commonly used in an amount effective to achieve the intended purpose. For use in treating or preventing disease symptoms, the bispecific antibody of the present invention, or its pharmaceutical composition thereof, is administered or applied in a therapeutically effective amount. Determining the therapeutically effective amount is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0333] For systemic administration, the therapeutically effective dose can first be estimated from an in vitro assay, such as a cell culture assay. Then, the IC (Induced Concentration) is determined in the cell culture. 50 To achieve a circulating concentration range that includes [specific concentration range], doses may be formulated in animal models. This information can then be used to more accurately determine an effective dose in humans.

[0334] The initial dosage can also be estimated from in vivo data, for example, from animal models, using techniques well known in the art. Those skilled in the art will be able to easily optimize the dosage to humans based on animal data.

[0335] Dosage and dosing intervals may be individually adjusted to provide plasma levels of bispecific antibodies sufficient to maintain therapeutic effect. Typical patient doses for administration by injection range from approximately 0.1 to 50 mg / kg / day, typically from approximately 0.5 to 1 mg / kg / day. Therapeutic plasma levels may be achieved by administering multiple doses each day. Plasma levels can be measured, for example, by HPLC.

[0336] In the case of local administration or selective uptake, the effective local concentration of a bispecific antibody may be independent of the plasma concentration. Those skilled in the art can optimize the therapeutically effective local dosage without excessive experimentation.

[0337] The therapeutically effective doses of the bispecific antibodies described herein generally provide therapeutic benefits without causing substantial toxicity. The toxicity and therapeutic efficacy of the fusion proteins can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal studies, LD 50 (Dose at which 50% of the population is lethal) and ED 50 The dose at which 50% of the population is therapeutically effective can be determined. The dose-to-toxicity ratio is the therapeutic index, which is the LD50. 50 / ED 50 This can be expressed as a ratio. Bispecific antibodies exhibiting a large therapeutic index are preferred. In one embodiment, the bispecific antibody of the present invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used when formulating a dosage range suitable for human use. The dosage is preferably within the range of circulating concentrations containing an ED50 with little to no toxicity. The dosage may vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, and the target symptoms. The exact formulation, route of administration, and dosage can be selected by individual physicians in terms of the patient's symptoms (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p.1, which is incorporated herein by reference in its entirety).

[0338] The attending physician of a patient treated with the bispecific antibody of the present invention knows how and when to discontinue, interrupt, or adjust the administration due to toxicity, organ failure, etc. Conversely, the attending physician also knows how to adjust the treatment to a higher level if the clinical response is insufficient (without causing toxicity). The size of the dose administered in the management of the disorder of interest will vary depending on the severity of the symptom being treated, the route of administration, etc. The severity of the symptom may be assessed, for example, in part by standard prognostic assessment methods. Furthermore, the dose and possibly the frequency of administration will also vary depending on the age, weight, and response of the individual patient.

[0339] Such other agents are preferably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of fusion protein used, the type of disorder or treatment, and other factors mentioned above. Bispecific antibodies are generally used in the same dosage and via the routes of administration described herein, or in approximately 1–99% of the dosages described herein, or in any dosage and via any route determined empirically / clinically appropriate.

[0340] Such combination therapies described above include combined administrations (where two or more therapeutic agents are included in the same or separate compositions) and separate administrations, in which case the administration of the bispecific antibody may be performed before, concurrently with, and / or after the administration of the additional therapeutic agent and / or adjuvant.

[0341] H.Manufactured products In another aspect of the present invention, a product is provided containing a substance useful for the treatment, prevention and / or diagnosis of the above-described disorders. The product comprises a container and a label or accompanying documentation affixed to or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The container may be formed from a variety of materials such as glass or plastic. The container holds a composition effective for the treatment, prevention and / or diagnosis of the symptoms, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). At least one activator in the composition is an anti-PD1 / anti-LAG3 antibody as described herein.

[0342] The label or accompanying information indicates that the composition is used to treat a selected condition. Furthermore, the product may comprise (a) a first container containing the composition, wherein the composition contains an anti-HLA-G / anti-CD3 bispecific antibody, and (b) a second container containing the composition, wherein the composition contains an anti-PD1 / anti-LAG3 antibody. The product in this embodiment of the present invention may further include accompanying information indicating that the composition can be used to treat a specific condition.

[0343] Alternatively, or in addition thereto, the product may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. This may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. [Table B] TIFF2026510584000003.tif255170TIFF2026510584000004.tif255170TIFF2026510584000005.tif255170TIFF20265105840 00006.tif255170TIFF2026510584000007.tif255170TIFF2026510584000008.tif255170TIFF2026510584000009.tif236170

[0344] General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of the antibody chain are numbered and referenced according to the numbering system by Kabat (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) as defined above.

[0345] Embodiments of the present invention Some aspects of the present invention are listed below.

[0346] 1. An anti-HLA-G / anti-CD3 bispecific antibody for use in methods of treating HLA-G expressing cancer, which is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody.

[0347] 2. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in item (item) 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.

[0348] 3. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in item 1 or 2, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain which is an IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions which reduce binding to the Fc receptor, particularly to the Fcγ receptor.

[0349] 4. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 3, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises the Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0350] 5. 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 is a VH domain. (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 3, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 6 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 4, including the above.

[0351] 6. The anti-PD1 / anti-LAG3 bispecific antibody contains a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain (a) A VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 13, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) containing a VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 16; or (b) VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 21, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 5, including the above.

[0352] 7. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method of any one of items 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 containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10.

[0353] 8. The anti-PD1 / anti-LAG3 bispecific antibody contains a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain (a) A VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, or (b) VH domain containing the amino acid sequence of SEQ ID NO: 25 and VL domain containing the amino acid sequence of SEQ ID NO: 26 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 7, including the above.

[0354] 9. The anti-PD1 / anti-LAG3 bispecific antibody contains a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain (a) A VH domain containing the amino acid sequence of SEQ ID NO: 27 and a VL domain containing the amino acid sequence of SEQ ID NO: 28, or (b) A VH domain containing the amino acid sequence of SEQ ID NO: 29 and a VL domain containing the amino acid sequence of SEQ ID NO: 30, or (c) A VH domain containing the amino acid sequence of SEQ ID NO: 31 and a VL domain containing the amino acid sequence of SEQ ID NO: 32, or (d) A VH domain containing the amino acid sequence of SEQ ID NO: 33 and a VL domain containing the amino acid sequence of SEQ ID NO: 34, or (e) VH domain containing the amino acid sequence of SEQ ID NO: 64 and VL domain containing the amino acid sequence of SEQ ID NO: 65 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1-5 or 7, including the above.

[0355] 10. Anti-PD1 / anti-LAG3 bispecific antibody, A first antigen-binding domain that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, A second antigen-binding domain that specifically binds to LAG3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, and An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 8, including the above.

[0356] 11. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 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.

[0357] 12. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 11, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a Fab fragment that specifically binds to PD1, and the variable domains VL and VH are substituted for each other such that VL is part of the heavy chain and VH is part of the light chain.

[0358] 13. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 12, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises monovalent conjugation to PD1 and monovalent conjugation to LAG3.

[0359] 14. Anti-PD1 / anti-LAG3 bispecific antibodies, (a) A first heavy chain containing the amino acid sequence of SEQ ID NO: 35, a first light chain containing the amino acid sequence of SEQ ID NO: 36, a second heavy chain containing the amino acid sequence of SEQ ID NO: 37, and a second light chain containing the amino acid sequence of SEQ ID NO: 38, or (b) An anti-HLA-G / anti-CD3 bispecific antibody for use in any one of items 1-8 and 10-13, comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 35, a first light chain containing the amino acid sequence of SEQ ID NO: 36, a second heavy chain containing the amino acid sequence of SEQ ID NO: 39, and a second light chain containing the amino acid sequence of SEQ ID NO: 40.

[0360] 15. An anti-HLA-G / anti-CD3 bispecific antibody for use in any one of items 1-8 and 10-14, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises a first heavy chain containing the amino acid sequence of SEQ ID NO: 35, a first light chain containing the amino acid sequence of SEQ ID NO: 36, a second heavy chain containing the amino acid sequence of SEQ ID NO: 37, and a second light chain containing the amino acid sequence of SEQ ID NO: 38.

[0361] 16. Anti-HLA-G / anti-CD3 bispecific antibodies have a heavy chain variable region (V H CD3) and light chain variable region (V L A first antigen-binding domain that specifically binds to CD3 (including CD3), and a heavy chain variable region (V H HLA-G) and light chain variable region (V L An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 15, comprising a second antigen-binding domain that specifically binds to HLA-G (including HLA-G).

[0362] 17. An 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 is a VH domain. (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 43, VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 46 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 15, including the above.

[0363] 18. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 17, wherein the first antigen-binding domain that specifically binds to CD3 comprises a VH domain containing the amino acid sequence of SEQ ID NO: 47 and a VL domain containing the amino acid sequence of SEQ ID NO: 48.

[0364] 19. The second antigen-binding domain, which specifically binds to HLA-G, a) VH domain, i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 51, VL domain, i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52, ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and iii) A VL domain comprising CDR-L3 containing the amino acid sequence of SEQ ID NO: 54; or b) VH domain, i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 91, ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 92, and iii) A VH domain containing CDR-H3 with the amino acid sequence of SEQ ID NO: 93, VL domain, i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 94, ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 95, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 95 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 18, including the above.

[0365] 20. The anti-HLA-G / anti-CD3 bispecific antibody contains a second antigen-binding domain that specifically binds to HLA-G, and the second antigen-binding domain (a) A VH domain containing the amino acid sequence of SEQ ID NO: 55 and a VL domain containing the amino acid sequence of SEQ ID NO: 56, or (b) An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 19, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 97 and a VL domain containing the amino acid sequence of SEQ ID NO: 98.

[0366] 21. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 20, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a third antigen-binding domain that binds to HLA-G.

[0367] 22. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method described in any one of items 1 to 21, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises an Fc domain having one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0368] 23. An anti-HLA-G / anti-CD3 bispecific antibody for use in any one of items 1 to 22, wherein the anti-HLA-G / anti-CD3 bispecific antibody comprises a first heavy chain containing the amino acid sequence of SEQ ID NO: 57, a second heavy chain containing the amino acid sequence of SEQ ID NO: 58, a first light chain containing the amino acid sequence of SEQ ID NO: 59, and a second light chain containing the amino acid sequence of SEQ ID NO: 60.

[0369] 24. An anti-HLA-G / anti-CD3 bispecific antibody for use in any one of items 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 the combination is administered at intervals of approximately 1 to 3 weeks.

[0370] 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 combination, sequential, or concurrent treatment of diseases, particularly cancer, particularly HLA-G expressing cancer.

[0371] 26. A pharmaceutical composition comprising an anti-HLA-G / anti-CD3 bispecific antibody and a pharmaceutically acceptable carrier, and a second pharmaceutical comprising an anti-PD1 / anti-LAG3 bispecific antibody and an optionally pharmaceutically acceptable carrier.

[0372] 27. HLA-G expressing cancers, especially lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer. The pharmaceutical composition described in item 26, for use in the treatment of cancer selected from one or more of the cancers described above, including, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, nasopharyngeal carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumor, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia (including refractory versions of any of the cancers described above), or combinations of one or more of the cancers described above.

[0373] 28. Use of combinations of anti-HLA-G / anti-CD3 bispecific antibodies and anti-PD1 / anti-LAG3 bispecific antibodies in the manufacture of pharmaceuticals for treating cancer, particularly HLA-G expressing cancers.

[0374] 29. A method for treating cancer, particularly HLA-G expressing cancer, in a subject, comprising administering an effective amount of anti-HLA-G / anti-CD3 antibody and an effective amount of anti-PD1 / anti-LAG3 bispecific antibody to the subject.

[0375] 30. The method according to item 29, wherein an anti-HLA-G / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody are administered together in a single composition or separately in two or more different compositions.

[0376] 31. The method according to item 29 or 30, wherein an anti-HLA-G / anti-CD3 bispecific antibody and an anti-PD1 / anti-LAG3 bispecific antibody are administered intravenously or subcutaneously.

[0377] 32. The method according to any one of items 29 to 31, wherein an anti-HLA-G / anti-CD3 bispecific antibody is administered simultaneously with, before, or after, an anti-PD1 / anti-LAG3 bispecific antibody.

[0378] 33. An anti-HLA-G / anti-CD3 bispecific antibody for use in a method for treating 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, and 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. The first antigen-binding domain that specifically binds to PD1 is a VH domain comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and a VL domain comprising (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6. The second antigen-binding domain that specifically binds to LAG3 is a VH domain comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, and a VL domain comprising (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 16. 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 that specifically binds to CD3 is a VH domain comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43, and a VL domain comprising (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46. An anti-HLA-G / anti-CD3 bispecific antibody comprising a second antigen-binding domain that specifically binds to HLA-G, the VH domain comprising i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 51, and a VL domain comprising i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52, ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 54.

[0379] 34. An anti-HLA-G / anti-CD3 bispecific antibody for use in methods of treating HLA-G expressing cancer, which is used in combination with an anti-PD1 / anti-LAG3 bispecific antibody. 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. The first antigen-binding domain that specifically binds to PD1 includes a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10. The second antigen-binding domain, which specifically binds to LAG3, includes a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18. 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, which specifically binds to CD3, comprises a VH domain containing the amino acid sequence of SEQ ID NO: 47 and a VL domain containing the amino acid sequence of SEQ ID NO: 48. An anti-HLA-G / anti-CD3 bispecific antibody in which the second antigen-binding domain, which specifically binds to HLA-G, comprises a VH domain containing the amino acid sequence of SEQ ID NO: 55 and a VL domain containing the amino acid sequence of SEQ ID NO: 56. [Examples]

[0380] Recombinant DNA technology DNA was manipulated using standard methods, as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulins is given below: Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242.

[0381] DNA sequencing The DNA sequence was determined by double-strand sequencing.

[0382] gene synthesis The desired gene segments were generated by PCR using appropriate templates or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products by Geneart AG (Regensburg, Germany). Where accurate gene sequences were unavailable, oligonucleotide primers were de...

Claims

1. An anti-HLA-G / anti-CD3 bispecific antibody for use in a method for treating 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 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 is a VH domain. (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, It is a VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) A VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 6 A bispecific anti-HLA-G / anti-CD3 antibody containing [specific ingredient].

2. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to claim 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.

3. An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to claim 1 or 2, wherein the anti-PD1 / anti-LAG3 bispecific antibody comprises an Fc domain which is an IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, and the Fc domain comprises one or more amino acid substitutions which reduce binding to the Fc receptor, particularly to the Fcγ receptor.

4. The anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) A VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 13, It is a VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) A VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 16 including; or (b) VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, It is a VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (iii) VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to any one of claims 1 to 3, comprising:

5. An anti-HLA-G / anti-CD3 bispecific antibody for use in the 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, comprising the VH domain comprising the amino acid sequence of SEQ ID NO: 9 and the VL domain comprising the amino acid sequence of SEQ ID NO:

10.

6. The anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, or (b) VH domain containing the amino acid sequence of SEQ ID NO: 25 and VL domain containing the amino acid sequence of SEQ ID NO: 26 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to any one of claims 1 to 5, comprising:

7. The anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 27 and a VL domain containing the amino acid sequence of SEQ ID NO: 28, or (b) A VH domain containing the amino acid sequence of SEQ ID NO: 29 and a VL domain containing the amino acid sequence of SEQ ID NO: 30, or (c) A VH domain containing the amino acid sequence of SEQ ID NO: 31 and a VL domain containing the amino acid sequence of SEQ ID NO: 32, or (d) A VH domain containing the amino acid sequence of SEQ ID NO: 33 and a VL domain containing the amino acid sequence of SEQ ID NO: 34, or (e) VH domain containing the amino acid sequence of SEQ ID NO: 64 and VL domain containing the amino acid sequence of SEQ ID NO: 65 An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to any one of claims 1 to 3 or 5, comprising:

8. The aforementioned anti-PD1 / anti-LAG3 bispecific antibody A first antigen-binding domain that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, A second antigen-binding domain that specifically binds to LAG3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, and An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to any one of claims 1 to 6, comprising:

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

38.

11. The anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) including 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. Ｈ CD3); and / or light chain variable region (V) including 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. Ｌ An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to any one of claims 1 to 10, comprising a first antigen-binding domain that specifically binds to CD3, including CD3.

12. The anti-HLA-G / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) including 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. Ｈ HLA-G); and / or light chain variable region (V) including 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. Ｌ An anti-HLA-G / anti-CD3 bispecific antibody for use in the method according to any one of claims 1 to 11, comprising a second antigen-binding domain that specifically binds to HLA-G.

13. A composition comprising an anti-PD1 / anti-LAG3 bispecific antibody for use in the treatment of cancer, particularly HLA-G expressing cancer, wherein the treatment comprises administration of the composition comprising the 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 is a VH domain, (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, It is a VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) A VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, A composition containing the following:

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

10.

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

16. The anti-PD1 / anti-LAG3 bispecific antibody comprises a second antigen-binding domain that specifically binds to LAG3, and the second antigen-binding domain is (a) A VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, or (b) VH domain containing the amino acid sequence of SEQ ID NO: 25 and VL domain containing the amino acid sequence of SEQ ID NO: 26 A composition according to any one of claims 13 to 15, comprising:

17. The aforementioned anti-PD1 / anti-LAG3 bispecific antibody A first Fab fragment that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, A second Fab fragment that specifically binds to LAG3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, and A composition according to any one of claims 13 to 16, comprising:

18. 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 combination, sequential, or simultaneous treatment of diseases, particularly cancer, particularly HLA-G expressing cancer.

19. Cancer, especially HLA-G expressing cancers, particularly lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer The pharmaceutical composition according to claim 18 for use in treating a disease selected from one or more of the above-mentioned cancers: cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, nasopharyngeal carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumor, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia (including refractory versions of any of the above-mentioned cancers), or one or more combinations of the above-mentioned cancers.

20. The 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 pharmaceutical 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 is a VH domain. (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, It is a VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) A VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, Includes, use.

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

22. The aforementioned anti-PD1 / anti-LAG3 bispecific antibody A first Fab fragment that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, A second Fab fragment that specifically binds to LAG3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, and The use according to claim 20 or 21, including the use described in claim 20 or 21.

23. A method for treating cancer, particularly HLA-G expressing cancer, comprising administering an effective amount of anti-HLA-G / anti-CD3 antibody and an effective amount of anti-PD1 / anti-LAG3 bispecific antibody to the subject, 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), and the first antigen-binding domain that specifically binds to PD1 is a VH domain. (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) A VH domain containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, It is a VL domain, (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) A VL domain containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, Methods that include...

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

25. The aforementioned anti-PD1 / anti-LAG3 bispecific antibody A first Fab fragment that specifically binds to PD1, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 9 and a VL domain containing the amino acid sequence of SEQ ID NO: 10, A second Fab fragment that specifically binds to LAG3, comprising a VH domain containing the amino acid sequence of SEQ ID NO: 17 and a VL domain containing the amino acid sequence of SEQ ID NO: 18, and The method according to claim 23 or 24, including the method described in claim 23 or 24.

26. The method according to any one of claims 23 to 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 according to any one of claims 23 to 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 according to any one of claims 23 to 27, wherein the anti-HLA-G / anti-CD3 bispecific antibody is administered simultaneously with, before, or after, the anti-PD1 / anti-LAG3 bispecific antibody.

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