CN117545770A

CN117545770A - anti-HLA-G antibodies and uses thereof

Info

Publication number: CN117545770A
Application number: CN202180084939.9A
Authority: CN
Inventors: A·布约泽克; A·卡皮-古铁雷斯-西洛斯; A·弗雷莫泽-格伦德斯科伯; C·哈格; T·霍费尔; S·基什内尔; M·马杰蒂; E·莫斯纳; C·诺依曼; C·斯皮克; G·蒂芬塔勒; T·温德尔
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2020-12-17
Filing date: 2021-12-15
Publication date: 2024-02-09

Abstract

The present invention relates to antibodies that bind to human HLA-G, multispecific antibodies thereof, their preparation, formulations, and methods of use thereof. In particular, specific variants of antibodies labeled HLA-G-0090 are provided having mutations comprising potential glycosylation sites (NSS) in the variable region (CDR-L1) of the light chain, CDR 1. Two particular variants showed improved binding properties, good expression capacity and stability, while no more N-glycosylation was shown at CDR-L1 of the light chain (thus Fab glycosylation could not be detected). In one embodiment, bispecific antibodies are disclosed that comprise variants of an HLA-G antibody and an antibody that binds human CD 3.

Description

anti-HLA-G antibodies and uses thereof

The present invention relates to anti-HLA-G antibodies, methods of making, formulations, and methods of using the antibodies.

Background

The human major tissue-compatible complex (class I, located on chromosome 6) is also known as human leukocyte antigen G (HLA-G), a protein encoded by the HLA-G gene in humans. HLA-G belongs to the class I heavy chain paralogs of HLA atypical. The class I molecule is a heterodimer (beta-2 microglobulin) consisting of one heavy chain and one light chain. The heavy chain is anchored in the membrane, but can also be shed/secreted.

The heavy chain consists of three domains: α1, α2, and α3. The α1 and α2 domains form a peptide binding groove flanked by two α helices. Similar to other MHC I proteins, small molecule peptides (about 9 monomer units) can bind to the groove.

The second chain is a beta-2 microglobulin, which binds to the heavy chain, similar to other MHC I proteins.

HLA-G has 7 isoforms, including 3 secreted and 4 membrane bound forms (as schematically shown in FIG. 1).

HLA-G can form a functionally active complex oligomeric structure (Kuroki, K et al Eur J Immunol.37 (2007) 1727-1729). Dimers connected by disulfide bonds are formed between Cys 42 of two HLA-G molecules. (Shiroishi M et al J Biol Chem 281 (2006) 10439-10447. Trimer and tetramer complexes have also been described in, for example, kuroki, K et al Eur J immunol.37 (2007) 1727-1729; allan D.S. et al J Immunol methods.268 (2002) 43-50; and T Gonen-Gross et al J Immunol 171 (2003) 1343-1351).

HLA-G is expressed mainly on the embryoid body of placenta. Several tumors, including pancreas, breast, skin, colorectal, gastric and ovarian, express HLA-G (Lin, A. Et al, mol Med.21 (2015) 782-791; amiot, L. Et al, cell Mol Life Sci.68 (2011) 417-431). It has also been reported that this expression is associated with pathological conditions such as inflammatory diseases, gvHD and cancer. HLA-G expression is reported to be associated with poor prognosis of cancer. Tumor cells induce immune tolerance/suppression via HLA-G expression to evade host immune surveillance.

HLA-G has a high degree of homology (> 98%) with other MHC I molecules, and thus it is difficult to generate a truly HLA-G specific antibody that is not cross-reactive with other MHC I molecules.

Some antibodies that interact with HLA-G in different ways were previously described: tissue anti-genes, 55 (2000) 510-518, are directed to monoclonal antibodies, such as 87G and MEM-G/9; neoplasma 50 (2003) 331-338 relates to certain monoclonal antibodies that recognize both intact HLA-G oligomeric complexes (e.g., 87G and MEM-G9) and heavy chains that do not contain HLA-G (e.g., 4H84, MEM-G/1, and MEM-G/2); hum immunol.64 (2003) 315-326 involves several antibodies tested on HLA-G expressing JEG3 tumor cells (e.g., MEM-G/09 and MEM-G/13.MEM-G/01 that specifically react with natural HLA-G1 molecules recognize (similar to 4H84 mAb) denatured HLA-G heavy chains of all isotypes, while MEM-G/04 recognizes selectively denatured HLA-G1, HLA-G2 and HLA-G5 isotypes); wiendl et al Brain 2003 176-85 are directed to different monoclonal HLA-G antibodies, e.g., 87G, 4H84, MEM-G/9.

The above publications report antibodies that bind to human HLA-G or to the human HLA-G-. Beta.2M MHC complex. However, due to the high polymorphism and high homology of the HLA family, most antibodies lack truly specific HLA-G binding properties and typically also bind or cross-react with other HLA family members (either as MHC complexes with β2m or in their β2m free form), or they do not inhibit the binding of HLA-gβ2m MHC complexes to their receptors ILT2 and/or ILT4 at all (considered non-antagonistic antibodies).

WO2019/202040 relates to HLA-G antibodies, including the antibody HLA-G-0090.WO 2019/202041 relates to multispecific HLA-G antibodies, including antibody HLA-G-0090.

Disclosure of Invention

The invention described herein provides an antibody that binds to human HLA-G comprising

A) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 1, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 2 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 3; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence SEQ ID NO:23, (ii) a CDR-L2 comprising the amino acid sequence SEQ ID NO:5 and (iii) a CDR-L3 comprising the amino acid sequence SEQ ID NO:6, or

B) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 1, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 2 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 3; and (b) a VL domain comprising (i) CDR-L1 comprising the amino acid sequence SEQ ID NO:25, (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acid sequence SEQ ID NO:6.

One embodiment of the present invention is an antibody that binds to human HLA-G, wherein the antibody

A) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 24; or (b)

B) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 26.

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

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

The antibodies have very valuable properties, such as their binding properties, their high specificity for HLA-G and no cross-reactivity to HLA-A and HLA MHC I complexes from other species. They can bind to HLA-G on cells and inhibit ILT2 and/or ILT4 from binding to HLA-G expressed on these cells. They are produced by HLA-G antibody HLA-G-0090.

Since the HLA-G antibody HLA-G-0090 described in WO 2019/202040 contains a glycosylation site in one of the CDRs (CDR-L1, which contains NSS motifs at amino acids 31, 32 and 33 of the Light Chain (LC)), its binding properties are affected by N-glycosylation, constituting a potential exploitability defect.

A homology model of the HLA-G-0090 variable region shows high solvent accessibility at positions 31 to 33 of the Light Chain (LC). Furthermore, the side chains of N31 and S32 are expected to point inward in the direction of CDR-H3, making them possible candidates for part of the paratope of an antibody. Indeed, many published antibody-antigen X-ray complex structures record that these residues interact chemically with the antigen. Thus, there is a high risk of a decrease in the binding affinity of the antibody resulting from the introduction of mutations at LC positions 31-33. Thus, various variants of antibody HLA-G-0090 were designed with mutations at LC positions 31, 32 and 33, but most variants had reduced binding properties or expression capacity. Surprisingly, it was found that of these various variants of HLAG-0090 with glycosylation sites removed, only two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A exhibited even improved binding properties, good expression capacity and stability, while no longer showing N-glycosylation at CDR-L1 of LC (thus Fab glycosylation could not be detected). Since all recently approved pharmaceutical antibody products are produced in mammalian cells, especially CHO cells (see, e.g., walsh g., nature Biotech (2018) 1136-1145), providing an antibody that lacks glycosylation sites in the binding regions (VH and VL, and especially CDRs) represents a valuable advantage, as these antibodies can be readily used in the production of mammalian expression systems without compromising (at least in part) the binding properties by glycosylation.

In another embodiment, the HLA-G antibodies of the invention comprise an Fc domain of human origin. In one embodiment, the Fc domain is of the IgG isotype, and in a preferred embodiment, of the IgG1 isotype.

In another embodiment, the HLA-G antibodies of the invention are bispecific antibodies, particularly bispecific antibodies that bind to human HLA-G and to human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD 3.

In one embodiment, the HLA-G antibody of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3,

wherein the first antigen-binding portion that binds to human HLA-G comprises

B) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 1, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 2 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 3; and (b) a VL domain comprising (i) CDR-L1 comprising the amino acid sequence SEQ ID NO:25, (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acid sequence SEQ ID NO:6;

and wherein the second antigen binding portion that binds to a T cell activating antigen binds to human CD3, the second antigen binding portion comprising

C) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 52, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 53 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 54; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence SEQ ID NO:55, (ii) a CDR-L2 comprising the amino acid sequence SEQ ID NO:56 and (iii) a CDR-L3 comprising the amino acid sequence SEQ ID NO:57, or

D) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 60, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 61 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 62; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence SEQ ID NO:63, (ii) a CDR-L2 comprising the amino acid sequence SEQ ID NO:64 and (iii) a CDR-L3 comprising the amino acid sequence SEQ ID NO:65, or

E) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 68, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 69 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 70; and (b) a VL domain comprising (i) CDR-L1 comprising the amino acid sequence SEQ ID NO:71, (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO:72 and (iii) CDR-L3 comprising the amino acid sequence SEQ ID NO:73.

One embodiment of the invention is a bispecific antibody,

wherein the first antigen binding portion

B) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 26,

and wherein the second antigen binding portion

C) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 58; and a VL domain comprising the amino acid sequence SEQ ID NO 59; or (b)

D) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 66; and a VL domain comprising the amino acid sequence SEQ ID NO 67; or (b)

E) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 74; and a VL domain comprising the amino acid sequence SEQ ID NO 75.

One embodiment of the invention is a bispecific antibody,

wherein the first antigen binding portion

Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 24;

and wherein the second antigen binding portion

Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 58; and a VL domain comprising the amino acid sequence SEQ ID NO 59.

One embodiment of the invention is a bispecific antibody,

wherein the first antigen binding portion

and wherein the second antigen binding portion

Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 66; and a VL domain comprising the amino acid sequence SEQ ID NO:67.

One embodiment of the invention is a bispecific antibody,

wherein the first antigen binding portion

and wherein the second antigen binding portion

Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 74; and a VL domain comprising the amino acid sequence SEQ ID NO 75.

Such bispecific antibodies binding to human HLA-G and to human CD3 exhibit, in addition to the properties of HLA-G antibodies, additional valuable properties such as ifnγ secretion mediated by T cell-induced antibodies on HLA-G expressing cells, activation of T cells in the presence of HLA-G expressing tumor cells, induction of T cell-mediated killing of tumor cells on HLA-G expressing cells, and in vivo anti-tumor efficacy and even tumor regression in different xenograft mouse models of cancer.

The present invention provides an isolated nucleic acid encoding an antibody or bispecific antibody as described herein.

The present invention provides a host cell comprising such nucleic acid.

The invention provides a method of producing an antibody comprising culturing a host cell to produce the antibody.

The invention provides such methods of producing an antibody, which further comprises recovering the antibody from the host cell.

The present invention provides an antibody produced by a host cell, wherein the host cell is a eukaryotic cell.

The present invention provides a pharmaceutical formulation comprising an antibody as described herein and a pharmaceutically acceptable carrier.

The invention provides an antibody as described herein for use as a medicament.

The invention provides antibodies described herein for use in the treatment of cancer.

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

The invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of an antibody described herein.

Drawings

Fig. 1: different isoforms of HLA-G

Fig. 2: fig. 2A: schematic representation of HLA-G molecules associated with β2m:

HLA-Gwt molecular schematic

Fig. 2B: schematic of HLA-G molecules associated with β2m: KIR2DL4 and ILT2/4 interactions are mediated from the crystal structure: HLA-G ILT4 complex structure (PDB code: 2 DYP). The KIR2DL1 structure was taken from PDB code 1IM9 (KIR 2DL1: HLA-Cw4 complex structure) and was located on HLA-G by superposition of HLA-Cw4 and HLA-G structures.

Fig. 2C: schematic diagram of HLA-G molecules binding to β2m: schematic of HLA-G chimeric molecule as counter:

HLA-G chimeric molecules are used as a schematic representation of the identified counter antigens for specific HLA-G binders. The white dots represent surface residues identified as unique to HLA. These residues are replaced by HLA consensus sequences in the chimeric molecule.

Fig. 2D: structure of HLA-G molecules associated with certain receptors: HLA-G structure in complexes with given receptors such as ILT4 and KIR2DL 1. ILT4 structure (PDB code: 2 DYP). The KIR2DL1 structure was taken from PDB code 1IM9 (KIR 2DL1: HLA-Cw4 complex structure) and was located on HLA-G by superposition of HLA-Cw4 with HLA-G structure. Receptors are shown in the band icons and HLA-G is shown in the molecular surface icons. HLA-G residues that are unique or remain in other HLA paralogues are colored white and gray, respectively. In chimeric antigen (counter antigen), unique surface residues are replaced with HLA consensus sequences.

Fig. 3: schematic antibody-antigen binding assay principle-relative Activity concentration of HLA-G antibodies to HLA-G binding (RAC)

Fig. 4A: n-glycosylation mass spectrometry of HLA-G-0090 indicates the presence of Fab-glycosylation

Fig. 4B: n-glycosylation mass spectrometry of HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A: fab-glycosylation was not detected

Fig. 5: exemplary FACS staining of anti-HLA-G antibodies HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A (2. Mu.g/ml) on SKOV3 cells (non-HLA-G expressing), JEG3 cells (HLA-G expressing) and SKOV3-HLA-G cells (HLA-G transfected SKOV3 cells)

Fig. 6: HLA-G antibodies HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A modify/inhibit the ability of recombinant soluble ILT2 (ILT 2Fc domain fusion) to interact and bind to HLA-G naturally expressed on JEG3 tumor cells. JEG3 cells were pre-incubated/pre-treated with HLA-G antibodies, thereby inhibiting/blocking ILT2 binding to JEG3 cells. Controls were performed using JEG3 cells that were not pre-treated with HLA-G antibodies (ILT 2-Fc only) and isotype antibodies.

Fig. 7: bispecific anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibody binding to CD3 expressed on T cells by antibodies P1AF7977, P1AF7978 and P1AF7979

Fig. 8: bispecific anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibodies P1AF7977, P1AF7978 and P1AF7979 showed binding to JEG3 cells and HLA-G transfected SKOV3 cells.

Fig. 9: bispecific anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibodies mediate/induce IFN gamma secretion by T cells through antibodies P1AF7977, P1AF7978 and P1AF7979

Fig. 10: bispecific anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibodies P1AF7977, P1AF7978 and P1AF7979 induce T cell mediated cytotoxicity/tumor cell killing.

Fig. 11: anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibody P1AF7977 anti-tumor efficacy in humanized NSG mice bearing SKOV3 human ovarian cancer transfected with recombinant HLA-G (SKOV 3 HLA-G), resulting in tumor regression figure 12: dose-remission studies of anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibody P1AF7977 in humanized NSG mice bearing human breast cancer PDX tumor (BC 004). Strong tumor growth inhibition was observed in mice treated with different doses until tumor regression.

Fig. 13: exemplary configurations of bispecific antigen binding molecules of the invention. (A, D) "1+1 CrossMab" molecule. (B, E) "2+1 IgG Crossfab" molecule, with an alternative sequence of Crossfab and Fab components ("inverted"). (C, F) "2+1 IgG cross fab" molecule. (G, K) "1+1 IgG Crossfab" molecule with an alternative sequence of Crossfab and Fab components ("reverse"). (H, L) "1+1 IgG cross fab" molecule. (I, M) "2+1 IgG Crossfab" has two crossFab representations. (J, N). Black dots: optional modifications in the Fc domain that promote heterodimerization (heteroodization). ++ - -): oppositely charged amino acids are optionally introduced in the CH1 and CL domains. Cross fab molecules are described as comprising an exchange of VH and VL domains, but may-in embodiments where no charge modification is introduced in the CH1 and CL domains-alternatively comprise an exchange of CH1 and CL domains.

Fig. 14: induction of T cell activation by bispecific anti-HLA-G/anti-CD 3 antibody P1AF7977 (HLA-G-0090-VL-S32P/CD 3P 035) in the presence of SKOV3 HLAG cells.

Fig. 15: original and optimized CD3 binder CD3 _orig CD3 _opt The relative binding activity (=p035-093 (P035)) to recombinant CD3, which was measured by SPR under stress-free conditions after 14 days at pH 6 at 40 ℃ or after 14 days at pH 7.4 at 37 ℃ (IgG form).

Fig. 16: original and optimized CD3 binder CD3 _orig And CD3 _opt (=p035-093 (P035)) to Jurkat NFAT cells, which were measured by flow cytometry (IgG format). Antibodies that bind to Jurkat NFAT cells are detected with a fluorescently labeled anti-human Fc specific secondary antibody.

Fig. 17: schematic of the CD3 activation assay used in example 8.

Fig. 18: using the original and optimised CD3 binder CD3 _orig And CD3 _opt Jurkat NFAT activation (IgG format) performed (=p035-093 (P035)). Jurkat NFAT reporter cells and CHO-expressing anti-PGLALA (CHO-PGLALA) cells in CD3 _orig Or CD3 _opt (=p035-093 (P035)) IgG PGLALA or CD3 _opt IgG wt (as negative control) was co-incubated in the presence of the same. CD3 activation was quantified by measuring luminescence after 24 hours.

Detailed Description

The terms "HLA-G", "human HLA-G (human HLA-G)", "HLAG" as used herein refer to HLA-G human major tissue compatibility complex class I G, also known as human leukocyte antigen G (HLA-G) (exemplary SEQ ID NO: 35). Typically, HLA-G forms an MHC class I complex with β2 microglobulin (B2M or β2m). In one embodiment, HLA-G refers to the MHC class I complex of HLA-G and beta 2 microglobulin. In a preferred embodiment, HLA-G refers to the cell surface binding MHC class I complex of HLA-G and beta 2 microglobulin, also known as HLA-G1 (see FIG. 1 of the present specification, and e.g., blaschitz et al Molecular Human Reproduction,11 (2005) 699-710, particularly FIG. 1).

As used herein, an antibody (monospecific, multispecific, or bispecific antibody) or antigen-binding portion that "binds to", "specifically binds to", "binds to", or "anti-HLA-G" refers to a polypeptide that binds to, or binds to, human HLA-G at 5.0x10 ^-8 K in mol/l or less _D Value binding affinity an antibody/antigen binding portion that specifically binds to a human HLA-G antigen or extracellular domain (ECD) thereof; in one embodiment, K _D The value was 1.0X10 ^-9 mol/l or less; in one embodiment, K _D The value was 5.0X10 ^-8 mol/l to 1.0X10 ^-13 mol/l. In one embodiment, the antibody binds to an HLA-Gβ2MMHC I complex comprising SEQ ID NO 39.

Binding affinity is measured using standard binding assays such as surface plasmon resonanceGE-Healthcare Uppsala, sweden) assays, for example, using constructs comprising an HLA-G extracellular domain (e.g., in its naturally occurring 3-dimensional structure). In one embodiment, binding affinity is determined using a standard binding assay, wherein an exemplary soluble HLA-G comprising MHC class I complex comprising SEQ ID NO 39 is used.

HLA-G has a regular MHC I fold and consists of two chains: chain 1 consists of three domains: α1, α2, and α3. The α1 and α2 domains form a peptide binding groove flanked by two α helices. Similar to other mhc i proteins, small molecule peptides (about 9 monomer units) can bind to the groove. Chain 2 is a β2 microglobulin (β2m) that is shared with various other mhc i proteins.

HLA-G can form a functionally active complex oligomeric structure (Kuroki, K et al Eur J Immunol.37 (2007) 1727-1729). Dimers connected by disulfide bonds are formed between Cys42 of two HLA-G molecules. (Shiroishi M et al J Biol Chem 281 (2006) 10439-10447. Trimer and tetramer complexes have also been described in, for example, kuroki, K et al Eur J immunol.37 (2007) 1727-1729; allan D.S. et al J Immunol methods.268 (2002) 43-50; and T Gonen-Gross et al J Immunol 171 (2003) 1343-1351). Unlike most other MHC class I molecules, HLA-G has several free cysteine residues. Boyson et al, proc Nat Acad Sci USA,99:16180 (2002) reported that recombinant soluble forms of HLA-G5 can form disulfide-linked dimers via intermolecular Cys42-Cys42 disulfide bonds. In addition, membrane-bound forms of HLA-G1 can also form disulfide-linked dimers on the cell surface of endogenous HLA-G expressing JEG3 cell lines. HLA-G1 and HLA-G5 in disulfide-linked dimer forms have also been found on the cell surface of endoderm cells (Apps, R., tissue Antigens,68:359 (2006)).

HLA-G has 7 isoforms, including 3 secreted and 4 membrane bound forms (as schematically shown in FIG. 1). The most important functional isoforms of HLA-G include HLA-G1 and HLA-G5 associated with b 2-microglobulin (. Beta.2M). However, the tolerogenic immune effects of these isoforms vary, depending on the form of the ligand (monomer, dimer) and the affinity of the ligand-receptor interaction.

HLA-G proteins can be produced using standard molecular biology techniques. Nucleic acid sequences of HLA-G isoforms are known in the art. See, e.g., GENBANK access number AY359818.

HLA-G isoform promotes signaling through ILT, particularly ILT2, ILT4, or a combination thereof.

ILT: ILT represents Ig-type activation and inhibition receptors that are involved in regulating immune cell activation and controlling immune cell function (Borges, L. Et al Curr Top Microbial Immunol,244:123-136 (1999)). ILT is divided into three groups: (i) An inhibitory ILT containing a cytoplasmic immunoreceptor complex amino acid inhibitory motif (ITIM) and conducting inhibitory signals (ILT 2, ILT3, ILT4, ILT5 and LIR 8); (ii) Activating ILT, which contains a short cytoplasmic tail and charged amino acid residues in the transmembrane domains (ILT 1, ILT7, ILT8 and lir6α) and delivers an activation signal via the cytoplasmic immunoreceptor amino acid activation motif (ITAM) of the related common gamma chain of Fc receptors; and (iii) a soluble molecule ILT6 lacking a transmembrane domain. Many recent studies have emphasized the immunomodulatory effects of ILT on the surface of Antigen Presenting Cells (APC). ILT2, ILT3 and ILT4 receptors are the most well characterized immunosuppressive receptors that are expressed on a variety of immune cells, including monocytes, B cells, dendritic cells, plasmacytoid dendritic cells and subsets of NK and T cells. ILT2 expressed on a subset of T cells has been shown to inhibit activation and proliferation of these cells when connected (Colonna M. Et al, J Immunol.20011,66:2514-2521,J Immunol 2000;165:3742-3755). ILT3 and ILT4 are upregulated by exposure of immature DCs to known immunosuppressive factors, including IL-10, vitamin D3 or inhibitory CD 8T cells (Chang, C.C. et al, nat Immunol,3:237-243 (2002)). Expression of ILT on DCs is tightly controlled by inflammatory stimuli, cytokines and growth factors, and is reduced upon DC activation (Ju, X.S. et al, gene,331:159-164 (2004)). Expression of ILT2 and ILT4 receptors is tightly regulated by histone acetylation, which helps tightly control gene expression that occurs only in myeloid lineage cells (Nakajima, h., J Immunol,171:6611-6620 (2003)).

The involvement of the inhibitory receptors ILT2 and ILT4 alters the cytokine and chemokine secretion/release characteristics of monocytes and inhibits Fc receptor signaling (Colonna, M.et al J Leukoc Biol,66:375-381 (1999)). The role and function of ILT3 on DC has been described precisely by the Sucif-Foca group (Sucif-Foca, N., int Immunopharmacol,5:7-11 (2005)). Although the ligand of ILT3 is unknown, ILT4 is known to bind to the third domain of HLA class I molecules (HLA-A, HLA-B, HLA-C and HLA-G) and to bind CD8 for MHC class I binding (Shiroishi, M., proc Natl Acad Sci USA,100:8856-8861 (2003)). The preferential ligand for several inhibitory ILT receptors is HLA-G. HLA-G plays a potential role in maternal tolerance and the mechanism by which tumor cells evade immune recognition and destruction (Hunt, J.S. et al, faseb J,19:681-693 (2005)). Modulation of DC function by HLA-G-ILT interactions is most likely an important pathway in DC biological mechanisms. Human monocyte-derived DCs that highly express ILT2 and ILT4 receptors have been identified that retain the potential to induce T cell anergy with stable tolerance-like phenotypes (CD 80low, CD86low, and HLA-DRlow) when treated with HLA-G and stimulated with allogeneic T cells (Ristinh, V. Et al, eur J Immunol,35:1133-1142 (2005)). Furthermore, the DC interaction of HLA-G with highly expressed ILT2 and ILT4 receptors results in down-regulation of several genes involved in the MHC class II presentation pathway. Lysosomal thiol reductase IFN-gamma inducible lysosomal thiol reductase (GILT) expressed in large amounts by professional APC is greatly reduced in HLA-G modified DCs. DC expression of GILT can affect the repertoire of sensitized CD4+ T cells (repertoire) because in vivo T cells respond to select antigens in animals lacking GILT following targeted gene disruption (Marie, M.et al, science,294:1361-1365 (2001)). HLA-G/ILT interactions on DCs interfere with MHC class II molecule assembly and transport to the cell surface, which may lead to reduced presentation or expression efficiency of structurally abnormal MHC class II molecules. HLA-G has been determined to significantly reduce transcription of the invariant chain (CD 74), HLA-DMA and HLA-DMB genes on human monocyte derived DC high expression ILT inhibitory receptors (Ristinh, V. Et al, eur J Immunol 35:1133-1142 (2005)).

Another receptor for HLA-G is KIR2DL4, because KIR2DL4 binds to cells expressing HLA-G (U.S. Pat. No. 3,79; cantoni, C.et al Eur J Immunol 28 (1998) 1980; rajagopalan, S.and E.O. Long. [ correction published in J Exp Med 191 (2000) 2027]J Exp Med 189 (1999) 1093; ponte, M.et al PNAS USA 96 (1999) 5674). KIR2DL4 (also known as 2DL 4) is a KIR family member (also known as CD158 d) that shares structural features with activating and inhibiting receptors (Selvakumar, a. Et al Tissue Antigens 48 (1996) 285). 2DL4 has cytoplasmic ITIM, indicating an inhibitory function, and has positively charged amino acids in the transmembrane region, which are typical features of activating KIR. Unlike other colonised KIRs, 2DL4 is transcribed by all NK cells (Valiante, n.m. et al Immunity 7 (1997) 739; cantoni, c. Et al Eur J Immunol 28 (1998) 1980; rajago palan, s. And e.o. long. [ correction published in J Exp Med 191 (2000) 2027]J Exp Med 189 (1999) 1093).

HLA-G has also been shown to interact with CD8 on cytotoxic T cells (Sanders et al, J.Exp.Med.,174 (1991), 737-740) and induce CD 95-mediated apoptosis in activated CD 8-positive cytotoxic T cells (Fourier et al, J.Immun.,164 (2000), 6100-6104). This mechanism of cytotoxic T cell depletion is reported to be one of the mechanisms of immune escape and tolerance induction in pregnancy, inflammatory diseases and cancer (Amodio g. Et al, tissue Antigens,84 (2014), 255-263).

As used herein, an anti-HLA-G antibody (monospecific antibody, multispecific antibody, or bispecific antibody) or antigen binding portion that "does not cross-react" or "does not (specifically) bind" to a modified human HLA-gβ2m MHC I complex refers to an anti-HLA-G antibody (monospecific antibody, multispecific antibody, or bispecific antibody) or antigen binding portion that does not substantially bind to any of these counterantigensWherein the HLA-G specific amino acid has been replaced with an HLA-A consensus amino acid, the complex comprising SEQ ID NO. 40; the mouse H2Kdβ2M MHC I complex comprises SEQ ID NO. 41; the rat RT1 A.beta.2M MHC I complex comprises SEQ ID NO. 43, and the human HLA-A 2.beta.2M MHC I complex comprises SEQ ID NO. 35 and SEQ ID NO. 33. In one embodiment, an anti-HLA-G antibody (monospecific antibody, multispecific antibody, or bispecific antibody) or antigen binding moiety that "does not cross-react" or "does not (specifically) bind" to a modified human HLA-gβ2m MHC I complex, wherein the HLA-G specific amino acid has been replaced with an HLA-a consensus amino acid, refers to an anti-HLA-G antibody (monospecific antibody, multispecific antibody, or bispecific antibody) or antigen binding moiety that does not exhibit significant binding/interaction in, for example, a surface plasmon resonance assay (as described in example 2), the complex comprising SEQ ID No. 40; the mouse H2Kdβ2M MHC I complex comprises SEQ ID NO. 41; the rat RT1 A.beta.2M MHC I complex comprises SEQ ID NO. 43 and/or the human HLA-A 2.beta.2M MHC I complex comprises SEQ ID NO. 35 and SEQ ID NO. 33. The binding/interaction is measured using standard binding assays such as surface plasmon resonance GE-Healthcare Uppsala, sweden) with corresponding antigen assay: a modified human HLA-gβ2m MHC I complex wherein an HLA-G specific amino acid has been replaced with an HLA-a consensus amino acid, the complex comprising SEQ ID No. 40; the mouse H2Kdβ2M MHC I complex comprises SEQ ID NO. 41; the rat RT1 A.beta.2M MHC I complex comprises SEQ ID NO. 43 and/or the human HLA-A 2.beta.2M MHC I complex comprises SEQ ID NO. 35 and SEQ ID NO. 33. The assay setup and construction/preparation of the antigen are as described in the examples.

The term "inhibition of ILT2 binding to HLA-G on JEG-3 cells (ATCC HTB 36)" refers to inhibition of the binding interaction of (recombinant) ILT2 in the assay described in example 5.

As used herein, "activated T cell antigen (activating T cell antigen)" refers to an epitope expressed on the surface of T lymphocytes (particularly cytotoxic T lymphocytes) that is capable of inducing T cell activation upon interaction with an antibody. In particular, the interaction of antibodies with activated T cell antigens can induce T cell activation by triggering a signaling cascade of T cell receptor complexes. In a specific embodiment, the activated T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt accession number P07766 (189 th edition), NCBI RefSeq accession number NP-000724.1, SEQ ID NO:88 for human sequences, or UniProt accession number Q95LI5 (49 th edition), NCBI GenBank accession number BAB71849.1, SEQ ID NO:108 for cynomolgus monkey sequences).

Unless otherwise indicated, "CD3" refers to any natural CD3 derived from any vertebrate, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). CD3 is an exemplary activated T cell antigen. The term "CD3" encompasses "full length" unprocessed CD3 as well as any form of CD3 produced by processing in a cell. The term also encompasses natural CD3 variants, such as splice variants or allelic variants. In one embodiment, CD3 is human CD3, specifically the epsilon subunit of human CD3 (CD 3 epsilon). The amino acid sequence of human CD3 ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 189) or NCBI (www.ncbi.nlm.nih.gov /) RefSeq NP-000724.1. See also SEQ ID NO. 88. The amino acid sequence of cynomolgus monkey [ Macaca fascicularis ] CD3 ε is shown in NCBI GenBank accession number BAB71849.1. See also SEQ ID NO. 89.

As used herein, an antibody (monospecific, multispecific, or bispecific antibody) or antigen-binding portion that "binds to human CD3," "specifically binds to human CD3," "binds to human CD3," or "anti-CD 3" refers to an antibody (monospecific, multispecific, or bispecific antibody) or antigen-binding portion that specifically binds to human CD3 antigen or its extracellular domain (ECD), which exhibits significant binding/interaction in a surface plasmon resonance assay. In one embodiment, K, which is indicative of binding affinity _D A value of 5.0X10-8 mol/l or less; in one embodiment, K _D The value was 1.0X10 ^- ⁹ mol/l or less; in one embodiment, K _D The value was 5.0X10 ^-8 mol/l to 1.0X10 ^-13 mol/l. In one embodiment, the antibody binds to CD3 comprising SEQ ID NO. 88.

Binding affinity is determined using standard binding assays such as surface plasmon resonance techniquesGE-Healthcare Uppsala, sweden) assays, for example, using constructs comprising an HLA-G extracellular domain (e.g., in its naturally occurring 3-dimensional structure). In one embodiment, binding affinity is determined using a standard binding assay, wherein an exemplary CD3 comprising SEQ ID NO 88 is used.

Thus, a multispecific or bispecific antibody that binds to human HLA-G and to human CD3 refers to an antibody that binds to a (first) antigen binding portion of human HLA-G as described herein and to another (second) antigen binding portion of human CD3 as described herein, comprising a first antigen binding portion that binds to human HLA-G and a second antigen binding portion that binds to human CD3.

As used herein, "T cell activation" refers to one or more cellular responses in T lymphocytes (particularly cytotoxic T lymphocytes) selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector release, cytotoxic activity and expression of activation markers. Suitable assays for determining T cell activation are known in the art and described herein.

The "acceptor human framework" is a framework derived from a human immunoglobulin framework or human consensus framework as defined below, comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework. The "derived from" human immunoglobulin framework or human consensus framework of the recipient human framework may comprise the same amino acid sequence as it, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical to the VL human immunoglobulin framework or the sequence of the human consensus framework.

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

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the 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 Bispecific antibodies formed from bifunctional antibodies, linear antibodies, single chain antibody molecules (e.g., scFv), and antigen fragments.

The term "bispecific" means that an antibody is capable of specifically binding to at least two different epitope sites. Typically, a bispecific antibody comprises two antigen binding sites or moieties, wherein each antigen binding site or moiety is specific for a different epitope. In certain embodiments, the bispecific antibody is capable of binding two epitopes simultaneously, in particular two epitopes expressed on two different cells.

As used herein, the term "valence" means the presence of a specified number of antigen binding sites in an antibody. Thus, the term "monovalent binding antigen (monovalent binding to an antigen)" means that there is one (and no more than one) antigen binding site in the antibody that is specific for the antigen.

The term "antigen binding site (antigen binding site)" is interchangeable with "antigen binding portion (antigen binding moiety)" and refers to a site, i.e., one or more amino acid residues, of an antibody that provides interaction with an antigen. For example, the antigen binding site of an antibody comprises amino acid residues from a Complementarity Determining Region (CDR). Untreated (native) immunoglobulin molecules typically have two antigen binding sites, and Fab molecules typically have a single antigen binding site. "antigen binding site" and "antigen binding portion" refer to a polypeptide molecule that specifically binds an epitope. In one embodiment, the antigen binding portion is capable of directing the entity to which it is attached (e.g., the second antigen binding portion) to a target site, e.g., to a particular type of tumor cell bearing an epitope. In another embodiment, the antigen binding portion is capable of activating signaling through its target antigen (e.g., T cell receptor complex antigen). Antigen binding portions include antibodies and fragments thereof as further defined herein. The specific antigen binding portion comprises an antigen binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding portion can include an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of five isoforms (isotype): alpha, delta, epsilon, gamma or mu. Useful light chain constant regions include either of two isoforms: kappa and lambda. In a preferred embodiment, such constant regions are of human origin.

As used herein, the term "epitope" (antigenic determinant) or "antigen" refers to a site on a polypeptide macromolecule to which an antigen binding moiety binds to form an antigen binding moiety-antigen complex. For example, useful epitope may be present on the surface of tumor cells, on the surface of cells infected with a virus, on the surface of other diseased cells, on the surface of immune cells, absent from serum, and/or present in the extracellular matrix (ECM).

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

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main classes of antibodies: igA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g. IgG ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha respectivelyDelta, epsilon, gamma and mu. In a preferred embodiment, such subclasses (isoforms) are of human origin. In a preferred embodiment, the antibodies of the invention are of the IgG isotype, and in another preferred embodiment, of the IgG1 isotype.

In one aspect, the antibody comprises a constant region of human origin. In one aspect, the antibody is an immunoglobulin molecule comprising a human constant region, in particular belonging to the IgG isotype, more particularly belonging to the IgG1 isotype, comprising human CH1, CH2, CH3 and/or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NO. 47 and SEQ ID NO. 48 (human kappa and lambda CL domains, respectively) SEQ ID NO. 49 (human IgG1 heavy chain constant domain CH1-CH2-CH 3) or SEQ ID NO. 50 (human IgG1 heavy chain constant region comprising mutations L234A, L235A and P329G). An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective to achieve a desired therapeutic or prophylactic effect over a desired dosage and period of time.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary somewhat, the Fc region of a human IgG heavy chain is generally defined as extending from Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by the host cell may undergo post-translational cleavage of one or more, particularly one or both, amino acids of the heavy chain C-terminus. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain, or may comprise a cut variant of a full-length heavy chain (also referred to herein as a "cut variant heavy chain"). This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the EU index of Kabat). Thus, the C-terminal lysine (Lys 447) or C-terminal glycine (Gly 446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise indicated, the amino acid sequence of the heavy chain comprising an Fc domain (or a subunit of an Fc domain as defined herein) is herein denoted as free of C-terminal glycine-lysine dipeptide. In one embodiment of the invention, the heavy chain (comprising subunits of the Fc domains specified herein) contained in an antibody or bispecific antibody as specified herein comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the invention, the heavy chain (comprising subunits of the Fc domains specified herein) contained in an antibody or bispecific antibody as specified herein comprises an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). The compositions/formulations of the invention, such as the pharmaceutical compositions/formulations described herein, comprise an antibody or bispecific antibody population of the invention. An antibody or bispecific antibody population may comprise molecules with full length heavy chains and molecules with cleaved variant heavy chains. The antibody or bispecific antibody population may consist of a mixture of molecules having full length heavy chains and molecules having cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies or bispecific antibodies have cleaved variant heavy chains. In one embodiment of the invention, a composition comprising an antibody or bispecific antibody population of the invention comprises an antibody or bispecific antibody comprising a heavy chain comprising a subunit of an Fc domain specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the invention, a composition comprising an antibody or bispecific antibody population of the invention comprises an antibody or bispecific antibody comprising a heavy chain comprising a subunit of an Fc domain specified herein and an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). In one embodiment of the invention, such a composition comprises an antibody or bispecific antibody population consisting of the following molecules: a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein; a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbered according to Kabat EU index); and a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is performed according to the EU numbering system (also known as the EU index) as described by Kabat et al (Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991) (see also above). As used herein, a "subunit" of an Fc domain refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising a C-terminal constant region capable of stabilizing a self-associated immunoglobulin heavy chain. For example, a subunit of an IgG Fc domain comprises IgG CH2 and IgG CH3 constant domains. In a preferred embodiment, such Fc domains are of human origin, in a preferred embodiment belonging to the IgG isotype, and in another preferred embodiment belonging to the IgG1 isotype.

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

The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region as defined herein.

"fusion" means that components (e.g., fab molecules and Fc domain subunits) are linked via peptide bonds either directly or via one or more peptide linkers.

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

"crosslever" Fab molecules (also referred to as "Crossfab") refer to Fab molecules in which the variable domains or constant domains of the Fab heavy and Fab light chains are exchanged (i.e. replaced with each other), i.e. the exchanged Fab molecules comprise a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1CH1 (VL-CH 1 in the N-to C-terminal direction) and a peptide chain consisting of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL in the N-to C-terminal direction). For clarity, in the swapped Fab molecules in which the variable domains of the Fab light and Fab heavy chains are swapped, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (swapped) Fab molecule. In contrast, in an exchangeable Fab molecule in which the constant domains of the Fab light and Fab heavy chains are exchanged, the peptide chain comprising the heavy chain variable domain VH is herein referred to as the "heavy chain" of the (exchangeable) Fab molecule.

In contrast, a "conventional" Fab molecule refers to a Fab molecule in its natural form (i.e., comprising a heavy chain consisting of a heavy chain variable domain and a constant domain (VH-CH 1 in the N-to C-terminal direction) and a light chain consisting of a light chain variable domain and a constant domain (VL-CL in the N-to C-terminal direction). The terms "host cell", "host cell line" and "host cell culture (host cell culture)" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including progeny cells of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells as well as progeny cells derived therefrom, regardless of the number of passages. The nucleic acid content of the daughter cells may not be exactly the same as the parent cell, but may contain mutations. Included herein are mutant daughter cells that have the same function or biological activity as selected or selected from the original transformed cells.

A "human antibody" is an antibody having an amino acid sequence corresponding to an amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing the human antibody lineage (antibody repertoire) or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.

"humanized" antibody refers to chimeric antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one (and typically two) variable domains, in which all or substantially all CDRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

The term "complementarity determining region (complementarity determining region)" or "CDR" as used herein refers to each region of an antibody variable domain that is highly variable in sequence and/or forms a structurally defined loop ("highly variable loop (hypervariable loop)") and/or comprises antigen contacting residues ("antigen contact"). Typically, an antibody comprises six CDRs: three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Herein, exemplary CDRs include:

(a) Highly variable loops (Chothia and Lesk, J.mol.biol.196:901-917 (1987)) present at amino acid residues 26-32 (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3), 26-32 (CDR-H1), 53-55 (CDR-H2) and 96-101 (CDR-H3);

(b) CDRs present at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2) and 95-102 (CDR-H3) (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991));

(c) Antigen-contacting points present at amino acid residues 27c-36 (CDR-L1), 46-55 (CDR-L2), 89-96 (CDR-L3), 30-35b (CDR-H1), 47-58 (CDR-H2) and 93-101 (CDR-H3) (MacCallum et al J.mol. Biol.262:732-745 (1996)); and

(d) Combinations of (a), (b) and/or (c) including CDR amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (vL 3), 31-35 (CDR-H1), 50-63 (CDR-H2) and 95-102 (CDR-H3).

CDR residues and other residues in the variable domains (e.g., FR residues) are numbered herein according to the methods described by Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991), unless otherwise indicated.

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

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

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. The isolated nucleic acid comprises a nucleic acid molecule that is normally contained in a cell comprising the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from the natural chromosomal location.

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

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

A "modification that facilitates association of a first subunit and a second subunit of an Fc domain" is an manipulation of the peptide backbone or post-translational modification of an Fc domain subunit that reduces or prevents association of a polypeptide comprising an Fc domain subunit with the same polypeptide to form a homodimer. Modification to facilitate association as used herein specifically includes individual modification to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to be associated, wherein the modifications are complementary to each other so as to facilitate association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both Fc domain subunits to render them sterically or electrostatically advantageous, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may differ in terms of components that are further fused to each subunit (e.g., antigen binding portion). In some embodiments, the modification that facilitates association includes an amino acid mutation, particularly an amino acid substitution, in the Fc domain. In a particular embodiment, the modification that facilitates association comprises a separate amino acid mutation, in particular an amino acid substitution, in each of the two subunits of the Fc domain. Such modifications that facilitate association of the first and second subunits of the Fc domain play an important role in heterodimerization of the multispecific or bispecific antibodies (see also exemplary multispecific anti-HLA-G/anti-CD 3 antibodies, e.g., below a.2).

"native antibody (Native antibodies)" refers to native immunoglobulin molecules having different structures. For example, an Ig natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains disulfide bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH 1, CH2 and CH 3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a light chain Constant (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be categorized into one of two types, called kappa (kappa) and lambda (lambda).

The term "package insert" is used to refer to instructions that are typically contained in commercial packages of therapeutic products, including information about the indication, usage, dosage, route of administration, combination therapy, contraindications and/or warnings for use of such therapeutic products.

"percent (%) amino acid sequence identity" as referred to with respect to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, with the greatest percentage of sequence identity being achieved after aligning the sequences and introducing differences (if necessary), and without regard to any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity may be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 was used to generate% amino acid sequence identity values. ALIGN-2 sequence comparison computer programs were written by Jian nan Dekker (Genntech, inc.), the source code has been deposited with the user files in the United states copyright office, washington, inc., 20559, and registered with the United states copyright registration number TXU 510087. ALIGN-2 programs are publicly available from Jiannan Dekker (Genntech, inc.) of san Francisco, calif., or compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including the digital UNIX V4.0D). All sequence comparison parameters were set by the ALIGN-2 program and did not change.

In the case of amino acid sequence comparison using ALIGN-2, the% amino acid sequence identity (which is alternatively expressed as given amino acid sequence A, which has or comprises a certain% amino acid sequence identity to, with, or relative to the given amino acid sequence B) for a given amino acid sequence A pair is calculated as follows:

100 times the fraction X/Y

Wherein X is the number of amino acid residues in the alignment program ALIGN-2 scored as identical matches in the alignment of A and B, and Y is the total number of amino acid residues in B. It will be appreciated that in the case where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. All% amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the previous paragraph, unless specifically indicated otherwise.

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

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

As used herein, "treatment" (and grammatical variants thereof, such as "treatment" or "treatment") refers to a clinical intervention that attempts to alter the natural course of a disease in a subject being treated, and may be performed prophylactically or during a clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating a disease state, alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the progression of a disease or to slow the progression of a disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, and each domain comprises four reserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). (see, e.g., kindt, T.J. et al Kuby Immunology, 6 th edition, W.H. Freeman and Co., N.Y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains can be used to isolate antibodies that bind a particular antigen from antibodies that bind the antigen, to screen libraries of complementary VL or VH domains, respectively. See, for example: portolano, S.et al, J.Immunol.150 (1993) 880-887; clackson, T.et al, nature 352 (1991) 624-628.

As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that are incorporated into the genome of the host cell into which they have been introduced. Certain vectors are capable of directing expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

I. Compositions and methods

In one aspect, the present invention is based in part on the discovery that it is surprising that of these various variants of HLA-G-0090 with glycosylation sites removed, only two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A exhibit even improved binding properties, good expression capacity and stability, while showing no more glycosylation at the CDR-L1 of the LC (thus Fab glycosylation cannot be detected). Since all recently approved pharmaceutical antibody products are produced in mammalian cells, especially CHO cells (see, e.g., walsh g., nature Biotech (2018) 1136-1145), providing an antibody that lacks glycosylation sites in the binding regions (VH and VL, and especially CDR) represents a valuable advantage, as these antibodies can then be readily used in the production of mammalian expression systems without compromising (at least in part) the binding properties by glycosylation. In particular for antibodies comprising an Fc domain, resistance in host cells lacking a glycosylation mechanism (such as a prokaryotic host cell) does not yield a product with comparable quality, because the N-glycan linked to amino acid residue ASN297 (numbered according to Kabat EU index) needs to remain soluble and thermostable in the Fc region and prevents aggregation of the antibody in aqueous solution (e.g., in a pharmaceutical composition).

A.1 exemplary anti-HLA-G antibodies

One embodiment of the present invention is an antibody that binds to human HLA-G, comprising

In embodiments, such an anti-HLA-G antibody has improved binding characteristics in terms of maximum binding (Rmax) and/or binding affinity (KD) compared to a (parent) antibody comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO:8 (as shown in example 2). In another embodiment, an HLA-G antibody of the invention comprises a human Fc domain; in one embodiment, the Fc domain belongs to the IgG isotype, and in a preferred embodiment, to the IgG1 isotype. In one embodiment, such a human IgG1 isotype Fc domain comprises the amino acid mutations L234A, L a and P329G ("P329G LALA", "PGLALA" or "lalag") (numbering according to the Kabat EU index).

In another embodiment, the HLA-G antibodies of the invention comprise a constant region of human origin, particularly of the IgG isotype, more particularly of the IgG1 isotype, comprising human CH1, CH2, CH3 and/or CL domains.

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

Such antibodies comprising an Fc domain typically may undergo N-glycosylation at the ASN-297 locus (numbered according to the Kabat EU index), for example when produced in eukaryotic cells such as mammalian cells, in particular CHO cells. N-glycosylation at the ASN-297 position (numbered according to the EU index (see Kabat)) represents a valuable contribution to such antibodies, e.g., high stability, low aggregation tendency, and/or good pharmacokinetics and other key mass properties (see e.g., zheng et al, mAbs (2011) 568-576; and Reusch et al, glycobiology (2015) 1325-133). Thus, such antibodies comprising an Fc domain of the invention are readily prepared for production in eukaryotic cells (e.g., mammalian cells, in particular CHO cells) without the risk of glycosylation of the binding region (which would interfere with its binding properties), but with the benefit of N-glycosylation at the ASN-297 position and valuable quality attributes.

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

In embodiments, the anti-HLA-G antibody

a) Does not cross-react with a modified human HLA-G.beta.2M MHC I complex, wherein HLA-G specific amino acids have been replaced by HLA-A consensus amino acids, the complex comprising SEQ ID NO:40; and/or

b) Does not cross-react with the mouse H2Kdβ2M MHC I complex comprising SEQ ID NO 41; and/or

c) Does not cross-react with the rat RT1A beta 2M MHC I complex comprising SEQ ID NO. 43.

In embodiments, the anti-HLA-G antibody

a) Inhibit ILT2 binding to JEG3 cells (ATCC accession No. HTB36) (HLA-G expressed thereon); or (b)

b) Binding to JEG3 cells (ATCC accession HTB 36) (HLA-G expressed thereon), and inhibiting ILT2 binding to JEG-3 cells (ATCC accession HTB 36) (HLA-G expressed thereon).

In another aspect, the invention relates to a multispecific antibody comprising an anti-HLA-G antigen binding portion. These multispecific antibodies (e.g., bispecific antibodies) as described herein use selected, improved anti-HLA-G antibodies as the first antigen binding portion/site. These anti-HLA-G antibodies bind to HLA-G with high specificity and affinity (improved binding properties, cross-reactivity with other species and human HLA-A consensus sequences), and have the ability to specifically inhibit ILT2 and/or ILT4 binding to HLA-G.

In one embodiment, the invention relates to multispecific (preferably bispecific) antibodies that bind to human HLA-G and to human CD 3. In one embodiment, the invention relates to a multispecific (preferably bispecific) anti-HLA-G/anti-CD 3 antibody, wherein the multispecific (preferably bispecific) antibody that binds to human HLA-G and to human CD3 comprises a first antigen binding portion that binds to human HLA-G and a second antigen binding portion that binds to human CD 3. The bispecific antibody as described herein binds to a specific second antigen binding portion/site to CD3, in particular CD3 epsilon, and is thus capable of attracting CD3 expressing T cells to HLA-G expressing tumor cells while inhibiting HLA-G induced immunosuppression in the tumor environment by blocking the binding of ILT2/4 to HLA-G. Thus, these bispecific anti-HLA-G/anti-CD 3 antibodies show strong in vivo tumor growth inhibition and tumor regression.

A.2 exemplary multispecific anti-HLA-G/anti-CD 3 antibodies

One embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3, wherein the first antigen-binding portion that binds to human HLA-G comprises

C) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 60, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 61 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 62; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence SEQ ID NO:63, (ii) a CDR-L2 comprising the amino acid sequence SEQ ID NO:64 and (iii) a CDR-L3 comprising the amino acid sequence SEQ ID NO:65, or

D) (a) a VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 68, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 69 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 70; and (b) a VL domain comprising (i) CDR-L1 comprising the amino acid sequence SEQ ID NO:71, (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO:72 and (iii) CDR-L3 comprising the amino acid sequence SEQ ID NO:73.

Another embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, comprising a first antigen-binding portion that binds to human HLA-G and a second antigen-binding portion that binds to human CD3,

wherein the first antigen binding portion

and wherein the second antigen binding portion

Wherein the first antigen binding portion

and wherein the second antigen binding portion

wherein the first antigen binding portion

and wherein the second antigen binding portion

Wherein the first antigen binding portion

and wherein the second antigen binding portion

In one embodiment, these bispecific antibodies are characterized by one or more of the following properties:

a) Inducing T cell mediated cytotoxicity/tumor cell killing in the presence of HLA-G expressing tumor cells, preferably in the presence of JEG3 cells (ATCC accession number HTB 36); and/or

b) Inducing ifnγ secretion by T cells in the presence of HLA-G expressing tumor cells, preferably JEG3 cells (ATCC accession number HTB 36); and/or

c) Inhibit tumor growth in vivo (in a mouse xenograft tumor model),

d) In vivo anti-tumor efficacy/tumor regression in humanized NSG mice bearing SKOV3 human ovarian cancer transfected with recombinant HLA-G (SKOV 3 HLA-G) humanized NSG mice (see example 13); and/or

e) In vivo anti-tumor efficacy/tumor of HLA-G CD 3T cells bispecific in humanized NSG mice bearing human breast cancer PDX tumor (BC 004) (see example 14).

In one embodiment, these bispecific antibodies are further characterized by one or more of the following properties: bispecific antibodies

a) Does not cross-react with a modified human HLA-G.beta.2M MHC I complex, wherein HLA-G specific amino acids have been replaced by HLA-A consensus amino acids, the complex comprising SEQ ID NO 44; and/or

b) Binding to JEG3 cells (ATCC accession HTB 36) (HLA-G expressed thereon), and inhibiting ILT2 binding to JEG3 cells (ATCC accession HTB 36) (HLA-G expressed thereon); and/or

Multispecific antibodies

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

Techniques for preparing multispecific, particularly bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs of different specificities (see Milstein and Cuello, nature 305:537 (1983)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al J.mol. Biol.270:26 (1997)). Multispecific antibodies can also be prepared by the following method: engineered electrostatic steering effects for the preparation of antibody Fc-heterodimer molecules (see, e.g., WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al Science,229:81 (1985)); bispecific antibodies are generated using leucine zippers (see, e.g., kostelny et al, j. Immunol.,148 (5): 1547-1553 (1992) and WO 2011/034605); the use of conventional light chain technology prevents the light chain mismatch problem (see, e.g., WO 98/50431); bispecific antibody fragments were prepared using the "diabody" technique (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA,90:6444-6448 (1993)); and single chain Fv (sFv) dimers (see, e.g., gruber et al, J.Immunol.,152:5368 (1994)); and the preparation of trispecific antibodies according to the method described, for example, by Tutt et al (J. Immunol.147:60 (1991)).

Also included herein are engineered antibodies having three or more antigen binding sites, including, for example, "octopus antibodies" (Octopus antibodies) or DVD-Ig (see, for example, WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies having three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Bispecific antibodies or antigen binding fragments thereof also comprise a "dual acting FAb" or "DAF" comprising antigen binding sites that bind to HLA-G and another different antigen or two different epitopes of HLA-G (see, e.g., US2008/0069820 and WO 2015/095539).

Multispecific antibodies may also be provided in asymmetric form comprising domains that cross in one or more binding arms of the same antigen specificity, i.e. by exchanging VH/VL domains (see e.g. WO 2009/080252 and WO 2015/150447), CH1/CL domains (see e.g. WO 2009/080253) or whole Fab arms (see e.g. WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS,108 (2011) 1187-1191, and Klein et al, MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be designed by introducing charged or uncharged amino acid mutations into the domain interface to guide correct Fab pairing. See, for example, WO 2016/172485.

Various other molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., spiess et al, mol Immunol 67 (2015) 95-106).

Also included herein are specific types of multispecific antibodies that are bispecific antibodies designed to bind simultaneously to a surface antigen on a target cell (e.g., a tumor cell) and to an activated invariant component (e.g., CD 3) complex of a T Cell Receptor (TCR) for recalibrating the T cell to kill the target cell. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one of the binding specificities is for HLA-G and the other binding specificity is for CD3.

Bispecific antibody formats useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules, in which two scFv molecules are fused by a flexible linker (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO2008/119567, nagorsen and nagorsen)Exp Cell Res317, 1255-1260 (2011)); diabodies (Holliger et al, prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("Tandab"; kipriyanov et al, J Mol Biol 293, 41-56 (1999)); "DART" (double affinity relocating) molecules, which are based on diabody forms but have a C-terminal disulfide bond for further stabilization (Johnson et al, J Mol Biol 399, 436-449 (2010)), and so-called tri-functional antibodies (triomab), which are intact mouse/rat IgG hybrid molecules (see for review by Seimetz et al: cancer Treat Rev36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in: WO 2013/026833; WO2013/026839; WO 2016/020309; and Bacac et al Oncoimmunology 5 (8) (2016) e1203498.

Bispecific antibodies that bind to HLA-G and to CD3

The invention also provides a bispecific antibody, i.e. an antibody comprising at least two antigen binding portions capable of specifically binding to two different epitope bits (a first antigen and a second antigen).

Based on the anti-HLA-G antigen binding portion and the anti-CD 3 antigen binding portion developed by the present inventors, the present inventors developed bispecific antibodies that bind to HLA-G and to CD 3.

As shown in the examples, these bispecific antibodies have a number of significant properties, including good potency and low toxicity.

Accordingly, in certain aspects, the present invention provides a bispecific antibody comprising (a) a first antigen-binding portion that binds to human HLA-G, and (b) a second antigen-binding portion that specifically binds to human CD3, wherein the bispecific antibody has any of the following characteristics: the bispecific antibodies of the invention specifically induce T cell mediated killing of HLA-G expressing cells. In some embodiments, bispecific antibodies of the invention specifically induce T cell-mediated killing of HLA-G expressing cells. In a more specific embodiment, the bispecific antibody specifically induces T cell mediated killing of HLA-G expressing cells.

In one embodiment, the bispecific antibody-induced T cell mediated killing is determined using HLA-G expressing cells.

In one embodiment, the activation of T cells by the bispecific antibody is determined by the following method: the expression of CD25 and/or CD69 of T cells after incubation with bispecific antibodies in the presence of HLA-G expressing cells is measured (particularly by flow cytometry).

In one specific embodiment, the bispecific antibody-induced T cell mediated killing is determined using the following method:

the ability of anti-HLA-G/anti-CD 3 TCB to activate T cells in the presence of HLA-G expressing tumor cells was tested on recombinant HLA-G transfected SKOV3 cells (SKOV 3 HLA-G). Activation of T cells was assessed by FACS analysis for cell surface activation marker CD25 and early activation marker CD69 on T cells. Briefly, peripheral Blood Mononuclear Cells (PBMC) were isolated from human peripheral blood by density gradient centrifugation using a lymphocyte separation medium tube (PAN#P04-60125). PBMCs were seeded to SKOV3HLA-G cells at a ratio of 10:1 to 96 well U-shaped bottom plates. The Co-cultures were then incubated with different concentrations of HLA-G-TCB and incubated at 37℃for 24 hours in an incubator containing 5% Co2 as described in example 12. The next day, the expression of CD25 and CD69 was measured by flow cytometry.

In flow cytometry, cells were stained with PerCP-Cy5.5 mouse anti-human CD8 (BD Pharmingen # 565310), PE mouse anti-human CD25 (eBioscience # 9012-0257) and APC mouse anti-human CD69 (BD Pharmingen # 555533) at 4 ℃. Briefly, antibodies were diluted to 2-fold concentration and 25 μl of antibody dilution was added to each well containing 25 μl of pre-washed co-culture. Cells were stained at 4℃for 30 min, washed twice with 200. Mu.l/Kong Ranse buffer and centrifuged at 300g for 5 min. The cell pellet was resuspended in 200. Mu.l staining buffer and stained with DAPI to distinguish between dead/surviving cells at a final concentration of 2. Mu.g/ml. The samples were then measured using a BD LSR flow cytometer. Data analysis was performed using FlowJo v.10.1 software.

The bispecific antibodies of the invention specifically activate T cells in the presence of HLA-G expressing cells. In some embodiments, the bispecific antibodies of the invention specifically activate T cells in the presence of cells expressing HLA-G. In a more specific embodiment, the bispecific antibody specifically activates T cells in the presence of cells expressing HLA-G.

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

As a specific embodiment of the invention, the antigen binding portion comprised in the bispecific antibody is a Fab molecule (i.e. an antigen binding domain consisting of a heavy chain and a light chain, each of which comprises a variable domain and a constant domain). In one embodiment, the first antigen binding portion and/or the second antigen binding portion is a Fab molecule. In one embodiment, the Fab molecule is a human Fab molecule. In a particular embodiment, the Fab molecule is a humanized molecule. In another embodiment, the Fab molecule comprises human heavy and light chain constant domains.

Preferably, at least one of the antigen binding portions is a cross-Fab molecule. Such modifications reduce the mismatch of the heavy and light chains from different Fab molecules, thereby increasing the yield and purity of the bispecific antibodies of the invention in recombinant production. In the specific exchange type Fab molecules used in the bispecific antibodies of the invention, the variable domains of the Fab light and Fab heavy chains (VL and VH, respectively) are exchanged. However, even with this domain exchange, the preparation of bispecific antibodies may contain certain byproducts due to the so-called Bence Jones-type interaction between mismatched heavy and light chains (see Schaefer et al, PNAS,108 (2011) 11187-11191). To further reduce the mismatches from the heavy and light chains of the different Fab molecules, thereby increasing the purity and yield of the desired bispecific antibody, oppositely charged amino acids may be introduced at specific amino acid positions in the CH1 and CL domains of the Fab molecule that binds to the first antigen (HLA-G) or the Fab molecule that binds to the second antigen (activated T cell antigen such as CD 3), as further described herein. Charge modification is performed in conventional Fab molecules comprised in bispecific antibodies (such as for example shown in fig. 13A to 13C, 13G to 13J) or in VH/VL exchanging Fab molecules comprised in bispecific antibodies (such as shown in fig. 13D to 13F, 13K to 13N but not both). In certain embodiments, the charge modification is performed in a conventional Fab molecule comprised in a bispecific antibody (which in certain embodiments binds to a first antigen, i.e. HLA-G).

In a particular embodiment as in the present invention, the bispecific antibody is capable of binding to both a first antigen (i.e.HLA-G) and a second antigen (e.g.an activated T cell antigen, in particular CD 3). In one embodiment, the bispecific antibody is capable of cross-linking T cells and target cells by binding to HLA-G and an activated T cell antigen. In an even more specific embodiment, such simultaneous binding results in lysis of target cells, particularly tumor cells expressing HLA-G. In one embodiment, such simultaneous binding results in T cell activation. In other embodiments, such simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector release, cytotoxic activity and expression of activation markers. In one embodiment, the bispecific antibody binds to an activated T cell antigen, particularly CD3, but not to HLA-G simultaneously, and does not result in T cell activation.

In one embodiment, the bispecific antibody is capable of redirecting the cytotoxic activity of T cells to target cells. In a particular embodiment, the redirecting is independent of MHC mediated peptide antigen presentation by the target cells and/or the specificity of the T cells.

In particular, the T cells according to any embodiment of the invention are cytotoxic T cells. In some embodiments, the T cell is CD4 ⁺ Or CD8 ⁺ Cells, in particular CD8 ⁺ T cells.

First antigen-binding portion that binds to human HLA-G

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

In particular embodiments, the antigen binding portion that binds to human HLA-G is a conventional Fab molecule. In such embodiments, the antigen binding portion that binds to the second antigen is a cross Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains or the constant domains CH1 and CL are swapped/replaced with each other.

In alternative embodiments, the antigen binding portion that binds to human HLA-G is an exchanged Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In such embodiments, the antigen binding portion that binds to the second antigen is a conventional Fab molecule.

The HLA-G binding moiety is capable of directing bispecific antibodies to target sites, e.g., to specific types of tumor cells that express human HLA-G.

Unless clearly unreasonable or impossible scientifically, the first antigen-binding portion of a bispecific antibody can bind any of the features described herein in connection with an antibody that binds HLA-G, alone or in combination.

Thus, in one aspect, the invention provides a bispecific antibody comprising a first antigen-binding portion that binds to a first antigen, wherein the first antigen is human HLA-G (in one embodiment, the antibody that binds to the HLA-Gβ2MMHC I complex comprises SEQ ID NO: 39), and the first antigen-binding portion comprises

(a) A VH domain comprising (i) a CDR-H1 comprising the amino acid sequence SEQ ID NO 1,

(ii) CDR-H2 comprising the amino acid sequence SEQ ID NO 2 and (iii) CDR-H3 comprising the amino acid sequence SEQ ID NO 3; and wherein the VH domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment 98% or 99% or 100%) sequence identity to amino acid sequence SEQ ID No. 7; and (b) a VL domain comprising (i) CDR-L1 comprising the amino acid sequence SEQ ID NO. 23, (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO. 5 and (iii) CDR-L3 comprising the amino acid sequence SEQ ID NO. 6; and wherein the VL domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment 98% or 99% or 100%) sequence identity to the amino acid sequence SEQ ID NO. 24.

Terms such as "VH domain comprising (i) CDR-H1 comprising the amino acid sequence SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence SEQ ID NO:2 and (iii) CDR-H3 comprising the amino acid sequence SEQ ID NO:3; and wherein the VH domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment 98% or 99% or 100%) sequence identity to amino acid sequence SEQ ID No. 7 "refers to a VH domain having amino acid sequence SEQ ID No. 7 wherein the 3 CDRs are unchanged (i.e. identical to SEQ ID No. 7) but zero, one, two, three, four or five amino acid residues in the framework region of the VH are changed/substituted by another amino acid, e.g. without affecting the binding properties of the VH to the antigen binding site. Since backbone residues with high probability of affecting binding properties are well known (see, e.g., foote J. And Winter G., J. Mol. Biol. (1992) 224, 487-499), substitutions may be selected that do not affect or affect very little residues. The same applies to similar terms used herein in relation to another VH or VL.

In one embodiment, the first antigen binding portion comprises: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 24.

In one embodiment, a first antigen-binding portion that binds to human HLA-G (in one embodiment, to an HLA-Gβ2M MHC I complex comprising SEQ ID NO: 39) comprises (a) a VH domain comprising (I) CDR-H1 comprising amino acid sequence SEQ ID NO:1, (ii) CDR-H2 comprising amino acid sequence SEQ ID NO:2 and (iii) CDR-H3 comprising amino acid sequence SEQ ID NO:3; and wherein the VH domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment 98% or 99% or 100%) sequence identity to amino acid sequence SEQ ID No. 7; and (b) a VL domain comprising (i) CDR-L1 comprising the amino acid sequence SEQ ID NO:25, (ii) CDR-L2 comprising the amino acid sequence SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acid sequence SEQ ID NO:6; and wherein the VL domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment 98% or 99% or 100%) sequence identity to the amino acid sequence SEQ ID NO. 26.

In one embodiment, the first antigen binding portion comprises: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 26.

Such anti-HLA-G antibodies exhibit very valuable properties because they are free of N-glycosylation at the antigen binding site (and CDR-L1) (as shown in example 2); has improved binding properties in terms of maximum binding (Rmax) and/or binding affinity (KD) compared to a (parent) antibody comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO:8 (as shown in example 2); non-cross-reactive with HLA-A MHC I complex or mouse or rat MHC I complex and binds to JEG3 cells (HLa-G expressed thereon) (ATCC accession No. HTB 36); and inhibits ILT2 binding to JEG-3 cells (HLA-G expressed thereon) (ATCC accession No. HTB36).

Second antigen binding portion that binds to human CD3

Bispecific antibodies of the invention comprise at least one antigen-binding portion (particularly a Fab molecule) that binds to human CD 3.

In a particular embodiment, the antigen binding portion that binds human CD3 is an exchangeable Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In such embodiments, the antigen binding portion that binds to human HLA-G is preferably a conventional Fab molecule. In embodiments where there is more than one antigen binding portion (particularly a Fab molecule) that binds to human CD3 contained in the bispecific antibody, the antigen binding portion that binds to human CD3 is preferably an exchangeable Fab molecule and the antigen binding portion that binds to human HLA-G is a conventional Fab molecule.

In an alternative embodiment, the antigen binding portion that binds to the second antigen is a conventional Fab molecule. In such embodiments, the antigen binding portion that binds to the first antigen (i.e., HLA-G) is an exchangeable Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In embodiments in which there is more than one antigen binding portion (particularly a Fab molecule) that binds to a second antigen contained in the bispecific antibody, the antigen binding portion that binds to human HLA-G is preferably an exchangeable Fab molecule and the antigen binding portion that binds to human CD3 is a conventional Fab molecule.

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

In particular embodiments, the second antigen is CD3, particularly human CD3 (SEQ ID NO: 88) or cynomolgus monkey CD3 (SEQ ID NO: 89), and most particularly CD3. In one embodiment, the second antigen binding portion cross-reacts with (i.e., specifically binds to) human and cynomolgus monkey CD3. In some embodiments, the second antigen is the epsilon subunit of CD3 (CD 3 epsilon).

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

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

In one embodiment, the second antigen binding portion that binds to human CD3 comprises: (a) A VH domain comprising (i) CDR-H1 comprising amino acid sequence SEQ ID No. 68, (ii) CDR-H2 comprising amino acid sequence SEQ ID No. 69 and (iii) CDR-H3 comprising amino acid sequence SEQ ID No. 70, and wherein the VH domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment 98% or 99% or 100%) sequence identity to amino acid sequence SEQ ID No. 74; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence SEQ ID NO:71, (ii) a CDR-L2 comprising the amino acid sequence SEQ ID NO:72 and (iii) a CDR-L3 comprising the amino acid sequence SEQ ID NO:73, and wherein the VL domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% (in a preferred embodiment 98% or 99% or 100%) sequence identity to the amino acid sequence SEQ ID NO: 75.

In one embodiment, the second antigen binding portion that binds to human CD3 comprises: a VH domain comprising the amino acid sequence SEQ ID No. 58; and a VL domain comprising the amino acid sequence SEQ ID NO 59.

In one embodiment, the second antigen binding portion that binds to human CD3 comprises: a VH domain comprising the amino acid sequence SEQ ID No. 66; and a VL domain comprising the amino acid sequence SEQ ID NO:67.

In one embodiment, the second antigen binding portion that binds to human CD3 comprises: a VH domain comprising the amino acid sequence SEQ ID No. 74; and a VL domain comprising the amino acid sequence SEQ ID NO 75.

Such CD3 antigen binding portions/sites exhibit very valuable properties (e.g., when provided as bispecific antibodies that bind to CD3 and HLA-G (wherein the HLA-G antigen binding portion is as described herein)). They show that: a (a)

a) Good thermal stability

b) Inducing ifnγ secretion by T cells in the presence of HLA-G expressing tumor cells, preferably in the presence of JEG3 cells (ATCC accession number HTB 36) (example 11); and/or

c) Inducing T cell mediated cytotoxicity/tumor cell killing in the presence of HLA-G expressing tumor cells, preferably in the presence of JEG3 cells (ATCC accession number HTB 36) (example 12); and/or

d) Inhibit tumor growth in vivo (in a mouse xenograft tumor model),

e) In vivo anti-tumor efficacy/tumor regression in SKOV3 human ovarian cancer humanized NSG mice (SKOV 3 HLA-G) humanized NSG mice harboring recombinant HLA-G transfection (see example 13); and/or

f) In vivo anti-tumor efficacy/tumor of HLA-G CD 3T cells bispecific in humanized NSG mice bearing human breast cancer PDX tumor (BC 004) (see example 14).

In some embodiments, the second antigen binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light and Fab heavy chains or the constant domains CL and CH1, in particular the variable domains VL and VH, are substituted for each other (i.e. according to such embodiments, the second antigen binding portion is a cross-Fab molecule, wherein the variable domains or constant domains of the Fab light and Fab heavy chains are exchanged). In one such embodiment, the first (and third, if any) antigen binding moiety is a conventional Fab molecule.

In one embodiment, no more than one antigen binding portion that binds to a second antigen (e.g., an activated T cell antigen such as CD 3) is present in the bispecific antibody (i.e., the bispecific antibody provides monovalent binding to the second antigen).

Charge modification

Bispecific antibodies of the invention may comprise amino acid substitutions in the Fab molecules contained therein that are particularly effective in reducing the mismatch of light chains with unmatched heavy chains (by-products of the Bence-Jones type), which may occur in the preparation of Fab-based multispecific antibodies, wherein VH/VL exchanges occur in one (or more if the molecule comprises more than two antigen-binding Fab molecules) of its binding arms (see also PCT publication No. WO 2015/150447, particularly examples thereof, the entire disclosure of which is incorporated herein by reference). The ratio of desired bispecific antibodies to undesired byproducts, particularly the Bence Jones type byproducts that occur in bispecific antibodies having VH/VL domain exchange in one of their binding arms, can be improved by introducing oppositely charged amino acids at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modifications").

Thus, in some embodiments, wherein the first and second antigen-binding portions of the bispecific antibody are each Fab molecules, and in one of the antigen-binding portions (particularly the second antigen-binding portion), the variable domains VL and VH of the Fab light and heavy chains are replaced with each other,

i) In the constant domain CL of the first antigen binding portion, the amino acid at position 124 is substituted with a positively charged amino acid (numbered according to Kabat) and wherein in the constant domain CH1 of the first antigen binding portion the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbered according to Kabat EU index); or (b)

ii) in the constant domain CL of the second antigen binding portion the amino acid at position 124 is substituted with a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH1 of the second antigen binding portion the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbering according to Kabat EU index).

Bispecific antibodies do not comprise the modifications mentioned under i) and ii). The constant domains CL and CH1 having VH/VL exchanged antigen-binding portions are not substituted for each other (i.e., remain in the un-exchanged state).

In a more specific embodiment of the present invention,

i) In the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first antigen binding portion, 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 according to Kabat EU index); or (b)

ii) in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen binding portion, 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 according to Kabat EU index).

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

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

In one particular embodiment, in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU numbering).

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

In an even more specific embodiment, in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted with arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is substituted with glutamic acid (E) (according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (according to Kabat EU index).

In a specific embodiment, the constant domain CL of the first antigen binding portion is of the kappa isotype if the amino acid substitutions according to the above embodiments occur in the constant domain CL and the constant domain CH1 of the first antigen binding portion.

Alternatively, amino acid substitutions according to the above embodiments may occur in the constant domain CL and the constant domain CH1 of the second antigen binding portion, but not in the constant domain CL and the constant domain CH1 of the first antigen binding portion. In certain such embodiments, the constant domain CL of the second antigen binding portion is a kappa isotype.

Thus, in one embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen binding portion, 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 according to Kabat EU index).

In another embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index).

In yet another embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU numbering).

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

In another embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted with lysine (K) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is substituted with glutamic acid (E) (according to Kabat EU numbering) and the amino acid at position 213 is substituted with glutamic acid (E) (according to Kabat EU numbering).

In a particular embodiment, the bispecific antibody of the present invention comprises:

i) A first antigen binding portion that binds to human HLA-G and is a Fab molecule comprising

A) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 32;24

B) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO 26;

and

II) a second antigen binding portion that binds to human CD3,

wherein the second antigen binding portion is a Fab molecule in which the variable domains VL and VH of the Fab light and heavy chains are replaced with each other comprising

C) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 58; and a VL domain comprising the amino acid sequence SEQ ID NO 59, or

D) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 66; and a VL domain comprising the amino acid sequence SEQ ID NO:67, or

E) Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 74; and a VL domain comprising the amino acid sequence SEQ ID NO 75;

and

III) wherein in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), in one particular embodiment, is independently substituted with lysine (K) or arginine (R), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), in one particular embodiment, is independently substituted with lysine (K) or arginine (R), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU numbering).

Bispecific antibody forms

The components of the bispecific antibodies of the invention may be fused to each other in various configurations. An exemplary configuration is shown in fig. 13.

In a particular embodiment, the antigen binding portion comprised in the bispecific antibody is a Fab molecule. In such embodiments, the first antigen binding portion, the second antigen binding portion, the third antigen binding portion, etc., may be referred to herein as a first Fab molecule, a second Fab molecule, a third Fab molecule, etc., respectively.

In one embodiment, the first antigen binding portion and the second antigen binding portion of the bispecific antibody are fused to each other, optionally via a peptide linker. In certain embodiments, the first antigen binding portion and the second antigen binding portion are each Fab molecules. In one such embodiment, the second antigen binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen binding portion at the C-terminus of the Fab heavy chain. In another such embodiment, the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion. In addition, in embodiments wherein (i) the second antigen binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen binding portion at the C-terminus of the Fab heavy chain or (ii) the first antigen binding portion is fused to the N-terminus of the Fab heavy chain of the second antigen binding portion, the Fab light chain of the first antigen binding portion may be fused to each other with the Fab light chain of the second antigen binding portion, optionally via a peptide linker.

Bispecific antibodies with a single antigen binding moiety (such as a Fab molecule) capable of binding to a target cell antigen such as HLA-G (e.g., as shown in fig. 13A, 13D, 13G, 13H, 13K, 13L) can be used, particularly where internalization of the target cell antigen is expected following binding of the high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety to a particular target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

However, in other cases, it would be advantageous to have a bispecific antibody (e.g., as shown in fig. 13B, 13C, 13E, 13F, 13I, 13J, 13M, or 13N) comprising two or more antigen binding moieties (such as Fab molecules) directed against a particular target cell antigen, e.g., to optimize targeting to the target or cross-linking the target cell antigen.

Thus, in a particular embodiment, a bispecific antibody as the invention comprises a third antigen binding moiety.

In one embodiment, the third antigen binding portion binds to the first antigen (i.e., HLA-G). In one embodiment, the third antigen binding portion is a Fab molecule.

In certain embodiments, the third antigen moiety is identical to the first antigen binding moiety.

Unless clearly unreasonable or impossible scientifically, the third antigen-binding portion of a bispecific antibody can bind any of the features described herein in connection with the first antigen-binding portion and/or antibody that binds HLA-G, alone or in combination.

In one embodiment, the third antigen binding portion

Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO. 24; or (b)

Comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO 26; or (b)

In a particular embodiment, the third and first antigen binding portions are each Fab molecules, and the third antigen binding portion is identical to the first antigen binding portion. Thus, in these embodiments, the first antigen binding portion and the third antigen binding portion comprise identical heavy and light chain amino acid sequences and have domains of identical arrangement (i.e., conventional or crossover). Furthermore, in these embodiments, the third antigen-binding portion comprises the same amino acid substitution (if any) as the first antigen-binding portion. For example, the "charge modified" amino acid substitutions described herein will be made in the constant domain CL and the constant domain CH1 of each of the first antigen binding portion and the third antigen binding portion. Alternatively, the amino acid substitutions may be made in the constant domain CL and constant domain CH1 of the second antigen binding portion (which in a specific embodiment is also a Fab molecule), but not in the constant domain CL and constant domain CH1 of the first and third antigen binding portions.

Similar to the first antigen binding portion, the third antigen binding portion is specifically a conventional Fab molecule. However, embodiments are also contemplated in which the first antigen binding portion and the third antigen binding portion are cross-Fab molecules (and the second antigen binding portion is a conventional Fab molecule). Thus, in a particular embodiment, the first antigen binding portion and the third antigen binding portion are each conventional Fab molecules, and the second antigen binding portion is a cross Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other embodiments, the first antigen binding portion and the third antigen binding portion are each cross Fab molecules, and the second antigen binding portion is a conventional Fab molecule.

If a third antigen binding portion is present, in a particular embodiment, the first antigen portion and the third antigen portion bind to human HLA-G and the second antigen binding portion binds to the second antigen human CD3, most particularly CD3 epsilon.

In a particular embodiment, the bispecific antibody comprises an Fc domain consisting of a first subunit and a second subunit. The first subunit and the second subunit of the Fc domain are capable of stable association.

Bispecific antibodies according to the invention may have different configurations, i.e. the first antigen binding portion, the second antigen binding portion (and optionally the third antigen binding portion) may be fused to each other and to the Fc domain in different ways. These components may be fused directly to each other or preferably through one or more suitable peptide linkers. In the case of a Fab molecule fused to the N-terminus of a subunit of the Fc domain, it is typically fused via an immunoglobulin hinge region.

In some embodiments, the first antigen binding portion and the second antigen binding portion are each Fab molecules, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit or the second subunit of the Fc domain. In such embodiments, the first antigen binding portion may be fused at the C-terminus of the Fab heavy chain to the other N-terminus of the Fab heavy chain or subunit of the Fc domain of the second antigen binding portion. In a particular such embodiment, the first antigen binding portion is a conventional Fab molecule and the second antigen binding portion is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other such embodiments, the first Fab molecule is a crossover Fab molecule and the second Fab molecule is a regular Fab molecule.

In one embodiment, the first antigen binding portion and the second antigen binding portion are each Fab molecules, the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit or the second subunit of the Fc domain, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion. In a specific embodiment, the bispecific antibody consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consisting of a first subunit and a second subunit, and optionally one or more peptide linkers present, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the first subunit or the N-terminus of the second subunit of the Fc domain. Such a configuration is schematically depicted in fig. 13G and 13K (in these examples, the second antigen binding domain is a VH/VL swapped Fab molecule). Additionally, alternatively, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In another embodiment, the first antigen binding portion and the second antigen binding portion are each Fab molecules, and the first antigen binding portion and the second antigen binding portion are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In a specific embodiment, the bispecific antibody consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consisting of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule and the second Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such a configuration is schematically depicted in fig. 13A and 13D (in these examples, the second antigen binding domain is a VH/VL swapped Fab molecule and the first antigen binding moiety is a conventional Fab molecule). The first Fab molecule and the second Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment, the first Fab molecule and the second Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In one embodiment, the immunoglobulin hinge region is a human IgG ₁ Hinge region, in particular, wherein the Fc domain is IgG ₁ An Fc domain.

In some embodiments, the first antigen binding portion and the second antigen binding portion are each Fab molecules, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit or the second subunit of the Fc domain. In such embodiments, the second antigen binding portion may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion or to the N-terminus of the other of the subunits of the Fc domain (as described above). In a particular such embodiment, the first antigen binding portion is a conventional Fab molecule and the second antigen binding portion is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other such embodiments, the first Fab molecule is a crossover Fab molecule and the second Fab molecule is a regular Fab molecule.

In one embodiment, the first antigen binding portion and the second antigen binding portion are each Fab molecules, the first antigen binding portion being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit or the second subunit of the Fc domain, and the second antigen binding portion being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In a specific embodiment, the bispecific antibody consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consisting of a first subunit and a second subunit, and optionally one or more peptide linkers present, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the first subunit or the N-terminus of the second subunit of the Fc domain. Such a configuration is schematically depicted in fig. 13H and 13L (in these examples, the second antigen binding domain is a VH/VL swapped Fab molecule and the first antigen binding moiety is a conventional Fab molecule). Additionally, alternatively, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In some embodiments, the third antigen binding portion, particularly the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit or the second subunit of the Fc domain. In a particular such embodiment, the first Fab molecule and the third Fab molecule are each conventional Fab molecules, and the second Fab molecule is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other such embodiments, the first Fab molecule and the third Fab molecule are each cross Fab molecules, and the second Fab molecule is a conventional Fab molecule.

In a particular such embodiment, the second antigen binding portion and the third antigen binding portion are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab moleculeAnd (5) fusion. In a specific embodiment, the bispecific antibody consists essentially of a first Fab molecule, a second Fab molecule, and a third Fab molecule, the Fc domain consisting of a first subunit and a second subunit, and optionally one or more peptide linkers present, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such a configuration is schematically depicted in fig. 13B and 13E (in these examples, the second antigen binding domain is a VH/VL swapped Fab molecule and the first and third antigen binding portions are conventional Fab molecules) and fig. 13J and 13N (in these examples, the second antigen binding domain is a conventional Fab molecule and the first and third antigen binding portions are VH/VL swapped Fab molecules). The second Fab molecule and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment, the second Fab molecule and the third Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In one embodiment, the immunoglobulin hinge region is a human IgG ₁ Hinge region, in particular, wherein the Fc domain is IgG ₁ An Fc domain. Additionally, alternatively, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In another such embodiment, the first antigen binding portion and the third antigen binding portion are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding portion. In a specific embodiment, the bispecific antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, the Fc domain consisting of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain,and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such a configuration is schematically depicted in fig. 13C and 13F (in these examples, the second antigen binding domain is a VH/VL swapped Fab molecule and the first and third antigen binding portions are conventional Fab molecules) and fig. 13I and 13M (in these examples, the second antigen binding domain is a conventional Fab molecule and the first and third antigen binding portions are VH/VL swapped Fab molecules). The first Fab molecule and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment, the first Fab molecule and the third Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In one embodiment, the immunoglobulin hinge region is a human IgG ₁ Hinge region, in particular, wherein the Fc domain is IgG ₁ An Fc domain. Additionally, alternatively, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In a configuration of a bispecific antibody in which a Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each of the subunits of the Fc domain via an immunoglobulin hinge region, the two Fab molecules, hinge region and Fc domain essentially form an immunoglobulin molecule. In a particular embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In an even more particular embodiment, the immunoglobulin is an IgG ₁ A subclass of immunoglobulins. In another embodiment, the immunoglobulin is an IgG ₄ A subclass of immunoglobulins. In another particular embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the immunoglobulin comprises a human constant region, in particular a human Fc region.

In some bispecific antibodies of the invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusion of the Fab light chains of the first Fab molecule and the second Fab molecule further reduces the mismatch of Fab heavy and light chains and also reduces the number of plastids required to express some bispecific antibodies of the invention.

The antigen binding portions may be fused directly to the Fc domain or to each other, or to the Fc domain or to each other via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are well known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example (G ₄ S) _n 、(SG ₄ ) _n 、(G ₄ S) _n Or G ₄ (SG ₄ ) _n Peptide linker. "N" is generally an integer from 1 to 10, in particular from 2 to 4. In one embodiment, the peptide linker is at least 5 amino acids in length; in one embodiment, from 5 to 100 amino acids in length; in another embodiment, from 10 to 50 amino acids in length. In one embodiment, the peptide linker is (GxS) _n Or (GxS) _n G _m Where g=glycine, s=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in another embodiment x=4 and n=2. In one embodiment, the peptide linker is (G ₄ S) ₂ . A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is (G ₄ S) ₂ . An exemplary peptide linker suitable for use in linking the Fab heavy chains of the first Fab fragment and the second Fab fragment comprises the sequences (D) - (G) ₄ S) ₂ . Another suitable such connector comprises a sequence (G ₄ S) ₄ . In addition, the connector may comprise (a portion of) an immunoglobulin hinge region. In particular, in the case where the Fab molecule is fused to the N-terminus of the Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof that comprises or lacks an additional peptide linker.

In certain embodiments, a bispecific antibody as the invention comprises: a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL ₍₂₎ -CH1 ₍₂₎ -CH2-CH3 (-CH 4)); and polypeptides wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₁₎ -CH1 ₍₁₎ -CH2-CH3 (-CH 4)). In some embodiments, the bispecific antibody further comprises: a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH) ₍₂₎ -CL ₍₂₎ ) And shares a carboxyl terminal peptide bond (VL) with the Fab light chain polypeptide of the first Fab molecule ₍₁₎ -CL ₍₁₎ ). In certain embodiments, the polypeptides are covalently linked by, for example, disulfide bonds.

In certain embodiments, a bispecific antibody as the invention comprises: a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₂₎ -CL ₍₂₎ -CH2-CH3 (-CH 4)); and polypeptides wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₁₎ -CH1 ₍₁₎ -CH2-CH3 (-CH 4)). In some embodiments, the bispecific antibody further comprises: a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL) with the Fab heavy chain constant region of the second Fab molecule ₍₂₎ -CH1 ₍₂₎ ) And shares a carboxyl terminal peptide bond (VL) with the Fab light chain polypeptide of the first Fab molecule ₍₁₎ -CL ₍₁₎ ). In certain embodiments, the polypeptides are covalently linked by, for example, disulfide bonds.

In some embodiments, the bispecific antibody comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule is constant with the Fab heavy chain of the second Fab molecule The constant region shares a carboxy-terminal peptide bond (i.e., the second Fab molecule comprises an exchangeable Fab heavy chain, wherein the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond (VL) with the Fc domain subunit ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ -CH2-CH3 (-CH 4)). In other embodiments, the bispecific antibody comprises a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4))。

In some of these embodiments, the bispecific antibody comprises an exchangeable Fab light chain polypeptide of a second Fab molecule, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ ) And shares a carboxyl terminal peptide bond (VL) with the Fab light chain polypeptide of the first Fab molecule ₍₁₎ -CL ₍₁₎ ). In other of these embodiments, the bispecific antibody further comprises: polypeptides wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond (VH ₍₂₎ -CL ₍₂₎ -VL ₍₁₎ -CL ₍₁₎ ) The method comprises the steps of carrying out a first treatment on the surface of the Or polypeptides, wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ ) (where appropriate).

Bispecific antibodies according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH 2-CH3 (-CH 4)), or (ii) a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond (VH) with the Fc domain subunit ₍₃₎ -CH1 ₍₃₎ -CH2-CH3 (-CH 4)), and shares a carboxy-terminal peptide bond (VL) with the Fab light chain polypeptide of the third Fab molecule ₍₃₎ -CL ₍₃₎ ). In certain embodiments, the polypeptides are covalently linked by, for example, disulfide bonds.

In some embodiments, the bispecific antibody comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond (VH) with the Fc domain subunit ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ -CH2-CH3 (-CH 4)). In other embodiments, the bispecific antibody comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises an exchanged Fab heavy chain wherein the heavy chain constant region is replaced with a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ -CH2-CH3(-CH4))。

In some of these embodiments, the bispecific antibody comprises an exchangeable Fab light chain polypeptide of a second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL) with the Fab heavy chain constant region of the second Fab molecule ₍₂₎ -CH1 ₍₂₎ ) And shares a carboxyl terminal peptide bond (VL) with the Fab light chain polypeptide of the first Fab molecule ₍₁₎ -CL ₍₁₎ ). In other of these embodiments, the bispecific antibody further comprises: a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond (VL) with the Fab light chain polypeptide of the first Fab molecule ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CL ₍₁₎ ) The method comprises the steps of carrying out a first treatment on the surface of the Or a polypeptide, wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the second Fab moleculeFab light chain constant regions share a carboxy-terminal peptide bond (VL ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ ) (where appropriate).

In all of the different configurations of bispecific antibodies according to the invention, the amino acid substitutions described herein (if present) may be in the CH1 and CL domains of the first antigen binding portion and (if present) the third antigen binding portion/Fab molecule, or in the CH1 and CL domains of the second antigen binding portion/Fab molecule. Preferably they are in the CH1 and CL domains of the first antigen binding portion and (if present) the third antigen binding portion/Fab molecule. According to the concepts of the present invention, if the amino acid substitutions described herein are made in the first antigen binding portion (and, if present, the third antigen binding portion/Fab molecule), then no such amino acid substitutions are present in the second antigen binding portion/Fab molecule. In contrast, if the amino acid substitutions described herein are made in the second antigen binding portion/Fab molecule, then such amino acid substitutions are not present in the first (and, if present), third antigen binding portion/Fab molecule. Amino acid substitutions are particularly made in bispecific antibodies comprising Fab molecules in which the variable domains VL and VH1 of the Fab light and Fab heavy chains are replaced with each other.

In a specific embodiment of a bispecific antibody as in the present invention, in particular, wherein the amino acid substitutions as described herein are performed in the first antigen binding moiety/Fab molecule (and the third antigen binding moiety/Fab molecule if present), the constant domain CL of the first Fab molecule (and the third Fab molecule if present) is of the kappa isotype. In other embodiments of bispecific antibodies as the invention, in particular, wherein the amino acid substitutions as described herein are made in the second antigen binding moiety/Fab molecule, the constant domain CL of the second antigen binding moiety/Fab molecule is the kappa isotype. In some embodiments, the constant domain CL of the first (and, if present, the third) antigen binding portion/Fab molecule and the constant domain CL of the second antigen binding portion/Fab molecule are of the kappa isotype.

In a particular aspect, the present invention provides a bispecific antibody comprising

a) A first antigen-binding portion and a third antigen-binding portion that bind to the first antigen; wherein the first antigen is HLA-G, and wherein the first antigen binding portion and the second antigen binding portion are each (conventional) Fab molecules comprising: a heavy chain variable region comprising the amino acid sequence SEQ ID NO. 7; and a light chain variable region comprising the amino acid sequence SEQ ID NO. 24;

b) A second antigen binding portion that binds to a second antigen; wherein the second antigen is CD3, and wherein the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light and Fab heavy chains are replaced with each other, comprising: (i) A heavy chain variable region comprising the amino acid sequence SEQ ID NO:58, and a light chain variable region comprising the amino acid sequence SEQ ID NO:59 or (ii) a heavy chain variable region comprising the amino acid sequence SEQ ID NO:66, and a light chain variable region comprising the amino acid sequence SEQ ID NO:67; or (iii) a heavy chain variable region comprising the amino acid sequence SEQ ID NO. 74, and a light chain variable region comprising the amino acid sequence SEQ ID NO. 75; and is also provided with

c) An Fc domain consisting of a first subunit and a second subunit;

wherein the method comprises the steps of

In the constant domain CL of the first and third antigen binding portions described under a), the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (more particularly arginine (R)), and wherein in the constant domain CH1 of the first and third antigen binding portions described under a), the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index);

And wherein further

The first antigen binding portion described under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding portion described under b), and the second antigen binding portion described under b) and the third antigen binding portion described under a) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described under C).

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

In another embodiment of these aspects of the invention, in the first subunit of the Fc domain, the serine residue at position 354 is in turn substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular, the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain, the serine residue at position 349 is in turn substituted with a cysteine residue (Y349C) (numbering according to the Kabat EU index).

In yet another embodiment of these aspects of the invention, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbered according to the Kabat EU index).

In yet another embodiment of these aspects of the invention, the Fc domain is a human IgG ₁ An Fc domain.

One specific embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, wherein the antibody comprises: a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID NO. 76; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID No. 77; a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID No. 78; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID No. 79 (wherein in the VH or VL framework region or in the constant region the amino acid is substituted without affecting the specific binding properties of such bispecific antibodies and the properties of the constant region).

One specific embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, wherein the antibody comprises: a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID NO. 76; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID No. 77; a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID No. 78; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence SEQ ID NO. 79,

and wherein the bispecific antibody has one or more of the following properties:

the bispecific antibody showed

a) Inhibition of ILT2 and/or ILT4 binding to HLA-G (see example 10); and/or

b) Antibody-mediated ifnγ secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV 3 HLA-G) and/or on JEG3 cells expressing endogenous HLA-G, wherein the ifnγ secretion is detected (by Luminex technology) (see example 11); and/or

c) T cell mediated cytotoxicity/tumor cell killing on SKOV3 cells transfected with recombinant HLA-G (SKOV 3 HLA-G) and/or JEG3 cells expressing endogenous HLA-G, wherein the cytotoxicity was detected by measuring caspase 8 activation in the cells following treatment with bispecific antibody (see example 12); and/or

In another specific embodiment, the bispecific antibody comprises: a polypeptide comprising the amino acid sequence SEQ ID NO 76; a polypeptide comprising the amino acid sequence SEQ ID NO. 77; a polypeptide comprising the amino acid sequence SEQ ID NO. 78; and a polypeptide comprising the amino acid sequence SEQ ID NO. 79.

Another specific embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, wherein the antibody comprises: a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to sequence SEQ ID No. 80; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to sequence SEQ ID NO. 81; a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID No. 82; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID No. 83 (wherein in the VH or VL framework region or in the constant region the amino acids are substituted without affecting the specific binding properties of such bispecific antibodies and the properties of the constant region).

One specific embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, wherein the antibody comprises: a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to sequence SEQ ID No. 80; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to sequence SEQ ID NO. 81; a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID No. 82; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence SEQ ID NO 83,

the bispecific antibody showed

a) Inhibition of ILT2 and/or ILT4 binding to HLA-G (see example 10); and/or

In another specific embodiment, the bispecific antibody comprises: a polypeptide comprising the amino acid sequence SEQ ID NO. 80; a polypeptide comprising the amino acid sequence SEQ ID NO. 81; a polypeptide comprising the amino acid sequence SEQ ID NO. 82; and a polypeptide comprising the amino acid sequence SEQ ID NO 83.

Another specific embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, wherein the antibody comprises: a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID No. 84; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID No. 85; a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID No. 86; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID No. 87 (wherein in the VH or VL framework region or in the constant region the amino acid is substituted without affecting the specific binding properties of such bispecific antibodies and the properties of the constant region).

One specific embodiment of the invention is a bispecific antibody that binds to human HLA-G and to human CD3, wherein the antibody comprises: a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to sequence SEQ ID No. 84; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence of SEQ ID No. 85; a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID No. 86; a polypeptide comprising an amino acid sequence which is at least 98% or 99% identical to the sequence SEQ ID NO. 87,

the bispecific antibody showed

a) Inhibition of ILT2 and/or ILT4 binding to HLA-G (see example 10); and/or

In another specific embodiment, the bispecific antibody comprises: a polypeptide comprising the amino acid sequence SEQ ID NO. 84; a polypeptide comprising the amino acid sequence SEQ ID NO. 85; a polypeptide comprising the amino acid sequence SEQ ID NO 86; and a polypeptide comprising the amino acid sequence SEQ ID NO. 87.

Fc domain

In a specific embodiment, a bispecific antibody of the invention comprises an Fc domain consisting of a first subunit and a second subunit. It is to be understood that the features of the Fc domains described herein in relation to bispecific antibodies may equally be applied to the Fc domains comprised in the monospecific anti-HLAG antibodies of the invention, in addition to those modifications related to Fc heterodimerization.

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

In one embodiment, the Fc domain of the bispecific antibody is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG ₁ An Fc domain. In another embodiment, the Fc domain is an IgG ₄ An Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ An Fc domain comprising an amino acid substitution at position S228 (numbered according to the Kabat EU index), in particular amino acid substitution S228P. The amino acid substitution reduces in vivo IgG ₄ Fab arm exchange of antibodies (see Stubenrauch et al Drug Metabolism and Disposition, 84-91 (2010)). In another specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG ₁ An Fc domain.

Fc domain modification to promote heterodimerization

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

Thus, in a particular embodiment, the Fc domain of a bispecific antibody as the invention comprises a modification that facilitates the association of the first subunit and the second subunit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the Fc domain of human IgG is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

There are a number of methods of modifying the CH3 domain of an Fc domain in order to enhance heterodimerization, which are well described in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or heavy chain comprising the CH3 domain) is no longer able to homodimerize with itself, but is forced to heterodimerize with other CH3 domains that are complementarily engineered (such that the first CH3 domain and the second CH3 domain heterodimer and no homodimer is formed between the two first CH3 domains or the two second CH3 domains). These different approaches for improving heavy chain heterodimerization are seen as different options for binding to heavy chain-light chain modifications (e.g., VH and VL exchanges/substitutions in one binding arm, and the introduction of substituents with oppositely charged amino acids in the CH1/CL interface) in bispecific antibodies, which reduce heavy chain/light chain mismatches and Bence Jones type byproducts.

In a specific embodiment, the modification that facilitates association of the first subunit and the second subunit of the Fc domain is a so-called "knob" modification that comprises a "knob" modification in one of the two subunits of the Fc domain and a "knob" modification in the other of the two subunits of the Fc domain.

The "pestle and mortar" technique is described, for example: US 5,731,168; US 7,695,936; ridgway et al, prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method comprises introducing a protrusion ("slug") at the interface of the first polypeptide and a corresponding cavity ("socket") in the interface of the second polypeptide, such that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusions are constructed by replacing smaller amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., amino acids or tryptophan). By replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a complementary cavity of the same or similar size as the protuberance is formed in the interface of the second polypeptide.

Thus, in one particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antibody, the amino acid residues are substituted with amino acid residues having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

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

Preferably, the amino acid residue with smaller side chain volume is selected from the group consisting of: alanine (a), serine (S), threonine (T) and valine (V).

Protrusions and cavities can be made by altering the nucleic acid encoding the polypeptide (e.g., by mutation to a specific site or by peptide synthesis).

In a specific embodiment, the threonine residue at position 366 is substituted with a tryptophan residue in the first subunit of the Fc domain ("the CH3 domain of the" subunit ") (T366W), and the tyrosine residue at position 407 is substituted with a valine residue in the second subunit of the Fc domain (" the "CH 3 domain of the" subunit ") (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is in turn substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the Kabat EU index).

In yet another embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is in turn substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain, the serine residue at position 349 is in turn substituted with a cysteine residue (Y349C) (numbered according to the Kabat EU index). The introduction of these two cysteine residues results in the formation of disulfide bonds between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248,7-15 (2001)).

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

In a particular embodiment, the antigen binding moiety that binds to a second antigen (e.g., an activated T cell antigen) is fused (optionally via a first antigen binding moiety and/or peptide linker that binds to HLA-G) to a first subunit of the Fc domain (comprising a "knob" modification). Without wishing to be bound by theory, fusion of the pestle-containing subunit of the Fc domain with the antigen-binding portion of a second antigen, such as an activated T cell antigen, will (further) minimize the production of antibodies comprising two antigen-binding portions that bind to the activated T cell antigen (spatial collision of two pestle-containing polypeptides).

Other techniques for CH3 modification for forced heterodimerization can be envisaged as alternatives to the present invention and are described in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment, the heterodimerization process described in EP 1870459 is alternatively used. The method is based on the introduction of oppositely charged amino acids at specific amino acid positions at the CH3/CH3 domain interface between two subunits of the Fc structure. A preferred embodiment of the bispecific antibody of the present invention is the amino acid mutations R409D and K370E in one of the two CH3 domains (of the Fc domain); and amino acid mutations D399K and E357K in the other of the CH3 domains of the Fc domain (numbered according to Kabat EU index).

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

In another embodiment, the bispecific antibody of the present invention comprises amino acid mutation S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutation Y349C, T366S, L368A, Y V in the CH3 domain of the second subunit of the Fc domain, or the bispecific antibody comprises amino acid mutation Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutation S354C, T366S, L368A, Y V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutation R409D, K E in the CH3 domain of the first subunit of the Fc domain and amino acid mutation D399K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to Kabat EU index).

In one embodiment, the heterodimerization process described in WO 2013/157953 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbered according to the Kabat EU index). In another embodiment, the first CH3 domain further comprises the amino acid mutation L351K. In another embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numbering according to the Kabat EU index).

In one embodiment, the heterodimerization process described in WO 2012/058768 is alternatively used. In one embodiment, the first CH3 domain comprises amino acid mutation L351Y, Y407A and the second CH3 domain comprises amino acid mutation T366A, K409F. In another embodiment, the second CH3 domain further comprises an amino acid mutation at position T411, D399, S400, F405, N390, or K392 selected from, for example: a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W; b) D399R, D399W, D399Y or D399K; c) S400E, S400D, S R or S400K; d) F405I, F405M, F405T, F405S, F V or F405W; e) N390R, N390K or N390D; f) K392V, K392M, K392R, K392L, K F or K392E (numbering according to the EU index of Kabat). In another embodiment, the first CH3 domain comprises amino acid mutation L351Y, Y407A and the second CH3 domain comprises amino acid mutation T366V, K409F. In another embodiment, the first CH3 domain comprises amino acid mutation Y407A and the second CH3 domain comprises amino acid mutation T366A, K409F. In another embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

In one embodiment, the heterodimerization process described in WO 2011/143545 is alternatively used, for example, with amino acid modifications at positions selected from 368 and 409 (numbered according to the Kabat EU index).

In one embodiment, the heterodimerization process described in WO 2011/090762 is alternatively used, which also uses the "mortar and pestle" technique described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbered according to the Kabat EU index).

In one embodiment, the bispecific antibody or Fc domain thereof belongs to the class of IgG ₂ Subclasses, and alternatively using the heterodimerization process described in WO 2010/129304.

In an alternative embodiment, the modification that facilitates association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering, e.g., as described in PCT publication WO 2009/089004. Typically, this approach involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues, thereby rendering homodimer formation electrostatically unfavorable, but heterodimerization electrostatically favorable. In one such embodiment, the first CH3 domain comprises amino acid substitutions of negatively charged amino acids (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D) for K392 and N392, and the second CH3 domain comprises amino acid substitutions of positively charged amino acids (e.g., lysine (K) or arginine (R), preferably D399K, E K, D K or E357K, and more preferably D399K and E356K) for D399, E356, D356 or E357). In another embodiment, the first CH3 domain further comprises an amino acid substitution of a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), more preferably K409D or R409D) for K409 or R409. In another embodiment, the first CH3 domain further or alternatively comprises amino acid substitutions of K439 and/or K370 (all numbered according to the Kabat EU index) by negatively charged amino acids, such as glutamic acid (E) or aspartic acid (D).

In yet another embodiment, the heterodimerization process described in WO 2007/147901 may alternatively be used. In one embodiment, the first CH3 domain comprises amino acid mutations K253E, D K282K and K322D, and the second CH3 domain comprises amino acid mutations D239K, E K and K292D (numbered according to the Kabat EU index).

In another embodiment, the heterodimerization process described in WO 2007/110205 is alternatively used.

In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbered according to the Kabat EU index).

Fc domain modification to reduce Fc receptor binding and/or effector function

The Fc domain confers the beneficial pharmacokinetic properties of a bispecific antibody (or antibody), comprising a longer serum half-life, which helps to obtain good accumulation and a beneficial tissue-blood partition ratio in the target tissue. At the same time, however, it may lead to undesired targeting of bispecific antibodies (or antibodies) to cells expressing Fc receptors, rather than preferred antigen bearing cells. Furthermore, co-activation of the Fc receptor signaling pathway can result in cytokine secretion/release, which in combination with T cell activation characteristics (e.g., in embodiments of bispecific antibodies in which the second antigen binding moiety binds to an activated T cell antigen) and long half-life of the bispecific antibody, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of immune cells other than T cells (bearing Fc receptors) may even reduce the efficacy of bispecific antibodies (in particular, bispecific antibodies in which the second antigen binding moiety binds to an activated T cell antigen) due to potential destruction of T cells (e.g. destruction by NK cells).

Thus, in certain embodiments, with native IgG ₁ Fc domains as bispecific antibodies of the invention exhibit reduced binding affinity for Fc receptors and/or reduced effector function compared to Fc domains. In one such embodiment, the Fc domain (or bispecific antibody comprising the Fc domain) is conjugated to a native IgG ₁ Fc domain (or comprising native IgG ₁ Bispecific antibodies of Fc domains) exhibit less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% binding affinity to Fc receptors, and/or to natural IgG ₁ Fc domain (or comprising native IgG ₁ Bispecific antibodies of Fc domains) exhibit less than 50%Preferably less than 20%, more preferably less than 10% and most preferably less than 5% of effector function. In one embodiment, the Fc domain (or bispecific antibody comprising the Fc domain) does not substantially bind to an Fc receptor and/or induces effector function. In a particular embodiment, the Fc receptor is an fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human fcγ receptor, more specifically human fcγriiia, fcγri or fcγriia, most specifically fcγriiia. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, with native IgG ₁ Fc domains exhibit substantially similar binding affinities for neonatal Fc receptors (FcRn) compared to Fc domains. When the Fc domain (or bispecific antibody comprising the Fc domain) exhibits greater than about 70%, specifically greater than about 80%, more specifically greater than about 90% of a native IgG ₁ Fc domain (or comprising native IgG ₁ Bispecific antibodies to Fc domains) to FcRn, substantially similar binding to FcRn is achieved.

In certain embodiments, the engineered Fc domain has reduced binding affinity and/or reduced effector function for Fc receptors as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of a bispecific antibody comprises one or more amino acid mutations that reduce the binding affinity and/or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of an amino acid to an Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least a factor of 10, at least a factor of 20, or even at least a factor of 50. In one embodiment, the bispecific antibody comprising a mutated Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% binding affinity to an Fc receptor as compared to a bispecific antibody comprising an un-engineered Fc domain. In a particular embodiment, the Fc receptor is an fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human fcγ receptor, more specifically human fcγriiia, fcγri or fcγriia, most specifically human fcγriiia. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components, in particular to C1q, is also reduced. In one embodiment, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved when the Fc domain (or bispecific antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the Fc domain (or bispecific antibody comprising the non-engineered form of the Fc domain) for FcRn, i.e., the binding affinity of the Fc domain for the receptor is maintained. The Fc domain or bispecific antibodies of the invention comprising the Fc domain may exhibit such affinity of greater than about 80% and even greater than about 90%. In certain embodiments, the Fc domain of the bispecific antibody is engineered to have reduced effector function as compared to a non-engineered Fc domain. Reduced effector functions may include, but are not limited to, one or more of the following: reducing Complement Dependent Cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), reducing Antibody Dependent Cellular Phagocytosis (ADCP), reducing cytokine secretion, reducing immune complex mediated antigen uptake by antigen presenting cells, reducing binding to NK cells, reducing binding to macrophages, reducing binding to monocytes, reducing binding to polymorphonuclear cells, reducing direct signaling-induced apoptosis, reducing cross-linking of target-bound antibodies, reducing dendritic cell maturation or reducing T cell priming. In one embodiment, the reduced effector function is selected from one or more of the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the non-engineered Fc domain (or bispecific antibody comprising a non-engineered Fc domain) induced ADCC.

In one embodiment, the amino acid that reduces the binding affinity and/or effector function of the Fc domain to the Fc receptor is mutated to an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from E233, L234, L235, N297, P331 and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group consisting of L234, L235, and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions of L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc domain is IgG ₁ Fc domain, in particular human IgG ₁ An Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitutions are P329A or P329G, in particular P329G (numbering according to the EU index of Kabat). In one embodiment, the Fc domain comprises an amino acid substitution at position P329, and another amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbered according to the Kabat EU index). In more specific embodiments, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "lalag"). Specifically, in particular embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L a and P329G (numbered according to the Kabat Eu index), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 The acid residue was substituted with an alanine residue (L235A) and the proline residue at position 329 was substituted with a glycine residue (P329G) (numbering according to the Kabat EU index).

In one such embodiment, the Fc domain is IgG ₁ Fc domain, in particular human IgG ₁ An Fc domain. The combination of amino acid-substituted "P329G LALA" almost completely eliminates human IgG ₁ Fcγ receptor (and complement) binding of the Fc domain is as described in PCT publication No. WO 2012/130831, incorporated herein by reference in its entirety. WO 2012/130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties (e.g., fc receptor binding or effector function).

IgG ₄ Antibodies and IgG ₁ Antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function as compared to antibodies. Thus, in some embodiments, the Fc domain of a bispecific antibody of the invention is IgG ₄ Fc domain, in particular human IgG ₄ An Fc domain. In one embodiment, the IgG ₄ The Fc domain comprises an amino acid substitution at position S228, in particular amino acid substitution S228P (numbering according to the EU index of Kabat). To further reduce its binding affinity to Fc receptors and/or its effector function, in one embodiment, the IgG ₄ The Fc domain comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E (numbering according to the EU index of Kabat). In another embodiment, the IgG ₄ The Fc domain comprises an amino acid substitution at position P329, in particular comprising amino acid substitution P329G (numbering according to the Kabat EU index). In a particular embodiment, the IgG ₄ The Fc domain comprises amino acid substitutions at positions S228, L235 and P329, in particular the amino acid substitutions S228P, L E and P329G (numbering according to the Kabat EU index). Such IgG ₄ Fc domain mutants and their fcγ receptor binding properties are described in PCT publication No. WO 2012/130831, which is incorporated herein by reference in its entirety.

In one particular embodiment, with native IgG ₁ Fc domains that exhibit reduced binding affinity for Fc receptors and/or reduced effector function as compared to Fc domains are packagesHuman IgG comprising amino acid substitution L234A, L235A and optionally P329G ₁ Fc domain or human IgG comprising the amino acid substitution S228P, L235E and optionally P329G (numbering according to the Kabat EU index) ₄ An Fc domain.

Mutant Fc domains may be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutations, PCR, gene synthesis, etc., of the coding DNA sequence. The correct nucleotide changes can be verified by, for example, sequencing.

Binding to the Fc receptor can be readily determined by ELISA, or by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instrument (GE Healthcare), and the Fc receptor can be obtained by, for example, recombinant expression. Alternatively, the binding affinity of an Fc domain or bispecific antibody comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor (e.g., human NK cells expressing fcγiiia receptor).

The effector function of an Fc domain or a bispecific antibody comprising an Fc domain can be determined by methods well known in the art. Examples of in vitro assays for assessing ADCC activity of a target molecule are described, for example: U.S. Pat. nos. 5,500,362; hellstrom et al Proc Natl Acad Sci USA, 83, 7059-7063 (1986); and Hellstrom et al Proc Natl Acad Sci USA, 1499-1502 (1985); U.S. Pat. nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive analysis methods (see, e.g., ACTI for flow cytometry ^TM Non-radioactive cytotoxicity assay (CellTechnology, inc.Mountain View, CA); cytoTox Non-radioactive cytotoxicity assay (Promega, madison, wis.). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or additionally, animal models may be disclosed in, for example, clynes et al, proc Natl Acad Sci USA, 652-656 (1998)In vivo, and the ADCC activity of the target molecule.

In some embodiments, the binding of the Fc domain to the complement component, in particular to C1q, is reduced. Thus, in some embodiments, wherein the Fc domain is engineered to have a reduced effector function comprising reduced CDC. A C1q binding assay may be performed to determine whether an Fc domain or bispecific antibody comprising an Fc domain is capable of binding C1q and thus has CDC activity. See, e.g., C1q and C3C binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays may be performed (see, e.g., gazzano-Santoro et al, J Immunol Methods, 163 (1996), cragg et al, blood 101, 1045-1052 (2003), and Cragg and Glennie, blood 103, 2738-2743 (2004)).

FcRn binding and in vivo clearance/half-life assays can also be performed using methods well known in the art (see, e.g., petkova, S.B. et al, int' l.Immunol.18 (12): 1759-1769 (2006); WO 2013/120929).

In yet another aspect, an anti-HLA-G antibody according to any of the above embodiments can bind, alone or in combination, any of the features set forth in sections 1-6 below:

1. affinity for antibodies

In certain embodiments, antibodies provided herein have a concentration of 1. Mu.M, 100nM, 10nM, 1nM, 0.1nM, 0.01nM or 0.001nM (e.g., 10) ^-8 M or less, e.g. 10 ^-8 M to 10 ^-13 M, e.g. 10 ^-9 M to 10 ^-13 M) dissociation constant KD.

In a preferred embodiment, surface Plasmon Resonance (SPR) is used (usingKD was measured at 25 ℃ with immobilized antigen CM5 chips at about 10 Reaction Units (RU). Briefly, carboxymethylated polyglucose biosensor chips (CM 5, BIACORE, inc.) were activated with N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of the supplier. With 10mM vinegarSodium acid (pH 4.8) dilutes the antigen to 5. Mu.g/ml (about 0.2. Mu.M) and then injects at a flow rate of 5. Mu.l/min to obtain about 10 Reaction Units (RU) of coupled protein. After antigen injection, 1M ethanolamines were injected to block unreacted groups. In kinetic measurements, double serial dilutions of Fab (0.78 nM to 500 nM) were injected at 25℃with a flow rate of about 25. Mu.l/min into a solution containing 0.05% polysorbate 20 (TWEEN-20) ^TM ) Surfactant (PBST) in PBS. Simple one-to-one Langmuir binding model (one-to-one Langmuir binding model) was used (Met)>Evaluation software version 3.2) the association rate (kon or ka) and dissociation rate (koff or kd) were calculated by fitting the association and dissociation sensor maps simultaneously. The equilibrium dissociation constant KD is calculated as the ratio KD/ka (koff/kon). See, e.g., chen et al, j.mol.biol.293 (1999) 865-881. If the association rate, as determined by the above surface plasmon resonance analysis, exceeds 106M-1s-1, then the association rate can be measured using fluorescence quenching techniques such as in a spectrometer (such as a flow stop equipped spectro-luminometer (Aviv Instruments) with stirred cuvette) or 8000-series SLM-AMINCO ^TM Fluorescence emission intensity (excitation = 295 nM) of 20nM anti-antigen antibody (Fab form) in PBS (pH 7.2) was measured at 25 ℃ in the presence of increased concentration of antigen measured in a spectro-luminometer (thermo spectronic); emission = 340nm,16nm bandpass), or decrease.

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, fab '-SH, F (ab') ₂ Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson, P.J. et al, nat.Med.9 (2003) 129-134. For reviews of scFv fragments, see, for example: plueckthun, A., incorporated: the Pharmacology of Monoclonal Antibodies, volume 113, rosenburg and Moore (Main plaited), springer-Verlag, new York (1994), pages 269-315; see also WO 93/16185; and U.S. Pat. nos. 5,571,894 and 5,587,458. With respect to the inclusion of salvage receptor binding antigens Fab and F (ab') which determine the position of residues and have increased half-life in vivo ₂ See U.S. Pat. No. 5,869,046 for discussion of fragments.

A bifunctional antibody is an antibody fragment having two antigen binding sites (which may be bivalent or bispecific). See, for example: EP 0 404 097; WO 1993/01161; hudson, P.J. et al, nat.Med.9 (2003) 129-134; and Holliger, P.et al, proc.Natl. Acad.Sci.USA 90 (1993) 6444-6448. Trifunctional and tetrafunctional antibodies are also described in Hudson, P.J. et al, nat.Med.9 (20039 129-134).

A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (domatis, inc., waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B1).

Antibody fragments can be made by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as described herein, and production of recombinant host cells (e.g., E.coli or phage).

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison, S.L. et al, proc.Natl. Acad.Sci.USA 81 (1984) 6851-6855). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In yet another example, the chimeric antibody is a "class switch" antibody in which the class or subclass has been altered compared to its parent antibody. Chimeric antibodies comprise antigen-binding fragments thereof.

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

Humanized antibodies and methods of making the same are reviewed in, for example, almagro, j.c. and Fransson, j., front. Biosci.13 (2008) 1619-1633, and further described, for example, in the following: riechmann, I.et al, nature 332 (1988) 323-329; queen, C. Et al, proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; kashmiri, S.V. et al Methods 36 (2005) 25-34 (describing SDR (a-CDR) transplantation); padlan, E.A., mol.Immunol.28 (1991) 489-498 (description "resurfacing"); dall' acquata, W.F. et al Methods 36 (2005) 43-60 (description "FR shuffling"); and Osbourn, j. Et al, methods 36 (2005) 61-68 and Klimka, a. Et al, br.j. Cancer 83 (2000) 252-260 (describing the "guided selection" method of FR shuffling).

Human framework regions useful for humanization include, but are not limited to: the framework regions selected using the "best match" approach (see, e.g., sims, M.J. et al, J.Immunol.151 (1993) 2296-2308; framework regions derived from consensus sequences of human antibodies of specific subsets of the light or heavy chain variable regions (see, e.g., carter, P. Et al, proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L.G. et al, J.Immunol.151 (1993) 2623-2632), human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., almagro, J.C. And Fransson, J., front. Biosci.13 (2008) 1619-1633), and framework regions derived from screening FR libraries (see, e.g., baca, M. Et al, J.biol. Chem.272 (1997)) 78-84; and Roso.M.618-19969 22611.

4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques well known in the art. Human antibodies are generally described in the following documents: van Dijk, m.a. and van de Winkel, J.G., curr.Opin.Pharmacol.5 (2001) 368-374; and Lonberg, n., curr.opin.immunol.20 (2008) 450-459.

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having human variable regions in response to antigen challenge. Such animals typically comprise all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, either extrachromosomal or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, n., nat. Biotech.23 (2005) 1117-1125. See also, for example: U.S. Pat. Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE) ^TM Technology; U.S. Pat. No. 5,770,429 (describingTechnology; U.S. Pat. No. 7,041,870 (describing K-M->Technology; U.S. patent application publication No. US2007/0061900 (describing +.>Technology). Human variable regions from whole antibodies produced by such animals may be further modified, for example by binding to different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., kozbor, D., J.Immunol.133 (1984) 3001-3005; brodeur, B.R. et al, monoclonal Antibody Production Techniques and Applications, marcel Dekker, inc., new York (1987), pp.51-63; and Boerner, P.et al, J.Immunol.147 (1991) 86-95) human antibodies generated via human B cell hybridoma techniques are also described in Li et al, proc.Natl. Acad. Sci.USA 103 (2006) 3557-3562. Other methods include those described in, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from a hybridoma cell line) and Ni, J., xiandai Mianyixue (2006) 265-268 (describing human-human hybridomas). Human fusion tumour technology (triple source fusion tumour technology) is also described in Vollmers, h.p. and Brandlein, s. Histology and Histopathology 20 (2005) 927-937 and Vollmers, h.p. and Brandlein, s. Methods and Findings in Experimental and Clinical Pharmacology27 (2005) 185-191.

Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a humanized phage display library. Such variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from a library of antibodies are described below.

5. Antibodies derived from libraries

Antibodies for use in the present invention may be isolated by screening a combinatorial library for antibodies having the desired activity or activities. For example, a variety of methods are well known in the art for generating phage display libraries and screening the libraries for antibodies having the desired binding properties. Such methods are reviewed in, for example, hoogenboom, H.R. et al, methods in Molecular Biology 178 (2001) 1-37, and further described in, for example, the following documents: mcCafferty, j. Et al, nature 348 (1990) 552-554; clackson, t. Et al, nature 352 (1991) 624-628; marks, J.D. et al, J.mol.biol.222 (1992) 581-597; marks, j.d. and braddury, a., methods in Molecular Biology 248 (2003) 161-175; sidhu, S.S. et al, J.mol.biol.338 (2004) 299-310; lee, C.V. et al, J.mol.biol.340 (2004) 1073-1093; fellouse, F.A., proc.Natl.Acad.Sci.USA 101 (2004) 12467-12472; and Lee, C.V. et al, J.Immunol. Methods 284 (2004) 119-132.

In some phage display methods, VH and VL gene profiles are cloned separately by Polymerase Chain Reaction (PCR) and randomly recombined in phage libraries, and antigen-binding phages can then be screened as described in the following documents: winter, G. Et al, ann. Rev. Immunol.12 (1994) 433-455. Phages typically display antibody fragments as single chain Fv (scFv) fragments or Fab fragments. Libraries from an immunogen can provide high affinity antibodies to the immunogen without the need to construct a hybridoma. Alternatively, the natural lineage (e.g., from a human) can be cloned without any immunization to provide a single source of antibodies to various non-self as well as self-antigens, as described by Griffiths, a.d. et al in EMBO j.12 (1993) 725-734. Finally, natural libraries can also be synthesized by cloning non-rearranged V gene fragments in stem cells and encoding high variability CDR3 regions using PCR primers comprising random sequences and finishing the rearrangement in vitro, as described in Hoogenboom, h.r. and Winter, g., j.mol.biol.227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example: us patent No. 5,750,373, us patent publication nos. 2005/007974, 2005/019455, 2005/0266000, 2007/017126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

An antibody or antibody fragment isolated from a human antibody repertoire is herein considered a human antibody or human antibody fragment.

6. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics, such as antigen binding characteristics.

a) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Substitution mutagenesis target sites contain CDRs and FR (in a preferred embodiment, framework residues are independent of the binding properties of the antibody (see, e.g., foote j. And Winter g., j. Mol. Biol. (1992) 224, 487-499). Exemplary alterations are provided under the "exemplary substitution" heading in table 1 and will be described further below with reference to the amino acid side chain classes.

TABLE 1

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity: n-leucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;

(3) Acid: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions require the exchange of members of one of these classes for members of another class.

One type of substitution variant involves substituting one or more CDRs of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further investigation will have modifications (e.g., improvements) in, and/or substantially retain, certain biological properties of the parent antibody (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody. Exemplary substitution variants are affinity matured antibodies that can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibody is displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in the CDRs to improve antibody affinity. Such changes may be made in CDR "hot spots" (i.e., residues encoded by codons that undergo high frequency mutations during somatic maturation) (see, e.g., chordhury, p.s., methods mol. Biol.207 (2008) 179-196) and/or SDR (a-CDRs), wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, in Hoogenboom, H.R. et al Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into variant genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A second library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity is the CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified by, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targets.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such modifications do not significantly reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the CDRs that do not substantially reduce binding affinity. Such changes may be outside of CDR "hot spots" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or comprises no more than one, two, or three amino acid substitutions.

One useful method for identifying antibody residues or regions that are likely to be mutagenized is known as "alanine scanning mutagenesis" as described by Cunningham, b.c. and Wells, j.a., science 244 (1989) 1081-1085. In this method, a recognition residue or group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. More substitutions can be introduced at amino acid positions, indicating good functional sensitivity to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be used to identify the point of contact between the antibody and the antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired property.

Amino acid sequence insertions include the length of amino and/or carboxy terminal fusions, ranging from one residue to polypeptides comprising one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include enzymes fused to the N-or C-terminus of the antibody (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antibody.

b) Variant Fc region

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

Antibodies with reduced effector function include antibodies in which one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 have been substituted (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described. ( See, for example: U.S. Pat. nos. 6,737,056; WO 2004/056312; and Shields, R.L. et al, J.biol.chem.276 (2001) 6591-6604 )

In one embodiment of the invention, such antibodies are IgG1 comprising mutations L234A and L235A or comprising mutations L234A, L235A and P329G. In another embodiment or in IgG4 comprising the mutations S228P and L235E or S228P, L E or/and P329G (numbering according to the Kabat EU index of Kabat et al: kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD, 1991).

Antibodies with longer half-lives and improved binding to neonatal Fc receptors (FcRn), which are responsible for transfer of maternal IgG to the fetus, see Guyer, R.L. et al, J.Immunol.117 (1976) 587-593 and Kim, J.K. et al, J.Immunol.24 (1994) 2429-2434, are described in US 2005/0014934. Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants having substitutions at one or more 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, substitution of Fc region residue 434 (U.S. patent No. 7,371,826).

See also Duncan, A.R. and Winter, G., nature 322 (1988) 738-740; U.S. Pat. No. 5,648,260; US 5,624,821; and WO 94/29351 relates to other examples of variants of the Fc region.

c) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are substituted with cysteine residues. In certain embodiments, the substitution residue occurs at an accessible site of the antibody. By substituting those residues with cysteines, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to bind the antibody to other moieties (e.g., drug moieties or linker-drug moieties) to form immunoconjugates as described further herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: v205 of light chain (Kabat numbering); a118 (EU numbering) of heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be produced, for example, as described in U.S. patent No. 7,521,541.

d) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers), dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be polybranched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular nature or function of the antibody to be improved, whether the antibody derivative will be used in a treatment under specified conditions, and the like.

In another embodiment, a conjugate of an antibody and a non-protein moiety that is selectively heatable by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam, N.W.et al., proc.Natl. Acad.Sci.USA 102 (2005) 11600-11605). The radiation may have any wavelength and includes, but is not limited to, wavelengths that do not damage general cells but heat the non-protein fraction to a temperature that kills cells in the vicinity of the antibody-non-protein fraction.

B. Recombinant methods and compositions

Recombinant methods and compositions can be used to make antibodies, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-HLA-G antibody as described herein is provided. Such nucleic acids encode amino acid sequences comprising a VL and/or amino acid sequences comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In this embodiment, the host cell comprises (e.g., has been transformed): (1) A vector comprising a nucleic acid encodes an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising a nucleic acid encodes an amino acid sequence comprising a VL of an antibody and a second vector comprising a nucleic acid encodes an amino acid sequence comprising a VH of an antibody. In a preferred embodiment, the host cell is a eukaryotic cell, e.g., a Chinese Hamster Ovary (CHO) cell, HEK293 cell, or lymphoid cell (e.g., Y0, NS0, sp20 cell). In one embodiment, a method of making an anti-HLA-G antibody or bispecific antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

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

The host cells most suitable for cloning or expressing the antibody-encoding vectors are eukaryotic cells, preferably mammalian cells, as described herein.

Vertebrate cells can be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be used. Other examples of useful mammalian host cell strains include: monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al, J. Gen Virol.36 (1977) 59-74); young murine kidney cells (BHK); mouse testis support cells (e.g., TM4 cells as described in Mather, J.P., biol.Reprod.23 (1980) 243-252); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., as described by Mather, J.P. et al, annals N.Y. Acad. Sci.383 (1982) 44-68); MRC 5 cells; and FS4 cells. The most suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells including DHFR ^- CHO cells (Urlaub, g. Et al, proc.Natl. Acad.sci.usa77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0, and Sp2/0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example: yazaki, p. And Wu, a.m., methods in Molecular Biology, volume 248, lo, b.k.c., master, humana Press, totowa, NJ (2004), pages 255-268.

Prokaryotic cells can also be used to some extent, but have the disadvantage that sometimes greater effort is required and more complex procedures are employed. For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. No. 5,648,237, U.S. Pat. No. 5, 5,789,199, and U.S. Pat. No. 5,840,523. (see also Charlton, K.A., in: methods in Molecular Biology, volume 248, lo, B.K.C. (main code), humana Press, totowa, NJ (2003), pages 245-254, which states the expression of antibody fragments in E.coli.) after expression, the antibodies can be separated from the soluble fraction in the bacterial cell paste and can be further purified.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing the production of antibodies in transgenic plants) ^TM Technology).

C. Measurement

The physical/chemical properties and/or biological activity of the anti-HLA-G antibodies provided herein can be identified, screened, or characterized using a variety of assays well known in the art.

1. Binding assays and other assays

In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA, western blot methods, and the like. Morris, G.E. presents detailed exemplary methods for mapping to antibody-binding epitopes. (Main editors), epitope Mapping Protocols, incorporated by reference in Methods in Molecular Biology, volume 66, humana Press, totowa, N.J. (1996).

2. Activity determination

In one aspect, an assay is provided for identifying the biological activity of an anti-HLA-G antibody. Biological activity may include, for example, the ability to enhance activation and/or proliferation of different immune cells (including T cells). For example, they enhance secretion of immune-modulating cytokines such as interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF alpha). Other immune-modulating cytokines that are or may be enhanced are, for example, IL1 beta, IL6, IL12, granzyme B, etc., that bind to different types of T cells. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, antibodies of the invention are tested for such biological activity as described in the examples below.

D. Methods and compositions for diagnosis and detection

In certain embodiments, any of the HLA-G antibodies provided herein can be used to detect the presence or absence of HLA-G in a biological sample. The term "detection" as used herein encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissue, such as immune cells or T cell infiltrates and/or tumor cells.

In one embodiment, an anti-HLA-G antibody is provided for use in a diagnostic or detection method. In another aspect, a method of detecting the presence or absence of HLA-G in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-HLA-G antibody as described herein under conditions that allow binding of the anti-HLA-G antibody to HLA-G, and detecting whether a complex is formed between the anti-HLA-G antibody and HLA-G. Such methods may be in vitro or in vivo. In one embodiment, the anti-HLA-G antibody is used to select a subject suitable for treatment with the anti-HLA-G antibody, e.g., wherein HLA-G is a biomarker for selecting a patient.

In certain embodiments, a labeled anti-HLA-G antibody is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent, chromogenic, electron dense, chemiluminescent, and radioactive labels), as well as moieties that are indirectly detected (e.g., by enzymatic reactions or molecular interactions), such as enzymes or ligands. Exemplary labels include, but are not limited to: radioisotope ³² P、 ¹⁴ C、 ¹²⁵ I、 ³ H and ¹³¹ i, a step of I; fluorophores such as rare earth chelates or luciferins and derivatives thereof; rose bengal and its derivatives; dansyl; umbelliferone; luciferases, such as firefly luciferases and bacterial luciferases (U.S. Pat. No. 4,737,456); fluorescein; 2, 3-dihydro-phthalide; horseradish peroxidase (HRP); alkaline phosphatase; beta-galactosidase; a glucoamylase; lysozyme; carbohydrate oxidase such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, are used in combination with enzymes that oxidize dye precursors (e.g., HRP, lactoperoxidase, or microperoxygenases) with hydrogen peroxide; biotin/avidin; rotating the mark; labeling phage; stable free radicals, and the like.

E. Pharmaceutical preparation

Pharmaceutical formulations of anti-HLA-G antibodies or anti-HLA-G/anti-CD 3 bispecific antibodies as described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16 th edition, osol, a. (ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, including, but not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents (e.g., EDTA); sugar, examples Such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20 #Baxter International, inc.). Certain exemplary shasegps and uses (including rhuPH 20) are described in U.S. patent publication nos. 2005/026086 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulations comprising histidine-acetate buffer.

The formulations described herein may also contain more than one active ingredient suitable for the particular indication being treated, preferably those having complementary activity that do not adversely affect each other. For example, further provision may be required. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethyl cellulose microcapsules or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, osol, a. (main code) (1980).

Can be prepared into sustained release preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by sterile filtration through a sterile filter membrane.

F. Therapeutic methods and compositions

Any of the anti-HLA-G antibodies or anti-HLA-G/anti-CD 3 bispecific antibodies provided herein can be used in a method of treatment.

In one aspect, an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody is provided for use as a medicament. In a further aspect, an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody is provided for use in the treatment of cancer. In certain embodiments, anti-HLA-G antibodies or anti-HLA-G/anti-CD 3 bispecific antibodies are provided for use in a method of treatment. In certain embodiments, the invention provides an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the anti-HLA-G/anti-CD 3 bispecific antibody.

In further embodiments, the invention provides anti-HLA-G antibodies or anti-HLA-G/anti-CD 3 bispecific antibodies for use as immunomodulators or for direct or indirect induction of proliferation and/or activation of immune cells such as T cells, B cells and bone marrow cells, including monocytes, macrophages, dendritic cells, plasmacytoid dendritic cells, which function, for example, by secretion of immunostimulatory cytokines such as TNFa (TNFa) and ifγ (IFNg) or further recruitment of immune cells. In certain embodiments, the invention provides an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody for use as an immunomodulator or in a method for directly or indirectly inducing proliferation, activation of immune cells, which acts, for example, by secretion of immunostimulatory cytokines such as TNFa and ifnγ or further recruitment of immune cells in an individual, comprising administering to the individual an effective amount of an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody for use as an immunomodulator or for directly or indirectly inducing proliferation, activation of immune cells, which acts, for example, by secretion of immunostimulatory cytokines such as TNFa and ifnγ or further recruitment of immune cells.

In further embodiments, the invention provides anti-HLA-G antibodies or anti-HLA-G/anti-CD 3 bispecific antibodies for use as immunostimulants or for stimulating secretion of tumor necrosis factor alpha (TNF alpha). In certain embodiments, the invention provides an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody for use in a method of immunomodulation to directly or indirectly induce proliferation, activation, e.g., by secretion of immunostimulatory cytokines such as TNFa and IFNg or further recruitment of immune cells in an individual, the method comprising administering to the individual an effective amount of an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody for immunomodulation to directly or indirectly induce proliferation, activation, e.g., by secretion of immunostimulatory cytokines such as TNFa and IFNg or further recruitment of immune cells.

The term "cancer" as used herein may be, for example, lung cancer, non-small cell lung cancer (NSCL), bronchioloalveolar lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer (stomach cancer), colorectal cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, prostate cancer, bladder cancer, renal cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, central Nervous System (CNS) tumors, spinal axis tumors, brain stem glioma, astrocytoma, neuroma, ependymoma, medulloblastoma, adenoma, meningioma, cell carcinoma, adenoma, lymphomas, squamous cell carcinoma, lymphomas including any one or a combination of the above.

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

In yet another aspect, the invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody. An "individual" according to any of the embodiments described above may be a human.

In a further aspect, the invention provides a method of inducing cell-mediated lysis of cancer cells in an individual having cancer. In one embodiment, the method comprises administering to the individual an effective amount of an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody to induce cell-mediated lysis of cancer cells in an individual having cancer. In one embodiment, the "individual" is a human.

In a further aspect, the invention provides a pharmaceutical formulation comprising any one of the anti-HLA-G antibodies or anti-HLA-G/anti-CD 3 bispecific antibodies provided herein for use in any one of the methods of treatment described above. In one embodiment, the pharmaceutical formulation comprises any one of the anti-HLA-G antibodies or anti-HLA-G/anti-CD 3 bispecific antibodies provided herein and a pharmaceutically acceptable carrier.

The antibodies of the invention (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if topical treatment is desired, intralesional administration may be employed. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on the short-term or long-term administration. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at various points in time, bolus administrations, and pulse infusion.

The antibodies of the invention will be formulated, administered and administered in a manner consistent with good medical practice. In this case, factors considered include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner. The antibody is not required but may optionally be formulated with one or more agents currently used to prevent or treat the disease. The effective amount of such other therapeutic agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or about 1% to 99% of the dosages described herein, or any dosages and by any route, as empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dosage of the antibodies of the invention (alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the administration of the antibody for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.5mg/kg-10 mg/kg) of antibody may be the initial candidate dose administered to the patient, e.g., by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may be in the range of about 1 μg/kg to 100mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of antibody will range from about 0.05mg/kg to about 10 mg/kg. Thus, one or more doses (or any combination thereof) of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg may be administered to the patient. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives about 2 to about 20, or e.g., about 6, doses of antibody). An initial higher load may be administered followed by one or more lower doses. An exemplary dosing regimen comprises administering an initial amount of carrier of about 4mg/kg followed by weekly administration of an antibody at a maintenance dose of about 2 mg/kg. However, other dosage regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays.

It will be appreciated that any of the above formulations or methods of treatment may be performed using the immunoconjugates of the invention instead of or in addition to an anti-HLA-G antibody or an anti-HLA-G/anti-CD 3 bispecific antibody.

II. product

In another aspect of the invention, articles of manufacture are provided that contain materials useful in the treatment, prevention and/or diagnosis of the above-described conditions. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container may contain the composition, either by itself or in combination with another composition effective to treat, prevent and/or diagnose the symptoms, and may have a sterile access (e.g., the container may be an intravenous solution bag or vial having a stopper that may be perforated by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition can be used to treat a selected condition. In addition, the article of manufacture may comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises other cytotoxic or other therapeutic agents. The article of manufacture in this embodiment of the invention may further comprise package insert indicating that the composition may be used to treat a particular disease. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer solution, and dextrose solution. From a commercial and user perspective, it may further comprise other materials including other buffers, diluents, filters, needles and syringes.

The following examples and drawings are provided to aid in the understanding of the invention, but the true scope of the invention is set forth in the appended claims. It will be appreciated that modifications may be made to the steps set forth without departing from the spirit of the invention.

Description of amino acid sequence

anti-HLA-G antibody/antigen binding portion (SEQ ID No of variable and Complementarity Determining Regions (CDRs):

SEQ ID NO. 1 heavy chain CDR-H1, HLA-G-0090

SEQ ID NO. 2 heavy chain CDR-H2, HLA-G-0090

SEQ ID NO. 3 heavy chain CDR-H3, HLA-G-0090

SEQ ID NO. 4 light chain CDR-L1, HLA-G-0090

SEQ ID NO. 5 light chain CDR-L2, HLA-G-0090

SEQ ID NO. 6 light chain CDR-L3, HLA-G-0090

SEQ ID NO. 7 heavy chain variable domain VH, HLA-G-0090

SEQ ID NO. 8 light chain variable domain VL, HLA-G-0090

SEQ ID NO. 9 light chain CDR-L1, HLA-G-0090-VL-N31D

SEQ ID NO. 10 light chain variable domain VL, HLA-G-0090-VL-N31D

11 light chain CDR-L1, HLA-G-0090-VL-N31L of SEQ ID NO

SEQ ID NO. 12 light chain variable domain VL, HLA-G-0090-VL-N31L

13 light chain CDR-L1, HLA-G-0090-VL-N31Q of SEQ ID NO

SEQ ID NO. 14 light chain variable domain VL, HLA-G-0090-VL-N31Q

15 light chain CDR-L1, HLA-G-0090-VL-N31S of SEQ ID NO

SEQ ID NO. 16 light chain variable domain VL, HLA-G-0090-VL-N31S

17 light chain CDR-L1, HLA-G-0090-VL-N31T of SEQ ID NO

18 light chain variable domain VL, HLA-G-0090-VL-N31T of SEQ ID NO

SEQ ID NO. 19 light chain CDR-L1, HLA-G-0090-VL-N31Y

SEQ ID NO. 20 light chain variable domain VL, HLA-G-0090-VL-N31Y

SEQ ID NO. 21 light chain CDR-L1, HLA-G-0090-VL-N31Y-N38Y

SEQ ID NO. 22 light chain variable domain VL, HLA-G-0090-VL-N31Y-N38Y

SEQ ID NO. 23 light chain CDR-L1, HLA-G-0090-VL-S32P

24 light chain variable domain VL, HLA-G-0090-VL-S32P of SEQ ID NO

SEQ ID NO. 25 light chain CDR-L1, HLA-G-0090-VL-S A

26 light chain variable domain VL, HLA-G-0090-VL-S33A of SEQ ID NO

SEQ ID NO. 27 light chain CDR-L1, HLA-G-0090-VL-S D

28 light chain variable domain VL, HLA-G-0090-VL-S33D of SEQ ID NO

SEQ ID NO. 29 light chain CDR-L1, HLA-G-0090-VL-S P

30 light chain variable domain VL, HLA-G-0090-VL-S33P of SEQ ID NO

Other sequences

SEQ ID NO. 31 exemplary human HLA-G

SEQ ID NO. 32 exemplary human HLA-G extracellular domain (ECD)

SEQ ID NO. 33 exemplary human beta.2M

34 modified human HLA-G (wherein HLA-G specific amino acids have been replaced by HLA-A consensus amino acids (=deiraft) HLA-G; see also FIG. 1) ECD)

SEQ ID NO. 35 exemplary human HLA-A2

SEQ ID NO. 36 exemplary human HLA-A2 ECD

Exemplary mouse H2Kd ECD of SEQ ID NO 37

SEQ ID NO. 38 exemplary rat RT1A ECD

Exemplary human HLA-Gβ2MMHC class I Complex of SEQ ID NO 39

SEQ ID NO. 40 exemplary modified human HLA-Gβ2M MHC class I complexes (wherein HLA-G specific amino acids have been replaced by HLA-A consensus amino acids (=to transplant HLA-G), see also FIG. 2)

Exemplary mouse H2Kdβ2M MHC class I Complex of SEQ ID NO 41

SEQ ID NO. 42 exemplary human HLA-G/mouse H2 Kd.beta.2M MHC class I complex (wherein a human HLA-G specific position is grafted onto a mouse H2Kd scaffold)

Exemplary rat RT1A beta 2M MHC class I complex of SEQ ID NO 43

SEQ ID NO. 44 exemplary human HLA-G/rat RT1A beta 2M MHC class I Complex (wherein a human HLA-G specific position is grafted onto a rat RT1A scaffold)

SEQ ID NO. 45 linker and His tag

SEQ ID NO. 46 peptide

47 human kappa light chain constant region of SEQ ID NO

48 human lambda light chain constant region of SEQ ID NO

SEQ ID NO. 49 shows human heavy chain constant region derived from IgG1

SEQ ID NO. 50 shows a human heavy chain constant region derived from IgG1 comprising the mutations L234A, L235A and P329G

SEQ ID NO. 51 shows human heavy chain constant region derived from IgG4

anti-CD 3 antibody/antigen binding portions (variable and Complementarity Determining Regions (CDRs)):

SEQ ID NO. 52 heavy chain CDR-H1, P035-093 (abbreviated as P035)

SEQ ID NO. 53 heavy chain CDR-H2, P035-093

54 heavy chain CDR-H3, P035-093 of SEQ ID NO

Light chain CDR-L1, P035-093 of SEQ ID NO. 55

SEQ ID NO. 56 light chain CDR-L2, P035-093

Light chain CDR-L3, P035-093 of SEQ ID NO. 57

58 heavy chain variable domain VH, P035-093 of SEQ ID NO

SEQ ID NO. 59 light chain variable domain VL, P035-093

SEQ ID NO. 60 heavy chain CDR-H1, clone 22 (abbreviated Cl 22)

SEQ ID NO. 61 heavy chain CDR-H2, clone 22

SEQ ID NO. 62 heavy chain CDR-H3 clone 22

SEQ ID NO. 63 light chain CDR-L1, clone 22

SEQ ID NO. 64 light chain CDR-L2 clone 22

SEQ ID NO. 65 light chain CDR-L3 clone 22

SEQ ID NO. 66 heavy chain variable domain VH, clone 22

SEQ ID NO. 67 light chain variable domain VL, clone 22

68 heavy chain CDR-H1, V9 of SEQ ID NO

69 heavy chain CDR-H2, V9 of SEQ ID NO

70 heavy chain CDR-H3, V9 of SEQ ID NO

71 light chain CDR-L1, V9 of SEQ ID NO

72 light chain CDR-L2, V9 of SEQ ID NO

73 light chain CDR-L3, V9 of SEQ ID NO

74 heavy chain variable domain VH, V9 of SEQ ID NO

75 light chain variable domain VL, V9 of SEQ ID NO

Bispecific anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibodies:

P1AF7977(HLA-G-0090-VL-S32P/CD3 P035-093(P035))：

76 light chain 1P1AF7977 of SEQ ID NO

77 light chain 2P1AF7977 of SEQ ID NO

78 heavy chain 1P1AF7977 of SEQ ID NO

79 heavy chain 2P1AF7977 of SEQ ID NO

P1AF7978 (HLA-G-0090-VL-S32P/CD 3 clone 22 (Cl 22)):

80 light chain 1P1AF7978 of SEQ ID NO

81 light chain 2P1AF7978 of SEQ ID NO

82 heavy chain 1P1AF7978

83 heavy chain 2P1AF7978

P1AF7979(HLA-G-0090-VL-S32P/CD3 V9)：

84 light chain 1P1AF7979

85 light chain 2P1AF7979 of SEQ ID NO

86 heavy chain 1P1AF7979

87 heavy chain 2P1AF7979 of SEQ ID NO

Other sequences

Exemplary human CD3 of SEQ ID NO 88

SEQ ID NO. 89 exemplary cynomolgus monkey CD3

SEQ ID NO 90 human CD3 epsilon handle-Fc (pestle) -Avi

91 human CD3 delta handle-Fc (mortar) -Avi

SEQ ID NO:92 CD3 _orig VH

SEQ ID NO:93 CD3 _orig VL

SEQ ID NO:94 CD3 _orig IgG HC

SEQ ID NO:95 P035 IgG HC

SEQ ID NO:96 CD3 _orig /P035 IgG LC

Amino acid sequence (variable region including Complementarity Determining Regions (CDRs) and CDRs) of HLA-G-0090 antibody Unmodified N-glycosylation sites in L1 (bold)):

SEQ ID NO. 7: heavy chain variable domain VH, HLA-G-0090:

QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSS

SEQ ID NO. 8: light chain variable domain VL, HLA-G-0090

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

Amino acid sequence of modified HLA-G-0090 antibody light chain variable region (containing complementarity determining region with bottom line) (CDR) and modified N-glycosyl groups in CDR-L1Sites of chemical conversion (bold)):

SEQ ID NO. 10: light chain variable domain VL, HLA-G-0090-N31D

DIVMTQSPDSLAVSLGERATINCKSSQSVLDSSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 12: light chain variable domain VL, HLA-G-0090-N31L

DIVMTQSPDSLAVSLGERATINCKSSQSVLLSSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 14: light chain variable domain VL, HLA-G-0090-N31Q

DIVMTQSPDSLAVSLGERATINCKSSQSVLQSSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 16: light chain variable domain VL, HLA-G-0090-N31S

DIVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 18: light chain variable domain VL, HLA-G-0090-N31T

DIVMTQSPDSLAVSLGERATINCKSSQSVLTSSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 20: light chain variable domain VL, HLA-G-0090-N31Y

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 22: light chain variable domain VL, HLA-G-0090-N31Y-N38Y

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 24: light chain variable domain VL, HLA-G-0090-S32P

DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 26: light chain variable domain VL, HLA-G-0090-S33A

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSANNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 28: light chain variable domain VL, HLA-G-0090- -S33D

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSDNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

SEQ ID NO. 30: light chain variable domain VL, HLA-G-0090-S33P

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSPNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK

The amino acid sequence of the anti-CD 3 binding moiety (variable region, comprising Complementarity Determining Regions (CDRs) with a bottom line)):

SEQ ID NO. 58 heavy chain variable domain VH, P035-093 (abbreviated as P035)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRASNFPASYVSYFAYWGQGTLVTVSS

SEQ ID NO. 59 light chain variable domain VL, P035-093 (P035)

QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL

SEQ ID NO. 66 heavy chain variable domain VH, clone 22 (abbreviated Cl 22)

EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSS

SEQ ID NO. 67 light chain variable domain VL, clone 22 (Cl 22)

74 heavy chain variable domain VH, V9 of SEQ ID NO

EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS

75 light chain variable domain VL, V9 of SEQ ID NO

DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

Amino acid sequence of bispecific anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibody:

P1AF7977(HLA-G-0090-VL-S32P/CD3 P035-093(P035))：

76 light chain 1P1AF7977 of SEQ ID NO

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRASNFPASYVSYFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

77 light chain 2P1AF7977 of SEQ ID NO

DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNNKNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

78 heavy chain 1P1AF7977 of SEQ ID NO

QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

79 heavy chain 2P1AF7977 of SEQ ID NO

QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

P1AF7978 (HLA-G-0090-VL-S32P/CD 3 clone 22 (Cl 22)):

80 light chain 1P1AF7978 of SEQ ID NO

EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

81 light chain 2P1AF7978 of SEQ ID NO

82 heavy chain 1P1AF7978

QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAV YYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

83 heavy chain 2P1AF7978

P1AF7979(HLA-G-0090-VL-S32P/CD3 V9)：

84 light chain 1P1AF7979

EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

85 light chain 2P1AF7979 of SEQ ID NO

86 heavy chain 1P1AF7979

87 heavy chain 2P1AF7979 of SEQ ID NO

QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

Specific examples of the invention are set forth below:

1. an antibody that binds to human HLA-G comprising

2. The antibody according to embodiment 1, wherein the antibody

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

4. The antibody according to any one of embodiments 1 or 2, wherein the antibody comprises a constant region of human origin, in particular of IgG isotype, more particularly of IgG1 isotype, comprising human CH1, CH2, CH3 and/or CL domains.

5. The antibody of any one of embodiments 1 to 4, wherein the antibody

a) Has improved binding properties in terms of maximum binding (Rmax) and/or binding affinity (KD) compared to a (parent) antibody comprising: a VH domain comprising the amino acid sequence SEQ ID No. 7; and a VL domain comprising the amino acid sequence SEQ ID NO:8 (as shown in example 2).

b) Does not cross-react with a modified human HLA-G.beta.2M MHC I complex, wherein HLA-G specific amino acids have been replaced with HLA-A consensus amino acids, the complex comprising SEQ ID NO:40 (as shown in example 2); and/or

c) Does not cross-react with the mouse H2Kdβ2M MHC I complex comprising SEQ ID NO 41 (shown in example 2); and/or

d) Does not cross-react with the rat RT1A beta 2M MHC I complex comprising SEQ ID NO 43 (as shown in example 2).

6. The antibody of any one of embodiments 1 to 3, wherein the antibody

a) Inhibiting binding of ILT2 to JEG3 cells (ATCC accession HTB 36) (up-expressed HLA-G) (as shown in example 5); or (b)

b) Bind to JEG3 cells (ATCC accession HTB 36) (HLA-G expressed thereon), and inhibit ILT2 binding to JEG3 cells (ATCC accession HTB 36) (HLA-G expressed thereon) (as shown in example 5).

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

8. The antibody of example 7, wherein the antibody is a bispecific antibody that binds to human HLA-G and to human CD 3.

9. The antibody of embodiment 7, wherein the antibody is a bispecific antibody that binds to human HLA-G and to human CD3, the bispecific antibody comprising a first antigen-binding moiety that binds to human HLA-G and a second antigen-binding moiety that binds to human CD3, wherein the first antigen-binding moiety that binds to human HLA-G comprises

10. The bispecific antibody according to example 9,

wherein the first antigen binding portion

And wherein the second antigen binding portion

11. The bispecific antibody according to example 10,

wherein the first antigen binding portion

and wherein the second antigen binding portion

12. The bispecific antibody of example 108,

wherein the first antigen binding portion

and wherein the second antigen binding portion

13. The bispecific antibody according to example 10,

wherein the first antigen binding portion

and wherein the second antigen binding portion

14. The bispecific antibody of any one of embodiments 8 to 13, wherein

The bispecific antibody showed

a) Inhibition of ILT2 and/or ILT4 binding to HLA-G (as shown in example 13); and/or

b) Antibody-mediated ifnγ secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV 3 HLA-G) and/or on JEG3 cells expressing endogenous HLA-G, wherein the ifnγ secretion is detected (by Luminex technology) (as shown in example 14); and/or

c) T cell mediated cytotoxicity/tumor cell killing on SKOV3 cells transfected with recombinant HLA-G (SKOV 3 HLA-G) and/or JEG3 cells expressing endogenous HLA-G, wherein the cytotoxicity was detected by measuring caspase 8 activation in the cells after treatment with bispecific antibody (as shown in example 15); and/or

d) In vivo anti-tumor efficacy/tumor regression in humanized NSG mice bearing SKOV3 human ovarian cancer transfected with recombinant HLA-G (SKOV 3 HLA-G) humanized NSG mice (as shown in example 16); and/or

e) HLA-G CD 3T cell bispecific in vivo anti-tumor efficacy/tumor in humanized NSG mice bearing human breast cancer PDX tumor (BC 004) (shown in example 17).

15. The bispecific antibody of any one of embodiments 9 to 14, wherein the first antigen binding moiety and the second antigen binding moiety are Fab molecules.

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

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

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

19. The bispecific antibody of any one of embodiments 9 to 18, comprising an Fc domain consisting of a first subunit and a second subunit.

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

21. the bispecific antibody according to embodiment 19 or 20, wherein the Fc domain is a human IgG Fc domain, in particular belonging to the IgG1 isotype.

22. The bispecific antibody of any one of embodiments 19 or 20, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to and/or effector function of an Fc receptor.

23. The bispecific antibody of example 22, wherein the antibody is an IgG1 isotype comprising mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

24. The bispecific antibody of any one of embodiments 19 to 23, wherein an amino acid residue in the CH3 domain of a first subunit of an Fc domain is substituted with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of a second subunit, and an amino acid residue in the CH3 domain of the second subunit of an Fc domain is substituted with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

25. The bispecific antibody of example 24, wherein the antibody belongs to the IgG1 isotype comprising the mutation T366W in a first subunit of the Fc domain and the mutations Y407V, T366S and L368A (numbered according to the Kabat EU index) in a second subunit of the Fc domain.

26. The bispecific antibody of example 25, wherein the antibody comprises an additional mutation S354C in a first subunit of the Fc domain and an additional mutation Y349C in a second subunit of the Fc domain (numbered according to the Kabat EU index).

27. The bispecific antibody of example 25, wherein the antibody comprises an additional mutation Y349C in a first subunit of the Fc domain and an additional mutation S354C in a second subunit of the Fc domain (numbered according to the Kabat EU index).

28. An isolated nucleic acid encoding the antibody of any one of embodiments 1-4 or the bispecific antibody of any one of embodiments 9-27.

29. A host cell, preferably a eukaryotic host cell, comprising a nucleic acid according to example 28.

30. A method of producing the antibody of any one of embodiments 1-4 or the bispecific antibody of any one of embodiments 9-227, the method comprising culturing the host cell of embodiment 29, thereby producing the antibody or bispecific antibody.

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

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

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

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

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

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

37. The use of embodiment 36, wherein the medicament is for treating cancer.

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

Examples

Recombinant DNA technology

DNA was manipulated using standard methods, as described in the following documents: sambrook, j et al Molecular cloning: A laboratory manual; cold Spring Harbor Laboratory Press, cold Spring Harbor, new York,1989. Molecular biological agents were used according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

The desired gene fragment was prepared by chemical synthesis in the Geneart GmbH (Regensburg, germany). The synthesized gene fragment is cloned into escherichia coli plastid for propagation/amplification. The DNA sequence of the subcloned fragments was verified by DNA sequencing. Alternatively, the synthetic short DNA fragments are assembled by annealing chemically synthesized oligonucleotides or via PCR. The corresponding oligonucleotides were prepared from the meta GmbH (Planegg-Martinsried, germany).

Basic/standard mammalian expression plastid description

For expression of the desired gene/protein (e.g. full length antibody heavy chain, full length antibody light chain or MHC class I molecules, e.g. HLA-G, or MHC class I molecules fused to peptides and beta-2 microglobulin, e.g. HLA-G fused to HLA-G binding peptides and/or beta-2 microglobulin), transcription units are used comprising the following functional elements:

Direct early enhancers and promoters from human cytomegalovirus (P-CMV) comprising intron A,

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

a murine immunoglobulin heavy chain signal sequence,

genes/proteins to be expressed (e.g. full length antibody heavy chain or MHC class I molecules),

and

Bovine growth hormone polyadenylation sequence (BGH pA).

In addition to the expression units/cassettes comprising the desired gene to be expressed, the basal/standard mammalian expression plastids also contain

An origin of replication from the vector pUC18, which allows the plastid to replicate in E.coli, and

-a β -lactamase gene conferring ampicillin resistance in e.

Protein assay

The protein concentration of the purified polypeptide was determined by determining the Optical Density (OD) at 280nm using the calculated molar extinction coefficient based on the amino acid sequence of the polypeptide.

Example 1

Production of HLA-G chimeric molecules

Immunization with HLA-G molecules resulted in the production of polyclonal serum, which consisted of a mixture of MHC-I cross-reactive antibodies and indeed HLA-G specific antibodies, due to high homology (> 98%) with other MHC I molecules.

There is currently no tool for selecting truly HLA-G specific antibodies without cross-reacting with other people's MHC-I (e.g. HLA-a) and for further selecting those antibodies with receptor blocking function.

We identified unique HLA-G positions and positions required to ensure structural identity and receptor interaction (ILT 2/4 and KIR2DL 4).

The unique proximal position of human HLA-G is then "grafted" onto MHC class I complex molecules from different rodent species (e.g., rat RT1A and mouse H2 kd) to generate a "chimeric" immunogen/screening antigen.

Stringent screening/testing for binding/specificity of antibodies produced (no cross-reactivity/no specificity for reverse antigen, respectively)

Antigen receiving binding test:

HLA-G, presented as a human HLA-Gβ2M MHC complex comprising SEQ ID NO 43

HLA-G specific sequences, grafted onto rat RT-1 and mouse H2Kd (SEQ ID NO:46: human HLA-G/mouse H2Kdβ2M MHC class I complex, wherein the site specific for human HLA-G is grafted onto mouse H2Kd scaffold, and SEQ ID NO:48: human HLA-G/rat RT1Aβ2M MHC class I complex, wherein the site specific for human HLA-G is grafted onto rat RT1A scaffold)

Cells expressing the natural HLA-G MHC class I complex (e.g. Jeg3 cells) or cell lines SKOV3 HLA-G+ and PA-TU-8902HLA-G+ transfected with human HLA-G

Inverse antigen subjected to cross-reactivity test:

-comprising other HLA-A sequences (HLA-A 2 and) Combinations of inverse antigens (MHC class I complexes) with different peptides (see, e.g., SEQ ID NO:35 (HLA-A 2) and SEQ ID NO:40HLA-A consensus sequence on HLA-G scaffold)

Inverse antigens (MHC class I complexes) from other species, such as rat RT-1 and mouse H2kd (SEQ ID NO:43 and SEQ ID NO: 41)

Unmodified cell lines SKOV3 and PA-TU-8902, characterized by the absence of HLA-G expression.

Chimeric HLA-G antigens were designed to determine specific binding of specific anti-HLA-G antibodies (see fig. 2):

chimeric rat MHC I molecules with HLA-G unique positions (RT 1-A) (SEQ ID NO: 44) were designed for use in binding assays:

unique HLA-G positions were determined by aligning the 2579HLA-A, 3283HLA-B, 2133HLA-C, 15HLA-E, 22HLA-F and 50HLA-G sequences from IMGT (available at 2014, 2, 6). Those residues in HLA-G that occur in less than 1% (mostly about 0%) of the sequences in any of the 3 sequence sets HLA-A, HLA-B, and HLA-C+HLA-E+HLA-F combinations are referred to as HLA-G unique positions.

The 4 core HLA-G unique positions (2 out of alpha-1 and 2 out of alpha-3) did not show polymorphism in the HLA-G sequence set, and none of the other HLA genes contained HLA-G specific residues at these positions (except 1 xHLA-A of M100, 1 xHLA-B of Q103, and 1 xHLA-C of Q103).

The crystal structure of rat RT1-A (Rudolph, M.G. et al J.mol.biol.324:975-990 (2002); PDB code: 1 KJM) was superimposed on the crystal structure of human HLA-G (Clements, C.S. et al PROC.NATL. ACAD.SCI.USA 102:3360-3365 (2005): PDB code: 1 YDP). The overall structure of the α -chain and related β -2-microglobulin is preserved.

By sequence comparison and structural alignment, unique positions of HLA-G were identified in the RT1-A structure. In a first step, unique HLA-G positions are identified which are exposed on the molecular surface of HLA-G and RT1-A, and thus accessible to antibodies. Unique positions hidden in protein folding are excluded from engineering. In a second step, the structurally proximal residues are identified which also need to be exchanged to form the corresponding region "HLA-G-like", i.e., to generate the actual HLA-G epitope containing the unique position, rather than to generate the HLA-G/rat RT1-A artificial chimeric epitope. All positions thus selected for mutation were analyzed and structural fits of individual residues from HLA-G were determined to avoid possible local interference of the molecular structure by mutation.

Chimeric murine MHC I molecules (H2 Kd) with HLA-G unique position (SEQ ID NO: 42) were similarly generated for binding assays.

Design of HLA-A based reverse antigen for cross-reactivity testing by "ungrafting" HLA-G unique positions of HLA-A consensus sequences (SEQ ID NO:40 = modified human HLA-Gβ2M MHC class I complex (where HLA-G specific amino acids have been replaced by HLA-A consensus amino acids (=ungrafted HLA-G))

Analysis was performed in the crystal structure of human HLA-G for unique positions derived from multiple sequence alignment (PDB code: 1 YDP). First, locations that are not exposed on the HLA-G surface and thus inaccessible to antibodies are excluded from engineering. Second, the exposed residue surface was analyzed for the feasibility of performing amino acid exchanges (i.e., excluding possible local perturbation of the molecular structure when mutations at the relevant positions). A total of 14 switch positions were verified. Amino acids at verified positions were mutated to HLA-A consensus sequences derived from multiple sequence alignments of 2579 HLA-A sequences downloaded from IMGT (available at 2014, 2, 6).

Generation of expression plastids for soluble classical and atypical MHC class I molecules

Recombinant MHC class I genes encode N-terminally extended fusion molecules consisting of peptides known to bind to corresponding MHC class I molecules, beta-2 microglobulin and corresponding MHC class I molecules.

The expression plasmid for the transient expression of soluble MHC class I molecules comprises, in addition to the soluble MHC class I molecule expression cassette, an origin of replication from the vector pUC18, which allows the plasmid to replicate in E.coli, and a beta-lactamase gene, which confers ampicillin resistance in E.coli.

The transcriptional unit of a soluble MHC class I molecule comprises the following functional elements:

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

a murine immunoglobulin heavy chain signal sequence,

n-terminally truncated staphylococcus aureus sortase A encoding nucleic acids

Bovine growth hormone polyadenylation sequence (BGH pA).

The amino acid sequences of mature soluble MHC class I molecules from different species are:

SEQ ID NO. 39: exemplary human HLA-Gβ2M MHC class I complexes

SEQ ID NO. 40: exemplary modified human HLA-Gβ2M MHC class I complexes (wherein HLA-G specific amino acids have been replaced with HLA consensus amino acids (=to transplant HLA-G, see also FIG. 2)

SEQ ID NO. 41: exemplary mouse H2Kdβ2M MHC class I Complex

SEQ ID NO. 42: exemplary human HLA-G/mouse H2Kd beta 2M MHC complexes (wherein a human HLA-G specific position is grafted onto a mouse H2Kd scaffold)

SEQ ID NO. 43: exemplary rat RT1A beta 2M MHC class I Complex

SEQ ID NO. 44: exemplary human HLA-G/rat RT1A beta 2M MHC complexes (wherein a human HLA-G specific position is grafted onto the rat RT1A scaffold)

For the exemplary HLA-A2 beta 2M MHC class I complex, the following components were used, and the complex was expressed in e.

MHCI complex HLA-A2/b2M (SEQ ID NO:35 and SEQ ID NO: 33) (each with an additional N-terminal methionine) + VLDFAPPGA peptide (SEQ ID NO: 46) +linker and His tag (SEQ ID NO: 45)

Example 2

Removal of N-glycosylation motifs in CDR-L1

CDR-L1 of the anti-HLA-G antibody HLA-G-0090 contains a typical N-glycosylation motif "NSS" comprising positions 31 to 33 of the Light Chain (LC). The motif is decided to be removed because it may constitute a potential developability defect. A homology model of the HLA-G-0090 variable region shows high solvent accessibility at LC positions 31 to 33. Furthermore, the side chains of N31 and S32 are expected to point inward in the direction of CDR-H3, making them possible candidates for part of the paratope of an antibody. Indeed, many published antibody-antigen X-ray complex structures record that these residues interact chemically with the antigen. Thus, the risk of causing a decrease in the binding affinity of the antibody upon introduction of mutations at LC positions 31-33 is quite high. To reduce risk, 11 different variants of the antibody HLA-G-0090 were designed and produced in the form of IgG1 in HEK293F cells, which contained mutations at positions 31, 32 and 33 in LC.

Note that the mutant variant HLA-G-0090-VL-N31Y-N38Y contains a second mutation (N38Y) outside the N-glycosylation motif, and is not related to its removal, to increase germline identity

Summary of anti-HLA-G antibody sequences (SEQ ID NO for variable region and CDR):

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the binding and other properties and biological activities of the obtained anti-HLA-G specific antibodies were determined as described in the examples below and compared to a known reference HLA-G-0090.

Expression and purification

Expression yields for 0.5L expression in HEK293F cells after purification by affinity chromatography (MabSelect Sure) and dialysis are shown in the following Table.

Variants comprising mutations involving position LC 31 (N31X) exhibit significantly reduced expressive valence and often compromise material quality, while LC 32 and LC33 position variants exhibit good to acceptable expressive valence and good material quality.

HLA-G binding

wt-HLA-G affinity/kinetics

The affinity of each 5nM anti-HLA-G antibody to the wt-HLA-G complex (SEQ ID NO: 39) was determined by capturing with anti-hFc (GE Healthcare BR-1008-39) on a CM3 sensor chip and injecting the wt-HLA-G antigen diluted with HBS-P+ (GE Healthcare) running buffer to a concentration of 11nM to 300nM, with a flow rate of 60 μl/min, a binding time of 120 seconds, and a dissociation time of 600 seconds. After each cycle, the surface was regenerated by washing with 3m MgCl2. Kinetic binding curves were assessed using T200 assessment software and binding characteristics were calculated using a 1:1 langmuir binding model.

RAC (relative Activity concentration) of HLAG antibody (assay protocol shown in FIG. 3)

The RAC of HLA-G binders for each anti-HLA-G antibody (10 nM solution in HBS-P+) was determined by capturing and injecting HBS-P+ (GE Healthcare) running buffer diluted to a concentration of 300nM on a CM3 sensor chip with anti-hFc (GE Healthcare BR-1008-39), at a flow rate of 10 μl/min, a binding time of 60 seconds, and a dissociation time of 600 seconds. After each cycle, the surface was regenerated by washing with 3m MgCl2. The final RAC and Rmax values were calculated from the "combined" report points and capture content. Rmax= (MW analyte/MW ligand) x RU capture ligand x stoichiometry of interaction.

11 variants were evaluated in SPR binding experiments in which antibodies were immobilized via the Fc portion on a CM3 chip and recombinant single-chain HLA-G monomer/human HLA-. Beta.2M MHC class I complex (SEQ ID NO: 39) was used as the analyte. Kinetic parameters were determined by single cycle kinetic measurements performed on a Biacore T200 device at 25 ℃.

Four variants (HLA-G-0090-VL-S32P, HLA-G-0090-VL-S33A, HLA-G-0090-VL-S33D, HLA-G-0090-VL-S33P) with acceptable expressive value were further evaluated in more accurate multicycle kinetic measurements using the same SPR apparatus and experimental setup.

	ka[1/ms]	kd[1/s]	t ¹ / ₂ [s]	Kd[nM]	Rmax[％]
						HLA-G-0090	1.30E+06	1.78E-03	389.9	1.4	87
HLA-G-0090-VL-S32P	1.27E+06	1.57E-03	440.6	1.2	99
						HLA-G-0090-VL-S33A	1.24E+06	1.49E-03	465.9	1.2	99
HLA-G-0090-VL-S33D	8.97E+05	6.67E-03	103.9	7.4	98
						HLA-G-0090-VL-S33P	1.36E+06	3.68E-03	188.1	2.7	97

The two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A have improved binding affinity compared to the parent antibody HLA-G-0090, while the binding affinity of the two variants HLA-G-0090-VL-S33D and HLA-G-0090-VL-S33P is reduced by a factor of 5 and 2, respectively. Unexpectedly, for the variants tested, removal of the N-glycosylation motif resulted in a higher Rmax value in the kinetic measurement, indicating that the proportion of complex formation was higher than that of N-glycosylated antibody.

Cross-reactivity of anti-HLA-G antibodies and variants to soluble human HLA-G, soluble shed human HLA-G with HLA-A consensus specific sequences, and rat/mouse homologs

To further investigate the binding properties of the two binding affinity improved variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A, SPR binding experiments were performed using four counter-screening constructs. These recombinant single chain peptide-MHC complex constructs are murine H2-K1 (SEQ ID NO: 41), rat RT1 (SEQ ID NO: 43) and human HLA-G.beta.2M MHC class I complexes, wherein HLA-G specific amino acids have been replaced by HLA-A consensus amino acids (SEQ ID NO: 40). The latter construct constitutes a form of HLA-G in which all HLA-G specific residues have been replaced with their HLA-A consensus counterpart. Again, antibodies were immobilized on CM3 chips and single-chain peptide-MHC constructs were used as analytes for evaluation on a Biacore T200 device at 25 ℃.

Stability under stress

The parent antibody, as well as the two derived variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A, were subjected to 13 days of stress treatment under two different conditions:

pH 6.0.20 mM His/HisCl,140mM NaCl; at 40 ℃ (His 40 ℃)

pH 7.4PBS; at 37℃in PBS 37 ℃.

The materials were then analyzed using SEC and SPR to investigate the possible effects of chemical degradation on target binding. For reference, the stress-treated material was compared with the material stored under the following storage conditions:

pH 6.0.20 mM His/HisCl,140mM NaCl; frozen at-80℃C (reference)

The results are listed in the following table (relative percentages compared to the reference):

although all three antibodies showed very similar stability characteristics, variant HLAG-0090_vl-S32P retained more HLA-G binding (SPR Relative Activity Concentration (RAC)) than the other two variants, including their parent antibodies, after stress treatment in PBS at 37 ℃.

Thermal stability test

In the thermal stability test of the purified proteins, the Uncle device (UNCHAINED LABS, boston, MA, USA) was used. The aggregation temperature (Tagg) and the deconstructation temperature (Tm) of the purified protein were determined accordingly using static light scattering at 266nm and 473nm and parallel intrinsic fluorescence. A temperature ramp from 30 ℃ to 90 ℃ was used at a step of 0.1 ℃/min. A glass cuvette with a concentration of 1mg/mL was used for each sample volume of 9. Mu.l, dissolved in 20mM histidine, 140mM NaCl, pH 6.0 buffer. Analysis uses UNcle analysis software (UNCHAINED LABS).

Mass spectrometry and N-glycosylation

Deconvolution mass spectrometry of the complete samples recorded the effect of removing the N-glycosylation sites/NSS motifs in HLA-G-0090_VL-S32P and HLA-G-0090_VL-S33A on N-glycosylation. Samples were prepared with PNGase F to remove all N-linked glycans and only the molecular weight of the antibodies was obtained. While PNGase F is fully specific for cleavage of N-linked Fc-glycans, it exhibits much lower potency when cleaving N-linked Fc-glycans. HLA-G-0090 shows a clear N-glycosylation pattern from incomplete deglycosylation, thus indicating the presence of Fab-glycosylation (see FIG. 4A). No sign of residual N-glycosylation of HLA-G-0090_VL-S32P and HLA-G-0090_VL-S33A was detected (see FIG. 4B).

Example 3

Inhibition of ILT2 and ILT4 binding by anti-HLA-G antibodies

ELISA was set up by coating Fc-labeled ILT2 and ILT4 onto Maxisorp microtiter plates, respectively. After incubation and washing steps, the corresponding antibodies were added at a concentration of 100 nM. Soluble His-tagged monomers, dimers or trimers HLA-G were added to the wells. After incubation and washing steps, bound receptors were detected by anti-His-antibody-POD conjugates. The percent (%) inhibition (100% binding = 0% inhibition) was calculated as compared to the values obtained from wells containing ILT2/4+ HLA-G (monomer, dimer or trimer) without anti-HLA-G or ILT2/4 antibodies and shown in the table.

Example 4

Binding of HLA-G antibodies to native or recombinant HLA-G expressed on cells (assessed by FACS analysis)

In flow cytometric analysis, cells were stained with anti-HLA-G mAb at 4 ℃. Briefly, each cell suspension was transferred to polypropylene tubes (2×10 ⁵ Individual cells/tube) and pre-cooled at 5 ℃ for 10 minutes. The cells were then washed with 2ml FACS buffer (4 ℃) and centrifuged at 300g for 5 min. The anti-HLA-G antibody HLAG-0090-VL-S32P, HLAG-0090-VL-S33A, HLAG-0090 was diluted in staining buffer to an initial concentration of 50. Mu.g/ml. The antibodies were serially diluted 5-fold to obtain final concentrations (10. Mu.g/ml, 2. Mu.g/ml, 0.4. Mu.g/ml, 0.08. Mu.g/ml, 0.016. Mu.g/ml, 0.0032. Mu.g/ml). The FACS buffer was then aspirated from the tube, and the cell pellet was resuspended in 100 μl antibody solution and incubated for 1 hour at 5 ℃. The cells were then washed once with 2ml staining buffer and centrifuged at 300g for 5 minutes.

For detection of fluorescence, labeled anti-species antibodies (goat anti-human IgG (h+l) conjugated to Alexa 488, # Life Technologies a 11013) were diluted to 10 μg/ml with staining buffer and the cell pellet was resuspended in 100 μl detection antibody. After incubation for 1 hour at 5 ℃, the cells were washed once again with 2ml of staining buffer, resuspended in 500 μl of staining buffer, and measured on FACS CELESTA.

Exemplary FACS of the anti-HLA-G antibodies HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S32P (10. Mu.g/ml) are shown in the FACS overlay of FIG. 5: the deglycosylated variants of HLA-G0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S32P all showed good binding to HLA-G expressing SKOV3 cells and JEG cells, but not to the parental SKOV3 cells. MFI values for the corresponding HLA-G antibodies are shown in the histogram.

Example 5

anti-HLA-G antibodies inhibit/regulate binding of ILT2 to HLA-G expressed on JEG3 cells in assays, JEG3 cells (ATCC HTB 36) were stained with ILT2-c-Myc-Fc fusion protein (control = no inhibition), with or without pre-incubation with anti-HLA-G antibodies.

Binding/inhibition of binding was determined according to the following method: recombinant ILT2-c-Myc-Fc protein was added to JEG3 cells pre-incubated with the anti-HLA-G mAb, or to untreated JEG3 cells (as reference). For pre-incubation with anti-HLA-G antibodies, 2X 10 ⁵ Individual cells were transferred to polypropylene tubes. The anti-HLA-G antibodies HLAG-0090-VL-S32P, HLAG-0090-VL-S33A and HLA-G-0090 were diluted with staining buffer to a concentration of 80. Mu.g/ml, and 25. Mu.l of the antibody solution was added to the prepared cells and incubated at 5℃for 1 hour. ILT2-c-Myc-Fc or (control human IgG (Jackson-immune-Research # 009-000-003)) was diluted to a 2-fold concentration of 20. Mu.g/ml with staining buffer and added to the prepared cells at a final concentration of 10. Mu.g/ml and incubated for 2 hours at 5 ℃. Cells were washed twice with 200 μl staining buffer. Human ILT2-c-Myc-Fc protein was detected with a fluorescent labeled anti-Myc label (9E 10) Alexa Fluor 647 (abcam; # ab 223895) at a dilution concentration of 10. Mu.g/ml in staining buffer. Cells were resuspended in 50 μl detection antibody dilution and incubated for 1 hour at 5 ℃. Cells were then stained with 2ml The color buffer was washed once and resuspended in 500 μl of staining buffer prior to FACS CELESTA measurement.

As a control, anti-HLA-G antibodies bound to JEG-3 pre-incubated cells were detected by using an anti-species antibody (goat anti-human IgG (h+l) bound to Alexa 488, life technologies #a11013), diluted to 10 μg/ml with staining buffer, and cell pellet resuspended in 100 μl/well detection antibody. After incubation for 1 hour at 5 ℃, the cells were washed once again with buffer, resuspended in 500 μl staining buffer, and measured on FACS CELESTA.

The histogram in FIG. 6 shows the ability of HLA-G antibodies to modify/inhibit the interaction and binding, respectively, of recombinant ILT2 with HLA-G naturally expressed on JEG3 tumor cells.

The following table summarizes the experimental results. Binding of anti-HLA-G antibodies to JEG3 cells described as + = weak binding- ++++ = strong and (5) combining. anti-HLA-G antibodies inhibit/block or increase the ability of ILT2 to bind to HLA-G expressing JEG3 cells. In the last column, binding of recombinant ILT2 to cells or inhibition/blocking thereof (staining of ILT2-c-Myc-Fc in the absence of anti-HLA-G antibodies was set to 100% binding = 0% inhibition) is shown/quantified:

Example 6

Production of optimized CD3 binders

Starting from the previously described CD3 binder, referred to herein as "CD3 _orig "(see, e.g., WO2014/131712, which is incorporated herein by reference) comprising VH and VL sequences of SEQ ID No. 92 and SEQ ID No. 93, the inventors aimed to optimize the properties of this binder by removing the two aspartyl deamidating sequence motifs at Kabat positions 97 and 100 of heavy chain CDR 3.

For this purpose, such a library of antibodies suitable for phage display heavy chains was generated, wherein the two aspartyl amines at Kabat positions 97 and 100 were removed, and furthermore CDRs H1, H2 and H3 were also randomly assigned to compensate for the loss of affinity caused by replacement of Asn97 and Asn100 via the affinity maturation process.

This pool was placed on filamentous phage via fusion with the minor coat protein p3 (Marks et al (1991) J Mol Biol 222, 581-597) and combined for binding to recombinant CD3 epsilon.

10 candidate clones were identified in a preliminary screen, exhibiting acceptable binding to recombinant antigen as measured by SPR as Fab fragments (produced in e.coli).

However, only one of these clones showed acceptable binding activity to CD3 expressing cells as measured by flow cytometry after conversion to IgG form.

The selected clone strain is referred to herein as P035-093 (P035) (= "CD 3) _opt ") and VH and VL sequences comprising SEQ ID NO:58 and SEQ ID NO:59, respectively, are further evaluated and converted to bispecific forms as described below.

Example 7

Optimizing binding of CD3 binders to CD3

Binding to recombinant CD3

Optimized CD3 binder P035-093 (P035) (= "CD 3") in the form of human IgG1 for both having the P329G L234A L a ("PGLALA", EU numbering) mutation in the Fc region _opt ") and the original CD3 binder" CD3 _orig "(SEQ ID NO:94 and SEQ ID NO:96 (CD 3) _orig ) SEQ ID NO 95 and SEQ ID NO 96 (P035 = CD 3) _opt ) The binding to recombinant CD3 was determined by Surface Plasmon Resonance (SPR).

To assess the effect of deamidation site removal and its effect on antibody stability, binding of the original and optimized CD3 binders to recombinant CD3 was tested after 14 days of stress at 37 ℃ or 40 ℃. Samples stored at-80℃were used as reference. The reference sample and the sample stressed at 40℃were in 20mM His, 140mM NaCl at pH 6.0, and the sample stressed at 37℃was in PBS at pH 7.4, all at concentrations of 1.2-1.3mg/ml. After the stress period (14 days), the samples in PBS were dialyzed back into 20mM His, 140mM NaCl, pH 6.0 for further analysis.

The Relative Activity Concentration (RAC) of the samples was determined by SPR as follows.

SPR was performed on a Biacore T200 instrument (GE Healthcare). anti-Fab capture antibodies (GE Healthcare, # 28958325) were immobilized on Series S Sensor Chip CM (GE Healthcare) using standard ammonia coupling chemistry, resulting in surface densities of 4000-6000 Resonance Units (RU). HBS-P+ (10mM HEPES,150mM NaCl pH 7.4,0.05% surfactant P20) was used as running and dilution buffer. CD3 antibody at a concentration of 2. Mu.g/ml was injected at a flow rate of 5. Mu.l/min for 60 seconds. CD3 antigen (see below) at a concentration of 10. Mu.g/ml was injected for 120 seconds and dissociation was monitored for 120 seconds at a flow rate of 5. Mu.l/min. The chip surface was regenerated by two consecutive injections of 10mM glycine, pH 2.1, for 60 seconds each. The bulk refractive index difference was corrected by subtracting the blank injection and by subtracting the response obtained from the blank control flow cell. For evaluation, the binding reaction was performed 5 seconds after the end of injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (signal (RU) obtained after capture of CD3 antibody on immobilized anti-Fab antibody). The relative activity concentration was calculated by comparing each temperature stress sample with the corresponding non-stress sample.

The antigens used were heterodimers of the CD3 delta and CD3 epsilon extracellular domains fused to a human Fc domain with a pestle-mortar modification and a C-terminal Avi-tag (see SEQ ID NOS: 90 and 91).

The results of this experiment are shown in fig. 15. As can be seen, the original CD3 binder CD3 _orig In contrast, optimization of the CD3 binder CD3 _opt P035-093(P035)(＝CD3 _opt ) It was shown that after a temperature stress (2 weeks of treatment at 37 ℃ at pH 7.4) the binding to CD3 was significantly improved. This result demonstrates that deamidating site removal is successful and correlated with in vivo half-life and formulation of antibodies at neutral pH yields antibodies with excellent stability characteristics.

Binding to CD3 on Jurkat cells

Both for mutations with P329G L234A L a ("PGLALA", EU numbering) in the Fc regionOptimized CD3 binder r P035-093 (P035) (=cd3) in the form of human el IgG1 _opt ) And original CD3 binder "CD3 _orig "(SEQ ID NO:94 and SEQ ID NO:96 (CD 3) _orig ) SEQ ID NO 95 and SEQ ID NO 96 (P035 = CD 3) _opt ) Binding to CD3 on human reporter T cell line Jurkat NFAT was determined by FACS.

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; promega#CS 176501) are human acute lymphoblastic leukemia reporter cell lines with NFAT promoter expressing human CD 3. Cells in RPMI1640, 2g/l glucose, 2g/l NaHCO ₃ Cultured in 10% FCS, 25mM HEPES, 2mM L-glutamylacid, 1 XNEAA, 1 Xsodium pyruvate, 10 ten thousand to 50 ten thousand cells per ml. Hygromycin B was added at a final concentration of 200 μg/ml each time the cells were passaged.

For binding assays, jurkat NFAT cells were collected, washed with PBS and resuspended in FACS buffer. Antibody staining was performed in a 96-well round bottom plate. Thus, 100'000 to 200'000 cells are seeded per well. The disc was centrifuged at 400x g for 4 minutes and the supernatant removed. The test antibodies were diluted in FACS buffer and 20 μl of antibody solution was added to the cells over 30 minutes at 4 ℃. To remove unbound antibody, cells were washed twice with FACS buffer before adding diluted secondary antibody (PE-bound AffiniPure F (ab') 2 fragment goat anti-human IgG Fcg fragment specificity; jackson ImmunoResearch # 109-116-170). After incubation at 4 ℃ for 30 minutes, unbound secondary antibodies were washed away. Prior to measurement, cells were resuspended in 200 μl FACS buffer and subsequently analyzed by flow cytometry using a BD Canto II device.

As shown in fig. 16, the CD3 binder P035-093 (P035) (=cd3) was optimized _opt ) And original CD3 binder "CD3 _orig "binds equally well to CD3 on Jurkat cells".

Example 8

Optimizing the functional Activity of the CD3 Binder

Optimization of the CD3 Binder "CD3 _opt "functional Activity was tested in Jurkat report cell assays and was compared to the originalCD3 binder "CD3 _orig "activity was compared. To test for functional activity of IgG, antibodies were prepared on CD3 _opt Human IgG1 PGLALA or CD3 _orig In the presence of an increased concentration of human IgG1 PGLALA, CHO cells expressing anti-PGLALA were co-cultured with Jurkat NFAT reporter cells. Activation of CD3 on Jurkat NFAT reporter cells after T cell cross-linking induces luciferase production and luminescence can be measured as an activation marker. Including CD3 _orig Human IgG1wt served as a negative control, which failed to bind to anti-PGLALA expressing CHO cells and thus failed to crosslink on Jurkat NFAT cells. A schematic of the analysis is provided in fig. 17.

The anti-PGLALA expressing CHO cells were CHO-K1 cells engineered to express specific binding to human IgG on their surface ₁ Antibodies to Fc (PGLALA) (see WO 2017/072210, incorporated herein by reference). These cells were cultured in DMEM/F12 medium containing 5% FCS+1% GluMax. Jurkat NFAT report cells are described and in example 7.

After simultaneous binding of CD3 huIgG1 PGLALA to CHO expressed on Jurkat-NFAT reporter cells and anti-PGLALA expressed on CD3, the NFAT promoter is activated and results in expression of active firefly luciferase. The intensity of the luminescence signal (obtained by addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat-NFAT reporter cells were grown in suspension and cultured in RPMI1640, 2g/L glucose, 2g/L NaHCO3, 10% FCS, 25mM HEPES, 2mM L-glutamine, 1 XNEAA, 1 Xsodium pyruvate, 10 to 50 ten thousand cells per ml, 200 μg per ml hygromycin. For analysis, CHO cells were collected and viability was determined using ViCell. 30,000 target cells/Kong Pingpu were added to CHO cells in flat bottom Bai Bi well plate (Greiner bio-one # 655098) and 100 μl of medium, and 50 μl/well of diluted antibody or medium (for control group) was added. Subsequently, jurkat-NFAT reporter cells were collected and viability was assessed using Vicell. Cells were resuspended in hygromycin B-free cell culture medium at 120 ten thousand cells/ml and added to CHO cells at 60 000 cells/well (50. Mu.l/well) to obtain a 2:1 final effector to target (E: T) ratio and a final volume of 200. Mu.l/well. Subsequently, 4 μl of GloSensor (promega#e1291) was added to each well (2% of final volume). Cells were incubated at 37℃for 24 hours in a humidified incubator. At the end of the incubation time, luminescence was detected using a TECAN Spark 10M.

As shown in fig. 18, in the case of CD3 _orig Following cross-linking, the optimized CD3 binder P035-093 (P035) (=cd3opt) had similar activity on Jurkat NFAT cells.

Example 9

Production of bispecific antibodies binding to human HLA-G and to human CD3 (anti-HLA-G/anti-CD 3 antibodies)

Recombinant DNA technology

Gene and oligonucleotide synthesis

Basic/standard mammalian expression plastid description

For expression of the desired gene/protein (e.g. antibody heavy chain or antibody light chain) transcription units are used comprising the following functional elements:

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

a murine immunoglobulin heavy chain signal sequence,

and

Bovine growth hormone polyadenylation sequence (BGH pA).

-a β -lactamase gene conferring ampicillin resistance in e.

Protein assay

Expression plastids for producing recombinant monoclonal bispecific antibodies

The recombinant monoclonal antibody genes encode the corresponding immunoglobulin heavy and light chains.

The expression plasmid for the transient expression of the monoclonal antibody molecule comprises, in addition to the immunoglobulin heavy or light chain expression cassette, an origin of replication from the vector pUC18, which allows the plasmid to replicate in e.coli, and a β -lactamase gene, which confers resistance to ampicillin in e.coli.

The transcriptional units of the heavy or light chain of the respective antibody comprise the following functional elements:

human heavy chain immunoglobulin 5 '-untranslated region (5' UTR),

-murine immunoglobulin heavy chain signal sequence

Bovine growth hormone polyadenylation sequence (BGH pA).

Transient expression and analytical characterization

Recombinant production was performed by transient transfection of HEK293 cells (of human embryonic kidney cell line 293 origin) cultured in F17 medium (Invitrogen corp.). To produce monoclonal antibodies, cells are co-transfected with plastids containing the corresponding immunoglobulin heavy and light chains. For transfection, the "293-Fectin" transfection reagent (Invitrogen) was used. Transfection was performed according to the manufacturer's instructions. Cell culture supernatants were harvested 3 to 7 days post transfection. The supernatant is stored at low temperature (e.g., -80 ℃).

For general information on recombinant expression of human immunoglobulins in e.g. HEK293 cells see: meissner, p. Et al, biotechnol. Bioeng.75 (2001) 197-203.

Using the methods described above for recombinant DNA technology, production of expression plastids for monoclonal antibodies, and transient expression and analytical characterization, the following bispecific antibodies binding to human HLA-G and to human CD3 were produced and analyzed:

Bispecific antibodies binding to human HLA-G and to human CD3 (anti-HLA-G/anti-CD 3 antibodies) (SEQ ID nos:

"clone 22 (abbreviated" Cl22 ")" is an optimized CD3 binder (see WO 2020/127619); "P035-093 (abbreviated" P035 ") is another optimized variant CD3 binder; v9 is another CD3 binder, which is described, for example: rodrigues et al Int J Cancer Suppl (1992) 7,45-50; and WO 1992/22653 (SEQ ID NO:20 and SEQ ID NO:17 for VH and VL sequences of WO 1992/22653).

Bispecific anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibodies:

P1AF7977(HLA-G-0090-VL-S32P/CD3 P035-093(P035))：

SEQ ID NO. 76 light chain 1P 1AF7977

SEQ ID NO. 77 light chain 2P 1AF7977

SEQ ID NO:78 heavy chain 1P 1AF7977

79 heavy chain 2P 1AF7977 of SEQ ID NO

P1AF7978 (HLA-G-0090-VL-S32P/CD 3 clone 22 (Cl 22)):

80 light chain 1P 1AF7978 of SEQ ID NO

81 light chain 2P 1AF7978 of SEQ ID NO

SEQ ID NO. 82 heavy chain 1P 1AF7978

83 heavy chain 2P 1AF7978 of SEQ ID NO

P1AF7979(HLA-G-0090-VL-S32P/CD3 V9)：

SEQ ID NO. 84 light chain 1P 1AF7979

85 light chain 2P 1AF7979 of SEQ ID NO

86 heavy chain 1P 1AF7979

SEQ ID NO. 87 heavy chain 2P 1AF7979

Example 10

Binding and stability of bispecific anti-HLA-G/anti-CD 3 antibodies (T cell bispecific (TCB) antibodies) to HLA-G

Stability under stress

Three TCB molecules with the same HLA-G targeting binder HLAG-0090-VL-S32P and three different CD3e binders were stress treated for 14 days under two different conditions:

pH 6.0.20 mM His/HisCl,140mM NaCl; at 40 ℃ (His 40 ℃)

pH 7.4PBS; at 37℃in PBS 37 ℃.

The material was then analyzed using CD-SDS, SEC, and Surface Plasmon Resonance (SPR) to investigate chemical degradation and possible effects on target binding. For reference, the stress-treated material was compared with the material stored under the following storage conditions:

pH 6.0.20 mM His/HisCl,140mM NaCl; frozen at-80℃C (reference)

The results are shown in the following table:

/>

all three TCBs showed acceptable stability characteristics with moderate loss of binding after stress treatment. Furthermore, thermal stability was measured by DLS (T _agg ) It is within the known normal range of human IgG. RAC binding was determined by surface plasmon resonance (Biacore) as described in example 2.

Example 11

Binding of bispecific anti-HLA-G/anti-CD 3 antibodies (T cell bispecific (TCB) antibodies) to CD3 expressed on T cells (as assessed by flow cytometry)

Briefly, 100ml of fresh blood was collected into an Erlenmeyer flask and mixed with 100ml of separation buffer (PBS containing 2% FBS and 2mM EDTA). 25ml of the suspension was then carefully transferred onto 15ml ficoll in a 50ml tube and centrifuged at 800g for 15 minutes without braking. The PBMC layer in ficoll gradient was then transferred to a new 50ml tube containing separation buffer and centrifuged at 300g for 10 min at 4 ℃. PBMCs were then washed twice and cells were pooled in 10ml of separation buffer. PBMCs were stored frozen at-80 ℃ until further use. Human T cell isolation kit Stem cell, # 17951) was used to isolate T cells from PBMCs according to the manufacturer's instructions. Binding of HLA-G TCB and T cells was then measured by flow cytometry. Briefly, 500. Mu. l T cells (5X 10) ⁵ Individual cells) were added to each FACS tube. T cells were washed with 2ml staining buffer (PBS with 2% FBS) and centrifuged at 300g for 5 min at 4 ℃. HLA-G TCB was diluted in medium to various concentrations ranging from 5. Mu.g/ml to 0.05. Mu.g/ml. T cells were then resuspended in 100 μl HLA-G TCB diluent and incubated at 4 ℃ for 30 minutes in the absence of light. After washing the cells once with 2ml staining buffer, they were centrifuged at 300g for 5 min and then resuspended in 100. Mu.l of secondary antibody dilution (Alexa Fluor 488-labeled anti-human IgG, 1:200) and incubated at 4℃for 30 min in the absence of light. T cells were washed twice with 2ml staining buffer and centrifuged at 300g for 5 min at 4 ℃. Finally, the cells were resuspended in 500. Mu.l of medium and HLA-G TCB binding to T cells was detected on BD LSR. HLA-G TCBs P1AF7977 (HLA-G-0090-VL-S32P/CD 3P 035); p1AF7978 (HLA-G-0090) The binding of VL-S32P/CD3 Cl 22) and P1AF7979 (HLA-G-0090-VL-S32P/CD 3V 9) to different concentrations of CD3 on T cells is shown in FIG. 7.

Example 12

Binding of bispecific anti-HLA-G/anti-CD 3 antibodies (T cell bispecific (TCB) antibodies) to native or recombinant HLA-G expressed on cells (as assessed by flow cytometry)

The ability of anti-HLA-G TCB mAbs to bind HLA-G expressed on different cells and cell lines was assessed by FACS analysis. Binding to JEG3 tumor cells or Skov3 transfectants naturally expressing HLA-G and corresponding parental untransfected cells is described.

In flow cytometric analysis, cells were stained with anti-HLA-G TCB mAb at 4 ℃. Briefly, 25 μl/well of each cell suspension (5×104 cells/well) was transferred to a polypropylene 96-well V-bottom plate and chilled in a refrigerator at 5 ℃ for 10 minutes. anti-HLA-G samples were diluted with staining buffer to a 2-fold starting concentration of 80. Mu.g/ml. Antibodies were serially diluted 4-fold, 25 μl/well of antibody solution was added to the prepared cells, and incubated at 5 ℃ for 1 hour. Cells were washed twice with 200 μl/well staining buffer and centrifuged at 300g for 5 min. The cell pellet was then resuspended in 25 μl staining buffer. For detection, fluorescently labeled anti-species antibodies (donkey anti-human IgG (H+L) conjugated to PE), jackson Immuno Research # 709-116-149) were diluted 1:100 with staining buffer, and then 25 μl/well of detection antibody was added to the cell suspension. After incubation for 1 hour at 5 ℃, the cells were washed twice again with staining buffer, resuspended in 70 μl staining buffer, and measured on FACS Canto II. Bispecific anti-HLA-G/anti-CD 3 antibody (T cell bispecific (TCB) antibody) P1AF7977 (HLA-G-0090-VL-S32P/CD 3P 035); p1AF7978 (HLA-G-0090-VL-S32P/CD 3 Cl 22) and P1AF7979 (HLA-G-0090-VL-S32P/CD 3V 9) showed binding to JEG3 cells and HLAG transfected SKOV3 cells (see FIG. 8). EC50 values for FACS binding are listed in the table below.

Example 13

Inhibition of ILT2 and ILT4 binding by bispecific anti-HLA-G/anti-CD 3 antibodies (T cell bispecific (TCB) antibodies)

ELISA was set up by coating Fc-labeled ILT2 and ILT4 onto Maxisorp microtiter plates, respectively. After incubation and washing steps, the corresponding antibodies were added at a concentration of 100 nM. Soluble His-tagged monomers, dimers or trimers HLA-G were added to the wells. After incubation and washing steps, bound receptors were detected by anti-His-antibody-POD conjugates. The percent (%) inhibition (100% binding = 0% inhibition) was calculated as compared to the values obtained from wells containing ILT2/4+ HLA-G (monomer, dimer or trimer) without anti-HLA-G or ILT2/4 antibodies and is shown in the table below.

% binding inhibition (133 nM)	ILT2	ILT4
			P1AF7977(HLA-G-0090-VL-S32P/CD3 P035)	100	92
P1AF7978(HLA-G-0090-VL-S32P/CD3 Cl22)	100	91
			P1AF7979(HLA-G-0090-VL-S32P/CD3 V9)	100	95

Example 14

Bispecific anti-HLA-G/anti-CD 3 antibody (T cell bispecific (TCB) antibody) -mediated IFN gamma secretion by T cells

The ability of anti-HLA-G TCB to induce IFN gamma secretion from T cells was tested in the presence of HLA-G expressing tumor cells using recombinant HLA-G transfected SKOV3 cells (SKOV 3 HLA-G) and endogenous HLA-G expressing JEG3 cells. Ifnγ secretion was detected by Luminex technology. To measure ifnγ secretion in T cells after TCB treatment, PBMCs and SKOV3HLA-G cells or JEG3 cells were co-cultured with anti-HLA-G TCB. Briefly, PBMC were isolated from human peripheral blood by density gradient centrifugation using lymphocyte separation medium tubes (PAN#P04-60125). PBMCs were seeded to SKOV3HLA-G cells at a ratio of 10:1 to 96 well U-shaped bottom plates. The Co-cultures were then incubated with different concentrations of HLA-G-TCB and incubated at 37℃for 24 hours in an incubator containing 5% Co2, as shown in the figure (FIG. 9). The following day, supernatants were collected and ifnγ secretion was measured using the Milliplex MAP kit (Luminex technology) following the manufacturer's instructions. Bispecific anti-HLA-G/anti-CD 3 (T cell bispecific (TCB)) antibody P1AF7977 (HLA-G-0090-VL-S32P/CD 3P 035); p1AF7978 (HLA-G-0090-VL-S32P/CD 3 Cl 22) and P1AF7979 (HLA-G-0090-VL-S32P/CD 3V 9) induced IFN gamma secretion by T cells (FIG. 9). EC50 values are listed in the following table.

Example 15

Bispecific anti-HLA-G/anti-CD 3 antibodies (T cell bispecific (TCB) antibodies) induce T cell mediated cytotoxicity/tumor cell killing

The ability of anti-HLA-G TCB to induce T cell mediated cytotoxicity was tested on SKOV3 cells transfected with recombinant HLA-G (SKOV 3 HLA-G) and JEG3 cells expressing endogenous HLA-G in the presence of tumor cells expressing HLA-G. Cytotoxicity was detected by measuring caspase 8 in cells treated with HLA-G TCB. To measure caspase 8 activation following HLA-G/anti-CD 3 antibody (TCB) treatment, a co-culture of PBMC and SKOV 3HLA-G cells or JEG3 cells was incubated with anti-HLA-G TCB for 24 hours, and caspase 8 activation was then measured using the caspase 8Glo kit (Promega, #G8200). Briefly, PBMC were isolated from human peripheral blood by density gradient centrifugation using lymphocyte separation medium tubes (PAN#P04-60125). PBMC and SKOV 3HLA-G or JEG3 cells were seeded into black clear bottom 96-well plates at a ratio of 10:1 (100 μl per well). The co-cultures were then incubated with different concentrations of HLA-G-TCB and incubated at 37℃for 24 hours or 48 hours in an incubator containing 5% CO2, as shown in the figure (FIG. 10). The next day, 100 μl of caspase 8Glo substrate was added to each well and placed on a shaker for 1 hour at room temperature. Luminescence was measured on a BioTek Synergy 2 instrument. The Relative Luminescence Units (RLU) corresponding to caspase 8 activation/cytotoxicity are plotted in the graph (fig. 10). Bispecific anti-HLA-G/anti-CD 3 (T cell bispecific (TCB)) antibody P1AF7977 (HLA-G-0090-VL-S32P/CD 3P 035); p1AF7978 (HLA-G-0090-VL-S32P/CD 3 Cl 22) and P1AF7979 (HLA-G-0090-VL-S32P/CD 3V 9) induced T cell-mediated cytotoxicity/tumor cell killing. EC50 values for tumor cell killing are listed in the table below.

Example 16

In vivo anti-tumor efficacy of bispecific anti-HLA-G/anti-CD 3 (T cell bispecific (TCB)) antibodies in humanized NSG mice (SKOV 3 HLA-G) bearing recombinant HLA-G transfected SKOV3 human ovarian cancer

Humanized NSG (NOD/scid/IL-2 rγnull, humanized by cd34+ cord blood cells, purchased from us Jackson Laboratories) mice (n=15) were subcutaneously injected 5×10 ⁶ The total volume of each SKOV3HLA-G cell was 100. Mu.l. The average tumor volume reaches 200mm ³ After that, mice were randomized and weekly using bispecific anti-HLA-G/anti-CD 3 (T cell bispecific (TCB)) antibodies (P1 AF7977 (HLA-G-0090-VL)-S32P/CD 3P 035)) (5 mg/kg). As a control, one group of mice received weekly intravenous histidine buffer (vehicle). Tumor volumes were measured twice weekly until the study was terminated. The experimental results are shown in FIG. 11. The results show tumor volume data (median and quarter bit distance (IQR)) measured by caliper in both study groups. The anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibody P1AF7977 exhibited strong tumor growth inhibition/tumor regression in SKOV3-HLA-G tumor models.

Example 17

Dose-remission studies of bispecific anti-HLA-G/anti-CD 3 (T cell bispecific (TCB)) antibodies in humanized NSG mice bearing human breast cancer PDX tumor (BC 004)

To humanized NSG (NOD/scid/IL-2 Rγnull, 1X 10 per mouse by intravenous injection) ⁵ Humanization of individual cd34+ umbilical cord blood cells) mice were injected 2 x 10 intraprostatic fat ⁶ The total volume of BC004 breast cancer cells was 50. Mu.L PBS. In the average tumor volume of about 200mm ³ Afterwards, mice were randomized (number of animals per group n=15) and treated weekly with three different doses (5 mg/kg,2.5mg/kg,0.5 mg/kg) of bispecific anti-HLA-G/anti-CD 3 (T cell bispecific (TCB)) antibodies (P1 AF7977 (HLA-G-0090-VL-S32P/CD 3P 035)). As a control, one group of mice received weekly intravenous histidine buffer (vehicle). Tumor volumes were measured twice weekly via caliper measurements. The experimental results shown in fig. 12 demonstrate tumor volume data (median and quarter bit distance (IQR)). All three doses of anti-HLA-G/anti-CD 3T cell bispecific (TCB) antibodies showed strong tumor growth inhibition/tumor regression in the BC004 tumor model. The highest dose (5 mg/kg) showed slightly higher efficacy compared to the 2.5mg/kg and 0.5mg/kg treatment groups.

Example 18

Testing for T cell activation induced by bispecific anti-HLA-G/anti-CD 3 antibodies (T cell bispecific (TCB) antibodies) in the presence of HLA-G expressing tumor cells on recombinant HLA-G transfected SKOV3 cells (SKOV 3 HLA-G)

The ability of anti-HLA-G/anti-CD 3 TCB to activate T cells in the presence of HLA-G expressing tumor cells was tested on recombinant HLA-G transfected SKOV3 cells (SKOV 3 HLA-G). Activation of T cells was assessed by FACS analysis for cell surface activation marker CD25 and early activation marker CD69 on T cells. Briefly, peripheral Blood Mononuclear Cells (PBMC) were isolated from human peripheral blood by density gradient centrifugation using a lymphocyte separation medium tube (PAN#P04-60125). PBMC and SKOV3 HLA-G cells were seeded at a ratio of 10:1 into 96-well U-shaped bottom plates. The Co-cultures were then incubated with anti-HLA-G/anti-CD 3 (T cell bispecific (TCB)) antibodies (P1 AF7977 (HLA-G-0090-VL-S32P/CD 3P 035)) (0.01 nM) and incubated at 37℃for 24 hours in an incubator containing 5% Co 2. The next day, the expression of CD25 and CD69 was measured by flow cytometry.

In flow cytometry, cells were stained with PerCP-Cy5.5 mouse anti-human CD8 (BD Pharmingen # 565310), PE-Cy7 mouse anti-human CD4 (biologed # 317414), FITC mouse anti-human CD25 (bioleged # 356106), and APC mouse anti-human CD69 (BD Pharmingen # 555533) at 4 ℃. Briefly, antibodies were diluted to 2-fold concentration and 25 μl of antibody dilution was added to each well containing 25 μl of pre-washed co-culture. Cells were stained at 4℃for 30 min, washed twice with 200. Mu.l/Kong Ranse buffer and centrifuged at 300g for 5 min. The cell pellet was resuspended in 200. Mu.l staining buffer and stained with DAPI to distinguish between dead/surviving cells at a final concentration of 2. Mu.g/ml. The samples were then measured using a BD LSR flow cytometer. Data analysis was performed using FlowJo v.10.1 software. FIG. 14 shows the induction of T cell activation by bispecific anti-HLA-G/anti-CD 3 antibody P1AF7977 (HLA-G-0090-VL-S32P/CD 3P 035) in the presence of SKOV3HLAG cells.

Sequence listing

<110> Haofu-Rogowski Co., ltd

<120> anti-HLA-G antibody and use thereof

<130> case number P36618-WO

<150> EP20214951.4

<151> 2020-12-17

<150> EP21203272.6

<151> 2021-10-18

<160> 96

<170> patent In version 3.5

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Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 11

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-N31L

<400> 11

Lys Ser Ser Gln Ser Val Leu Leu Ser Ser Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 12

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-N31L

<400> 12

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Leu Ser

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 13

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-N31Q

<400> 13

Lys Ser Ser Gln Ser Val Leu Gln Ser Ser Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 14

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-N31Q

<400> 14

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Gln Ser

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 15

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-N31S

<400> 15

Lys Ser Ser Gln Ser Val Leu Ser Ser Ser Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 16

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-N31S

<400> 16

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Ser Ser

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 17

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-N31T

<400> 17

Lys Ser Ser Gln Ser Val Leu Thr Ser Ser Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 18

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-N31T

<400> 18

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Thr Ser

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 19

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-N31Y

<400> 19

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 20

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-N31Y

<400> 20

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 21

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-N31Y-N38Y

<400> 21

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 22

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-N31Y-N38Y

<400> 22

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 23

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-S32P

<400> 23

Lys Ser Ser Gln Ser Val Leu Asn Pro Ser Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 24

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-S32P

<400> 24

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Pro

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 25

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-S33A

<400> 25

Lys Ser Ser Gln Ser Val Leu Asn Ser Ala Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 26

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-S33A

<400> 26

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser

20 25 30

Ala Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 27

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-S33D

<400> 27

Lys Ser Ser Gln Ser Val Leu Asn Ser Asp Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 28

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-S33D

<400> 28

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser

20 25 30

Asp Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 29

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, HLA-G-0090-VL-S33P

<400> 29

Lys Ser Ser Gln Ser Val Leu Asn Ser Pro Asn Asn Lys Asn Asn Leu

1 5 10 15

Ala

<210> 30

<211> 113

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, HLA-G-0090-VL-S33P

<400> 30

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser

20 25 30

Pro Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 31

<211> 314

<212> PRT

<213> Chile person

<400> 31

Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly

1 5 10 15

Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg

35 40 45

Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr

50 55 60

Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr

65 70 75 80

Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln

85 90 95

Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly

100 105 110

Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu

115 120 125

Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys

130 135 140

Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys

165 170 175

Glu Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His

180 185 190

Pro Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe

195 200 205

Tyr Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln

210 215 220

Thr Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg

245 250 255

Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu

260 265 270

Arg Trp Lys Gln Ser Ser Leu Pro Thr Ile Pro Ile Met Gly Ile Val

275 280 285

Ala Gly Leu Val Val Leu Ala Ala Val Val Thr Gly Ala Ala Val Ala

290 295 300

Ala Val Leu Trp Arg Lys Lys Ser Ser Asp

305 310

<210> 32

<211> 274

<212> PRT

<213> Chile person

<400> 32

Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly

1 5 10 15

Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg

35 40 45

Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr

50 55 60

Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr

65 70 75 80

Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln

85 90 95

Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly

100 105 110

Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu

115 120 125

Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys

130 135 140

Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys

165 170 175

Glu Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His

180 185 190

Pro Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe

195 200 205

Tyr Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln

210 215 220

Thr Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg

245 250 255

Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu

260 265 270

Arg Trp

<210> 33

<211> 99

<212> PRT

<213> Chile person

<400> 33

Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu

1 5 10 15

Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro

20 25 30

Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys

35 40 45

Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu

50 55 60

Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys

65 70 75 80

Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp

85 90 95

Arg Asp Met

<210> 34

<211> 275

<212> PRT

<213> Artificial work

<220>

<223> modified human HLA-G (wherein HLA-G specific amino acid

Has been replaced by HLA-A consensus amino acid (= transplantation HLA-G)

ECD

<400> 34

Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly

1 5 10 15

Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Met Glu Pro Arg

35 40 45

Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Glu Glu Thr

50 55 60

Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Val Asn Leu Gly Thr

65 70 75 80

Leu Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln

85 90 95

Trp Met Ile Gly Cys Asp Val Gly Ser Asp Gly Arg Leu Leu Arg Gly

100 105 110

Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu

115 120 125

Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys

130 135 140

Arg Lys Cys Glu Ala Ala His Val Ala Glu Gln Arg Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys

165 170 175

Glu Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His

180 185 190

Pro Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe

195 200 205

Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln

210 215 220

Thr Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg

245 250 255

Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu

260 265 270

Arg Trp Lys

275

<210> 35

<211> 341

<212> PRT

<213> Chile person

<400> 35

Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly

1 5 10 15

Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg

35 40 45

Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly Glu Thr

50 55 60

Arg Lys Val Lys Ala His Ser Gln Thr His Arg Val Asp Leu Gly Thr

65 70 75 80

Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Val Gln

85 90 95

Arg Met Tyr Gly Cys Asp Val Gly Ser Asp Trp Arg Phe Leu Arg Gly

100 105 110

Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu

115 120 125

Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr Lys

130 135 140

His Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys

165 170 175

Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His

180 185 190

Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe

195 200 205

Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln

210 215 220

Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg

245 250 255

Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu

260 265 270

Arg Trp Glu Pro Ser Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Ile

275 280 285

Ala Gly Leu Val Leu Phe Gly Ala Val Ile Thr Gly Ala Val Val Ala

290 295 300

Ala Val Met Trp Arg Arg Lys Ser Ser Asp Arg Lys Gly Gly Ser Tyr

305 310 315 320

Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu

325 330 335

Thr Ala Cys Lys Val

340

<210> 36

<211> 275

<212> PRT

<213> Chile person

<400> 36

Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly

1 5 10 15

Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro Arg

35 40 45

Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly Glu Thr

50 55 60

Arg Lys Val Lys Ala His Ser Gln Thr His Arg Val Asp Leu Gly Thr

65 70 75 80

Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Val Gln

85 90 95

Arg Met Tyr Gly Cys Asp Val Gly Ser Asp Trp Arg Phe Leu Arg Gly

100 105 110

Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu

115 120 125

Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr Lys

130 135 140

His Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys

165 170 175

Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His

180 185 190

Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Ser Phe

195 200 205

Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln

210 215 220

Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg

245 250 255

Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu

260 265 270

Arg Trp Glu

275

<210> 37

<211> 275

<212> PRT

<213> mice

<400> 37

Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly

1 5 10 15

Leu Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ala Asp Asn Pro Arg Phe Glu Pro Arg

35 40 45

Ala Pro Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Gln Thr

50 55 60

Gln Arg Ala Lys Ser Asp Glu Gln Trp Phe Arg Val Ser Leu Arg Thr

65 70 75 80

Ala Gln Arg Cys Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Phe Gln

85 90 95

Arg Met Phe Gly Cys Asp Val Gly Ser Asp Trp Arg Leu Leu Arg Gly

100 105 110

Tyr Gln Gln Phe Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu

115 120 125

Asp Leu Lys Thr Trp Thr Ala Ala Asp Thr Ala Ala Leu Ile Thr Arg

130 135 140

Arg Lys Trp Glu Gln Ala Gly Asp Ala Glu Tyr Tyr Arg Ala Tyr Leu

145 150 155 160

Glu Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Leu Gly Asn

165 170 175

Glu Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr Tyr His

180 185 190

Pro Arg Ser Gln Val Asp Val Thr Leu Arg Cys Trp Ala Leu Gly Phe

195 200 205

Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu

210 215 220

Thr Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Leu Gly Lys Glu Gln Asn

245 250 255

Tyr Thr Cys His Val His His Lys Gly Leu Pro Glu Pro Leu Thr Leu

260 265 270

Arg Trp Lys

275

<210> 38

<211> 274

<212> PRT

<213> rat

<400> 38

Gly Ser His Ser Leu Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly

1 5 10 15

Leu Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Glu

20 25 30

Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Met Glu Pro Arg

35 40 45

Ala Arg Trp Met Glu Arg Glu Gly Pro Glu Tyr Trp Glu Gln Gln Thr

50 55 60

Arg Ile Ala Lys Glu Trp Glu Gln Ile Tyr Arg Val Asp Leu Arg Thr

65 70 75 80

Leu Arg Gly Cys Tyr Asn Gln Ser Glu Gly Gly Ser His Thr Ile Gln

85 90 95

Glu Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Ser Leu Leu Arg Gly

100 105 110

Tyr Arg Gln Asp Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu

115 120 125

Asp Leu Lys Thr Trp Thr Ala Ala Asp Phe Ala Ala Gln Ile Thr Arg

130 135 140

Asn Lys Trp Glu Arg Ala Arg Tyr Ala Glu Arg Leu Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu Ser Arg Tyr Leu Glu Leu Gly Lys

165 170 175

Glu Thr Leu Leu Arg Ser Asp Pro Pro Glu Ala His Val Thr Leu His

180 185 190

Pro Arg Pro Glu Gly Asp Val Thr Leu Arg Cys Trp Ala Leu Gly Phe

195 200 205

Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu

210 215 220

Thr Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu Gly Lys Glu Gln Asn

245 250 255

Tyr Thr Cys Arg Val Glu His Glu Gly Leu Pro Lys Pro Leu Ser Gln

260 265 270

Arg Trp

<210> 39

<211> 440

<212> PRT

<213> Chile person

<400> 39

Arg Ile Ile Pro Arg His Leu Gln Leu Gly Cys Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys Ile Gln

20 25 30

Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn

35 40 45

Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu

50 55 60

Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe

65 70 75 80

Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro

85 90 95

Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser

100 105 110

Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg

145 150 155 160

Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe

165 170 175

Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg Ala

180 185 190

Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr Arg

195 200 205

Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr Leu

210 215 220

Arg Gly Cys Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln Trp

225 230 235 240

Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr

245 250 255

Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp

260 265 270

Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg

275 280 285

Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu

290 295 300

Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu

305 310 315 320

Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His Pro

325 330 335

Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr

340 345 350

Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr

355 360 365

Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe

370 375 380

Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr

385 390 395 400

Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg

405 410 415

Trp Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp

420 425 430

His Glu His His His His His His

435 440

<210> 40

<211> 441

<212> PRT

<213> Artificial work

<220>

<223> exemplary modified human HLA-Gbeta 2M MHC I complexes (wherein

HLA-G specific amino acids have been modified by HLA-A

Common amino acid substitution (=transplantation HLA-G)

<400> 40

Arg Ile Ile Pro Arg His Leu Gln Leu Gly Cys Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys Ile Gln

20 25 30

Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn

35 40 45

Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu

50 55 60

Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe

65 70 75 80

Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro

85 90 95

Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser

100 105 110

Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg

145 150 155 160

Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe

165 170 175

Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Met Glu Pro Arg Ala

180 185 190

Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Glu Glu Thr Arg

195 200 205

Asn Thr Lys Ala His Ala Gln Thr Asp Arg Val Asn Leu Gly Thr Leu

210 215 220

Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln Trp

225 230 235 240

Met Ile Gly Cys Asp Val Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr

245 250 255

Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp

260 265 270

Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg

275 280 285

Lys Cys Glu Ala Ala His Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu

290 295 300

Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys Glu

305 310 315 320

Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His Pro

325 330 335

Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr

340 345 350

Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr

355 360 365

Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe

370 375 380

Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr

385 390 395 400

Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Thr Leu Arg

405 410 415

Trp Lys Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu

420 425 430

Trp His Glu His His His His His His

435 440

<210> 41

<211> 441

<212> PRT

<213> mice

<400> 41

Thr Tyr Gln Arg Thr Arg Ala Leu Val Gly Cys Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln

20 25 30

Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn

35 40 45

Cys Tyr Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu

50 55 60

Lys Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser Phe

65 70 75 80

Ser Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro

85 90 95

Thr Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys His Asp Ser Met Ala

100 105 110

Glu Pro Lys Thr Val Tyr Trp Asp Arg Asp Met Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly Leu

145 150 155 160

Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln Phe

165 170 175

Val Arg Phe Asp Ser Asp Ala Asp Asn Pro Arg Phe Glu Pro Arg Ala

180 185 190

Pro Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Gln Thr Gln

195 200 205

Arg Ala Lys Ser Asp Glu Gln Trp Phe Arg Val Ser Leu Arg Thr Ala

210 215 220

Gln Arg Cys Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Phe Gln Arg

225 230 235 240

Met Phe Gly Cys Asp Val Gly Ser Asp Trp Arg Leu Leu Arg Gly Tyr

245 250 255

Gln Gln Phe Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu Asp

260 265 270

Leu Lys Thr Trp Thr Ala Ala Asp Thr Ala Ala Leu Ile Thr Arg Arg

275 280 285

Lys Trp Glu Gln Ala Gly Asp Ala Glu Tyr Tyr Arg Ala Tyr Leu Glu

290 295 300

Gly Glu Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Leu Gly Asn Glu

305 310 315 320

Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr Tyr His Pro

325 330 335

Arg Ser Gln Val Asp Val Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr

340 345 350

Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu Thr

355 360 365

Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe

370 375 380

Gln Lys Trp Ala Ala Val Val Val Pro Leu Gly Lys Glu Gln Asn Tyr

385 390 395 400

Thr Cys His Val His His Lys Gly Leu Pro Glu Pro Leu Thr Leu Arg

405 410 415

Trp Lys Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu

420 425 430

Trp His Glu His His His His His His

435 440

<210> 42

<211> 441

<212> PRT

<213> Artificial work

<220>

<223> exemplary human HLA-G/mouse H2 Kdbeta 2M MHC I complex, wherein

Specific positions for human HLA-G were transplanted into mice

On H2Kd frame

<400> 42

Thr Tyr Gln Arg Thr Arg Ala Leu Val Gly Cys Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln

20 25 30

Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn

35 40 45

Cys Tyr Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu

50 55 60

Lys Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser Phe

65 70 75 80

Ser Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro

85 90 95

Thr Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys His Asp Ser Met Ala

100 105 110

Glu Pro Lys Thr Val Tyr Trp Asp Arg Asp Met Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly Leu

145 150 155 160

Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln Phe

165 170 175

Val Arg Phe Asp Ser Asp Ser Ala Ser Pro Arg Phe Glu Pro Arg Ala

180 185 190

Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Gln Thr Gln

195 200 205

Arg Ala Lys Ser Asp Glu Gln Trp Phe Arg Met Ser Leu Gln Thr Ala

210 215 220

Arg Gly Cys Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Phe Gln Arg

225 230 235 240

Met Phe Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr

245 250 255

Gln Gln Phe Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu Asp

260 265 270

Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Leu Ile Thr Lys Arg

275 280 285

Lys Trp Glu Ala Ala Asn Asp Ala Glu Tyr Tyr Arg Ala Tyr Leu Glu

290 295 300

Gly Glu Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu

305 310 315 320

Met Leu Gln Arg Thr Asp Ser Pro Lys Ala His Val Thr His His Pro

325 330 335

Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr

340 345 350

Pro Ala Glu Ile Ile Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu Thr

355 360 365

Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe

370 375 380

Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Lys Glu Gln Asn Tyr

385 390 395 400

Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg

405 410 415

Trp Lys Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu

420 425 430

Trp His Glu His His His His His His

435 440

<210> 43

<211> 440

<212> PRT

<213> rat

<400> 43

Ala Gln Phe Ser Ala Ser Ala Ser Arg Gly Cys Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln

20 25 30

Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Phe Leu Asn

35 40 45

Cys Tyr Val Ser Gln Phe His Pro Pro Gln Ile Glu Ile Glu Leu Leu

50 55 60

Lys Asn Gly Lys Lys Ile Pro Asn Ile Glu Met Ser Asp Leu Ser Phe

65 70 75 80

Ser Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro

85 90 95

Thr Glu Thr Asp Val Tyr Ala Cys Arg Val Lys His Val Thr Leu Lys

100 105 110

Glu Pro Lys Thr Val Thr Trp Asp Arg Asp Met Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Ser His Ser Leu Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly Leu

145 150 155 160

Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Glu Phe

165 170 175

Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Met Glu Pro Arg Ala

180 185 190

Arg Trp Met Glu Arg Glu Gly Pro Glu Tyr Trp Glu Gln Gln Thr Arg

195 200 205

Ile Ala Lys Glu Trp Glu Gln Ile Tyr Arg Val Asp Leu Arg Thr Leu

210 215 220

Arg Gly Cys Tyr Asn Gln Ser Glu Gly Gly Ser His Thr Ile Gln Glu

225 230 235 240

Met Tyr Gly Cys Asp Val Gly Ser Asp Gly Ser Leu Leu Arg Gly Tyr

245 250 255

Arg Gln Asp Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu Asp

260 265 270

Leu Lys Thr Trp Thr Ala Ala Asp Phe Ala Ala Gln Ile Thr Arg Asn

275 280 285

Lys Trp Glu Arg Ala Arg Tyr Ala Glu Arg Leu Arg Ala Tyr Leu Glu

290 295 300

Gly Thr Cys Val Glu Trp Leu Ser Arg Tyr Leu Glu Leu Gly Lys Glu

305 310 315 320

Thr Leu Leu Arg Ser Asp Pro Pro Glu Ala His Val Thr Leu His Pro

325 330 335

Arg Pro Glu Gly Asp Val Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr

340 345 350

Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu Thr

355 360 365

Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe

370 375 380

Gln Lys Trp Ala Ser Val Val Val Pro Leu Gly Lys Glu Gln Asn Tyr

385 390 395 400

Thr Cys Arg Val Glu His Glu Gly Leu Pro Lys Pro Leu Ser Gln Arg

405 410 415

Trp Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp

420 425 430

His Glu His His His His His His

435 440

<210> 44

<211> 440

<212> PRT

<213> Artificial work

<220>

<223> exemplary human HLA-G/rat RT1A beta 2M MHC I complex, wherein

Specific positions for human HLA-G were transplanted into rats

On the RT1A frame

<400> 44

Ala Gln Phe Ser Ala Ser Ala Ser Arg Gly Cys Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln

20 25 30

Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn Phe Leu Asn

35 40 45

Cys Tyr Val Ser Gln Phe His Pro Pro Gln Ile Glu Ile Glu Leu Leu

50 55 60

Lys Asn Gly Lys Lys Ile Pro Asn Ile Glu Met Ser Asp Leu Ser Phe

65 70 75 80

Ser Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro

85 90 95

Thr Glu Thr Asp Val Tyr Ala Cys Arg Val Lys His Val Thr Leu Lys

100 105 110

Glu Pro Lys Thr Val Thr Trp Asp Arg Asp Met Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Ser His Ser Leu Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly Leu

145 150 155 160

Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Glu Phe

165 170 175

Val Arg Phe Asp Ser Asp Ser Ala Ser Pro Arg Met Glu Pro Arg Ala

180 185 190

Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Gln Gln Thr Arg

195 200 205

Ile Ala Lys Glu Trp Glu Gln Ile Tyr Arg Met Asp Leu Gln Thr Leu

210 215 220

Arg Gly Cys Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Ile Gln Glu

225 230 235 240

Met Tyr Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr

245 250 255

Arg Gln Asp Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu Asp

260 265 270

Leu Arg Ser Trp Thr Ala Ala Asp Phe Ala Ala Gln Ile Thr Lys Arg

275 280 285

Lys Trp Glu Ala Ala Asn Tyr Ala Glu Arg Leu Arg Ala Tyr Leu Glu

290 295 300

Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu

305 310 315 320

Met Leu Gln Arg Ala Asp Pro Pro Glu Ala His Val Thr His His Pro

325 330 335

Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr

340 345 350

Pro Ala Glu Ile Ile Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu Thr

355 360 365

Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe

370 375 380

Gln Lys Trp Ala Ser Val Val Val Pro Ser Gly Lys Glu Gln Asn Tyr

385 390 395 400

Thr Cys Arg Val Gln His Glu Gly Leu Pro Lys Pro Leu Met Leu Arg

405 410 415

Trp Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp

420 425 430

His Glu His His His His His His

435 440

<210> 45

<211> 33

<212> PRT

<213> Artificial work

<220>

<223> linker and his-tag

<400> 45

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Leu Asn Asp

1 5 10 15

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu His His His His His

20 25 30

His

<210> 46

<211> 9

<212> PRT

<213> Artificial work

<220>

<223> peptide

<400> 46

Val Leu Asp Phe Ala Pro Pro Gly Ala

1 5

<210> 47

<211> 107

<212> PRT

<213> Chile person

<400> 47

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 48

<211> 105

<212> PRT

<213> Chile person

<400> 48

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 49

<211> 328

<212> PRT

<213> Chile person

<400> 49

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 50

<211> 328

<212> PRT

<213> Chile person

<400> 50

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 51

<211> 325

<212> PRT

<213> Chile person

<400> 51

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu

325

<210> 52

<211> 6

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H1, P035-093 (abbreviated as P035)

<400> 52

Ser Tyr Ala Met Asn Trp

1 5

<210> 53

<211> 18

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H2, P035-093

<400> 53

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys

<210> 54

<211> 14

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H3, P035-093

<400> 54

Ala Ser Asn Phe Pro Ala Ser Tyr Val Ser Tyr Phe Ala Tyr

1 5 10

<210> 55

<211> 14

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, P035-093

<400> 55

Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 56

<211> 7

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L2, P035-093

<400> 56

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 57

<211> 9

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L3, P035-093

<400> 57

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 58

<211> 125

<212> PRT

<213> Artificial work

<220>

<223> heavy chain variable domain VH, P035-093

<400> 58

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg Ala Ser Asn Phe Pro Ala Ser Tyr Val Ser Tyr Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 59

<211> 109

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, P035-093

<400> 59

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 60

<211> 5

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H1, clone 22 (abbreviated as Cl 22)

<400> 60

Ser Tyr Ala Met Asn

1 5

<210> 61

<211> 18

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H2, clone 22

<400> 61

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys

<210> 62

<211> 14

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H3, clone 22

<400> 62

His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr Gly Tyr

1 5 10

<210> 63

<211> 14

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L1, clone 22

<400> 63

Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 64

<211> 7

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L2, clone 22

<400> 64

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 65

<211> 9

<212> PRT

<213> Artificial work

<220>

<223> light chain CDR-L3, clone 22

<400> 65

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 66

<211> 125

<212> PRT

<213> Artificial work

<220>

<223> heavy chain variable domain VH, clone 22

<400> 66

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr

100 105 110

Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 67

<211> 109

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, clone 22

<400> 67

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 68

<211> 5

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H1, V9

<400> 68

Gly Tyr Thr Met Asn

1 5

<210> 69

<211> 17

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H2, V9

<400> 69

Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 70

<211> 13

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-H3, V9

<400> 70

Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

1 5 10

<210> 71

<211> 11

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-L1, V9

<400> 71

Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn

1 5 10

<210> 72

<211> 7

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-L2, V9

<400> 72

Tyr Thr Ser Arg Leu Glu Ser

1 5

<210> 73

<211> 9

<212> PRT

<213> Artificial work

<220>

<223> heavy chain CDR-L3, V9

<400> 73

Gln Gln Gly Asn Thr Leu Pro Trp Thr

1 5

<210> 74

<211> 122

<212> PRT

<213> Artificial work

<220>

<223> heavy chain variable domain VH, V9

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 75

<211> 107

<212> PRT

<213> Artificial work

<220>

<223> light chain variable Domain VL, V9

<400> 75

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 76

<211> 232

<212> PRT

<213> Artificial work

<220>

<223> light chain 1P 1AF7977

<400> 76

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg Ala Ser Asn Phe Pro Ala Ser Tyr Val Ser Tyr Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 77

<211> 220

<212> PRT

<213> Artificial work

<220>

<223> light chain 2P 1AF7977

<400> 77

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Pro

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 78

<211> 449

<212> PRT

<213> Artificial work

<220>

<223> heavy chain 1P 1AF7977

<400> 78

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Arg Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 79

<211> 674

<212> PRT

<213> Artificial work

<220>

<223> heavy chain 2P 1AF7977

<400> 79

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Arg Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 80

<211> 232

<212> PRT

<213> Artificial work

<220>

<223> light chain 1P 1AF7978

<400> 80

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr

100 105 110

Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 81

<211> 220

<212> PRT

<213> Artificial work

<220>

<223> light chain 2P 1AF7978

<400> 81

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Pro

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 82

<211> 449

<212> PRT

<213> Artificial work

<220>

<223> heavy chain 1P 1AF7978

<400> 82

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Arg Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 83

<211> 674

<212> PRT

<213> Artificial work

<220>

<223> heavy chain 2P 1AF7978

<400> 83

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Arg Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 84

<211> 229

<212> PRT

<213> Artificial work

<220>

<223> light chain 1P 1AF7979

<400> 84

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro

115 120 125

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

130 135 140

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

145 150 155 160

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

165 170 175

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

180 185 190

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

195 200 205

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

210 215 220

Asn Arg Gly Glu Cys

225

<210> 85

<211> 220

<212> PRT

<213> Artificial work

<220>

<223> light chain 2P 1AF7979

<400> 85

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Pro

20 25 30

Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 86

<211> 449

<212> PRT

<213> Artificial work

<220>

<223> heavy chain 1P 1AF7979

<400> 86

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Arg Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 87

<211> 672

<212> PRT

<213> Artificial work

<220>

<223> heavy chain 2P 1AF7979

<400> 87

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Arg Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Asp Ile Gln Met Thr

225 230 235 240

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

245 250 255

Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln

260 265 270

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg

275 280 285

Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

290 295 300

Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

305 310 315 320

Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly

325 330 335

Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

435 440 445

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

450 455 460

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

465 470 475 480

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

485 490 495

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

500 505 510

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

515 520 525

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

530 535 540

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

545 550 555 560

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

565 570 575

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

580 585 590

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

595 600 605

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

610 615 620

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

625 630 635 640

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

645 650 655

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 88

<211> 207

<212> PRT

<213> Chile person

<400> 88

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met

115 120 125

Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu

130 135 140

Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys

145 150 155 160

Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn

165 170 175

Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg

180 185 190

Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 89

<211> 198

<212> PRT

<213> macaque

<400> 89

Met Gln Ser Gly Thr Arg Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Ile Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr

20 25 30

Gln Thr Pro Tyr Gln Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Ser Gln His Leu Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys

50 55 60

Asn Lys Glu Asp Ser Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu

65 70 75 80

Met Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro

85 90 95

Glu Asp Ala Ser His His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn

100 105 110

Cys Met Glu Met Asp Val Met Ala Val Ala Thr Ile Val Ile Val Asp

115 120 125

Ile Cys Ile Thr Leu Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys

130 135 140

Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly

145 150 155 160

Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn

165 170 175

Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly

180 185 190

Leu Asn Gln Arg Arg Ile

195

<210> 90

<211> 360

<212> PRT

<213> Artificial work

<220>

<223> human CD3 epsilon handle-Fc (pestle) -Avi

<400> 90

Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro

20 25 30

Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp

35 40 45

Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys

50 55 60

Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg

65 70 75 80

Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg

85 90 95

Val Ser Glu Asn Cys Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser

100 105 110

Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

115 120 125

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

130 135 140

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

145 150 155 160

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

165 170 175

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

180 185 190

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

195 200 205

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

210 215 220

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

225 230 235 240

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

245 250 255

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

260 265 270

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

275 280 285

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

290 295 300

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

305 310 315 320

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

325 330 335

Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu

340 345 350

Ala Gln Lys Ile Glu Trp His Glu

355 360

<210> 91

<211> 325

<212> PRT

<213> Artificial work

<220>

<223> human CD3 delta handle-Fc (mortar) -Avi

<400> 91

Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys

1 5 10 15

Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser

20 25 30

Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly

35 40 45

Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr

50 55 60

Val Gln Val His Tyr Arg Met Cys Arg Ser Glu Gln Leu Tyr Phe Gln

65 70 75 80

Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

85 90 95

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

100 105 110

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

115 120 125

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

130 135 140

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

145 150 155 160

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

165 170 175

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

180 185 190

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

195 200 205

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

210 215 220

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

225 230 235 240

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

245 250 255

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

260 265 270

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

275 280 285

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

290 295 300

Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys

305 310 315 320

Ile Glu Trp His Glu

325

<210> 92

<211> 125

<212> PRT

<213> Artificial work

<220>

<223> CD3orig VH

<400> 92

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 93

<211> 109

<212> PRT

<213> Artificial work

<220>

<223> CD3orig VL

<400> 93

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 94

<211> 453

<212> PRT

<213> Artificial work

<220>

<223> CD3orig IgG HC

<400> 94

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro

450

<210> 95

<211> 453

<212> PRT

<213> Artificial work

<220>

<223> P035 IgG HC

<400> 95

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr

100 105 110

Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro

450

<210> 96

<211> 216

<212> PRT

<213> Artificial work

<220>

<223> CD3orig / P035 IgG LC

<400> 96

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. An antibody that binds to human HLA-G comprising

A) (a) 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 (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, or

B) (a) a VH domain comprising (i) 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 (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

2. The antibody of claim 1, wherein the antibody

A) Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 7, and a VL domain comprising the amino acid sequence of SEQ ID No. 24; or (b)

B) Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 7, and a VL domain comprising the amino acid sequence of SEQ ID No. 26.

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

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

5. The antibody of any one of claims 1 to 4, wherein the antibody

a) Does not cross-react with a modified human HLA-G beta 2M MHC I complex, wherein HLA-G specific amino acids have been replaced by HLA-A consensus amino acids, said complex comprising SEQ ID NO:40; and/or

6. The antibody of any one of claims 1 to 5, wherein the antibody

a) Inhibiting ILT2 binding to JEG3 cells (ATCC accession HTB 36) (HLA-G expressed thereon); or (b)

b) Binds to JEG3 cells (ATCC No. HTB36) (HLA-G expressed thereon) and inhibits ILT2 binding to JEG-3 cells (HLA-G expressed on ATCC No. HTB36).

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

8. The antibody of any one of claims 1 to 4, wherein the antibody is a bispecific antibody that binds to human HLA-G and to human CD 3.

9. The antibody of claim 5, wherein the antibody is a bispecific antibody that binds to human HLA-G and to human CD3, the bispecific antibody comprising a first antigen binding moiety that binds to human HLA-G and a second antigen binding moiety that binds to human CD3, wherein the first antigen binding moiety that binds to human HLA-G comprises

B) (a) 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 (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:25, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6;

and wherein said second antigen binding portion that binds to a T cell activating antigen binds to human CD3, comprising

C) (a) a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID No. 52, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID No. 53, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID No. 54; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:55, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:56, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:57, or

D) (a) a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID No. 60, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID No. 61, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID No. 62; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:63, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:64, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:65, or

E) (a) a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID No. 68, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID No. 69, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID No. 70; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:71, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:72, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73.

10. The bispecific antibody according to claim 9,

wherein the first antigen binding portion

B) Comprising: a VH domain comprising the amino acid sequence of SEQ ID NO. 7, and a VL domain comprising the amino acid sequence of SEQ ID NO. 26,

And wherein the second antigen binding portion

C) Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 58, and a VL domain comprising the amino acid sequence of SEQ ID No. 59; or (b)

D) Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 66, and a VL domain comprising the amino acid sequence of SEQ ID No. 67; or (b)

E) Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 74, and a VL domain comprising the amino acid sequence of SEQ ID No. 75.

11. The bispecific antibody according to claim 10,

wherein the first antigen binding portion

Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 7, and a VL domain comprising the amino acid sequence of SEQ ID No. 24;

and wherein the second antigen binding portion

Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 58, and a VL domain comprising the amino acid sequence of SEQ ID No. 59.

12. The bispecific antibody according to claim 10,

wherein the first antigen binding portion

And wherein the second antigen binding portion

Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 66, and a VL domain comprising the amino acid sequence of SEQ ID No. 67.

13. The bispecific antibody according to claim 10,

wherein the first antigen binding portion

and wherein the second antigen binding portion

Comprising: a VH domain comprising the amino acid sequence of SEQ ID No. 74, and a VL domain comprising the amino acid sequence of SEQ ID No. 75.

14. The bispecific antibody of any one of claims 7 to 13, wherein the bispecific antibody exhibits a binding activity of

a) Inhibition of ILT2 and/or ILT4 binding to HLA-G; and/or

b) Antibody-mediated ifnγ secretion by T cells on SKOV3 cells transfected with recombinant HLA-G (SKOV 3 HLA-G) and/or on JEG3 cells expressing endogenous HLA-G, wherein the ifnγ secretion is detected; and/or

c) T cell mediated cytotoxicity/tumor cell killing on SKOV3 cells transfected with recombinant HLA-G (SKOV 3 HLA-G) and/or JEG3 cells expressing endogenous HLA-G, wherein the cytotoxicity is detected by measuring caspase 8 activation in the cells following treatment with bispecific antibody; and/or

d) In vivo anti-tumor efficacy/tumor regression in humanized NSG mice bearing SKOV3 human ovarian cancer transfected with recombinant HLA-G (SKOV 3 HLA-G) humanized NSG mice; and/or

e) HLA-G CD 3T cell bispecific in vivo anti-tumor efficacy/tumor in humanized NSG mice bearing human breast cancer PDX tumor (BC 004).

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

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

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

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

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

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

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

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

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

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

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