Anti-HLA-g antibodies

Abstract

FIELD: biotechnology.SUBSTANCE: antibody or its antigen-binding fragment that specifically binds to HLA-G, as well as to a method for producing it. Also an isolated polynucleotide encoding the above antibody or fragment thereof, as well as a cell and vector containing it are disclosed.EFFECT: producing a medicinal product for use in the treatment of a disease characterized by overexpression of HLA-G.34 cl, 21 dwg, 36 tbl, 28 ex

Description

[0001] FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention relates to antibodies directed against HLA-G and compositions containing them. The invention also relates to the use of anti-HLA-G antibodies and compositions in therapy, particularly for the treatment of solid cancers.

[0003] STATE OF THE ART

[0004] Human leukocyte antigens class I (HLA-I) contain classical antigens, HLA-A, HLA-B, and HLA-C, as well as non-classical antigens, HLA-E, HLA-F, and HLA-G. Human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule expressed in the human body and encoded by the HLA-G gene. HLA-G is a heterodimeric molecule containing a heavy chain that has 3 globular domains (α1, α2, and α3) associated with a light chain, namely, beta-2-microglobulin (B2m).

[0005] Seven HLA-G isoforms have been identified, four membrane-bound (HLA-G1, HLA-G2, HLA-G3, HLA-G4) and three soluble (HLA-G5, HLA-G6 and HLA-G7), which result from alternative splicing of the primary HLA-G transcript.

[0006] HLA-G is typically expressed on placental cytotrophoblasts. HLA-G expression has been reported to be associated with pathological conditions such as inflammatory diseases and cancer. Notably, HLA-G is a tolerogenic molecule whose activity is specifically increased in solid cancers and associated with a poor prognosis.

[0007] HLA-G is known to exhibit immunoregulatory activity through binding to at least three receptors expressed on various myeloid and lymphoid cells:

[0008] - Inhibitory receptor LILRB1 (for leukocyte immunoglobulin-like receptor B1), also called ILT2 or CD85j, expressed on lymphoid (B cells, some T cells and NK cells) and myeloid cells (monocytes, macrophages and dendritic cells).

[0009] - Inhibitory receptor LILRB2 (for leukocyte immunoglobulin-like receptor B2), also called ILT4 or CD85d, expressed on myeloid cells (monocytes, macrophages and dendritic cells); and

[0010] - Regulatory receptor KIR2DL4 or CD158d expressed on NK cells.

[0011] HLA-G inhibits immune cell function by directly binding to its inhibitory receptors. HLA-G has been reported to exert tolerogenic function, which is primarily mediated by the interaction of the α3 domain of HLA-G with ILT2 and ILT4. ILT2 recognizes only HLA-G molecules bound to B2m, whereas ILT4 recognizes both B2m-bound and B2m-free HLA-G molecules.

[0012] Due to this immune-inhibitory function, HLA-G expression by tumors can induce an immunosuppressive environment, allowing the tumor to evade immune surveillance and ultimately reducing patient survival. Thus, antibody-mediated HLA-G blockade may be an effective strategy for attenuating localized tumor immune suppression, promoting the development of anti-cancer immunity, and ensuring long-term therapy.

[0013] Although the antitumor effect of an HLA-G blocking antibody could theoretically be enhanced by the inclusion of an active Fc component capable of interacting with FcγR on immune effector cells, thereby enabling direct killing of HLA-G+ tumor cells, reports of HLA-G mRNA and protein expression in a number of normal tissues, including the pancreas and pituitary gland, suggest that the use of an active Fc may result in unacceptable toxicity, precluding its use in therapy.

[0014] HLA-G has high similarity to other HLA-I molecules (i.e., HLA-A, HLA-B, HLA-C, HLA-E, and HLA-F). Of the 338 amino acid positions in the HLA-G protein, only 20 have residues that are unique to HLA-G and not present at the same position in any other of the approximately 5,000 human HLA-I molecules. This greatly complicates the task of producing highly specific antibodies to HLA-G without cross-reactivity with other HLA-I molecules.

[0015] Currently, commercially available HLA-G antibodies are available. However, some of them have been reported to lack specificity for HLA-G (e.g., they cross-react with other HLA-I molecules), and all of them contain epitopes in the α1 and α2 domains of HLA-G, distant from the ILT2 / 4-binding site of HLA-G in the α3 domain and, therefore, are not expected to block the interaction between HLA-G and ILT2 and / or ILT4. It has also been reported that antibody 87G, which interacts with an epitope in the α1 domain of HLA-G, attenuates the effect of HLA-G in inhibiting immune cell function. However, there are no reports that 87G and other commercially available anti-HLA-G antibodies are capable of blocking the interaction of HLA-G with ILT2 / 4. Due to the lack of specificity and / or blocking activity, commercial antibodies are not suitable for the development of therapeutic anti-HLA-G antibodies.

[0016] Other antibodies binding to HLA-G have been reported in WO19202040 and WO2020069133, and although such antibodies appear to modulate one or more activities of HLA-G, their binding site (i.e., epitope) on HLA-G has not been characterized.

[0017] To date, the efficacy of anti-HLA-G antibodies has not been demonstrated in patients, particularly for the treatment of solid cancer.

[0018] Therefore, there remains a need to develop antibodies that bind to HLA-G and have biological properties useful in therapy, such as improved pharmacokinetic properties and / or improved biological functions (e.g., specificity, binding affinity, neutralization and / or cellular cytotoxicity and phagocytosis) and / or reduced toxicity to humans.

[0019] SUMMARY OF THE INVENTION

[0020] The present invention aims to address the above need by providing novel anti-HLA-G antibodies useful in therapy, especially for the treatment of solid cancer, with the structural and functional properties disclosed herein, in particular, with high specificity for HLA-G, the ability to block the interaction of HLA-G with its receptors ILT2 and ILT4. Evidence is provided that the epitope recognized by the antibodies of the invention is not present in normal tissues, including the pituitary gland and pancreas, thereby confirming the first use of an antibody format containing an active Fc capable of directly killing tumor cells in patients.

[0021] In particular, the present invention relates to an antibody that specifically binds to human HLA-G, comprising:

[0022] a. a light chain variable region comprising:

[0023] CDR-L1 containing SEQ ID NO:1,

[0024] CDR-L2 containing SEQ ID NO: 2, and

[0025] CDR-L3 comprising SEQ ID NO: 3; and

[0026] b. a heavy chain variable region comprising:

[0027] CDR-H1 containing SEQ ID NO: 4,

[0028] CDR-H2 containing SEQ ID NO: 5, and

[0029] CDR-H3 containing SEQ ID NO: 6.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Fig. 1: HLA-G isoforms (Fig. 1B, Carosella et al., Blood, Vol. 111, No. 10, 2008).

[0032] Fig. 2. Humanization of the rabbit variable light chain sequence of antibody 12389. Grafts 12389gL1, gL2, and gL3 are humanized grafts of the rabbit variable light chain of antibody 12389, where the human germline IGKV1D-13 is used as the acceptor scaffold. CDRs are shown in bold / underlined. Donor residues are shown in bold / italics and shaded in gray: V3 and Q70.

[0033] Fig. 3. Humanization of the rabbit heavy chain variable region sequence of antibody 12389. Grafts 12389gH1, gH4, gH5, gH6, gH8, gH9, gH11, gH12, gH13, gH14, gH15 and gH16 are humanized grafts of the rabbit heavy chain variable region of antibody 12389, where the human germline IGHV3-66 was used as the acceptor scaffold. CDRs are shown in bold / underlined. Donor residues are shown in bold / italics and shaded in gray: V24, I48, G49, K71, S73, V78 and G96.

[0034] Fig. 4. HLA-G02 specificity in PBMCs from 50 different donors. Results are expressed as MFI of each CD4+ cell population for each donor and antibody.

[0035] Fig. 5. Specificity of HLA-G02; binding to HLA-G1, HLA-G2, HLA-G3 and HLA-G4 expressed on cells (Fig. 5A). Binding to HLA-G2 (Fig. 5B).

[0036] Fig. 6. Percentage of Epcam+ GFP+ depleted HCT116 target cells after treatment with different anti-HLA-G antibodies or IgG1 antibody as an isotype control. Each antibody was tested at two different concentrations: 1 μg / mL (white bars) or 0.01 μg / mL (striped bars). The E:T ratio was 3.5:1. Each bar represents the mean (and range) of three data points, and each dot / square represents an individual copy. Data are from one representative donor. HLA-G01 to HLA-G08 are represented by the corresponding donor identification (ID) numbers (01 to 08).

[0037] Fig. 7. Percentage of Epcam+ GFP+ depleted HCT116 target cells after treatment with anti-HLA-G antibodies HLA-G01 and HLA-G02 or IgG1 antibody as an isotype control from three separate experiments (3 different donors). Antibodies were tested at a concentration of 1 μg / mL (Fig. 7A) or 0.01 μg / mL (Fig. 7B). The E:T ratio ranged from 2.5 to 3:1. Each bar represents the mean (and range) of data obtained in a separate experiment, and each dot, square, or triangle represents an individual replicate.

[0038] Fig. 8. Percent depletion of Epcam+ GFP+ HCT116 cells (vertical axis) after titration with anti-HLA-G antibodies HLA-G01 and HLA-G02 compared with IgG1 antibody as an isotype control (horizontal axis: antibody concentration in μg / mL). The E:T ratio was 4:1. Each point represents the mean (and range) of three replicates. The data shown are from one representative donor.

[0039] Fig. 9A. Percent depletion of JEG3 cells (vertical axis) after treatment with conventional IgG1 HLA-G02 (solid line) or afucosylated IgG1 HLA-G02 (“aF HLA-G02”, dotted line). Horizontal axis: antibody concentration in μg / mL. The E:T ratio was 10:1. Each point represents the mean (and range) of two replicates. Data are shown for one representative donor.

[0040] Fig. 9B. Percent depletion of Epcam+ GFP+ HCT116 cells (vertical axis) after treatment with conventional IgG1 HLA-G02 (solid line) or afucosylated IgG1 HLA-G02 (“aF HLA-G02”, dashed line) compared with isotype control IgG1. Horizontal axis: antibody concentration in μg / mL. The E:T ratio was 5:1. Each point represents the mean (and range) of three replicates. Data are shown for one representative donor.

[0041] Fig. 10. Titration of HLA-G-specific phagocytosis activity of HLA-G02 on mock-transfected (Fig. 10A) and HLA-G / B2m-transfected K562 target cells (Fig. 10B) compared with anti-CD47 antibody (“aCD47”) and isotype control IgG1. Vertical axis: percentage of double-positive CTY+CD11b+ cells; horizontal axis: antibody concentration in μg / mL.

[0042] Fig. 11. Titration of HLA-G-specific phagocytosis activity of conventional and afucosylated (aF) HLA-G02 formats on mock-transfected (Fig. 11A) and HLA-G-expressing K562 target cells (Fig. 11B) compared with anti-CD47 antibody and IgG1 isotype control. Vertical axis: percentage of double-positive CTY+CD11b+ cells; horizontal axis: antibody concentration in μg / mL.

[0043] Fig. 12. VR12389 (the surface area of ​​which is shown in black) blocks the interaction of HLA-G with ILT-2 and ILT4. Fig. 12A: The crystal structure (PDB ID 6AEE) of ILT2 (white image) in complex with HLA-G (gray image) and β2M (gray mesh image). Fig. 12B: Superposition with the crystal structure of VR12389 in complex with HLA-G and β2M shows that VR12389 (the surface area of ​​which is shown in black) blocks the interaction of ILT2 with HLA-G. Fig. 12C: The crystal structure (PDB ID 2DYP) of ILT4 (white image) in complex with HLA-G (gray mesh image) and β2M (gray mesh image). Fig. 12D: Superposition of the crystal structure of VR12389 in complex with HLA-G and β2M shows that VR12389 (surface shown in black) blocks the interaction of ILT4 with HLA-G.

[0044] Fig. 13. Tumor cell killing assay. Fig. 13A: data obtained with anti-PDL1 antibody from RCC. Fig. 13B: data obtained with anti-PDL1 antibody from CRC. Fig. 13C: data obtained with HLA-G02 from RCC. Fig. 13D: data obtained with HLA-G02 from CRC. Data are presented as % dead cells, where light gray indicates cultures treated with isotype control, dark gray indicates cultures treated with anti-PDL1 or HLA-G02, and black indicates cultures treated with anti-PDL1 antibody or HLA-G02, which showed a 1.5-fold or greater increase in cell death.

[0045] Fig. 14. HLA-G depletion of transfected HCT116 cells at different effector:target ratios 2.5-3 hours after treatment with normal or afucosylated HLA-G02. The number of viable HLA-G GFP+ HCT116 cells was determined by flow cytometry, and the percentage of depletion (vertical axis) was calculated relative to the no-antibody control. Horizontal axis: E:T ratio (effector:target ratio). Data shown are from a single donor (1). Each data point represents the mean of three replicates. Error bars represent 95% confidence intervals.

[0046] Fig. 15. Specificity of afucosylated HLA-G02 (“aF HLA-G02”) in PBMCs from 10 different donors. Results are expressed as MFI of each CD4+ cell population for each donor and antibody.

[0047] Fig. 16. CDC mediated by afucosylated HLA-G02. Fig. 16A: Effect of serum activity on HLAG-β2m-Reh cell lysis (concentration-response curve depicting aF HLA-G02-mediated CDC of HLAG-β2m-Reh cells in the presence of active or heat-inactivated serum. Vertical axis: lysis (%) normalized to the minimum and maximum controls (N=1, 4-PL match); horizontal axis: antibody concentration [M]). Fig. 16B: HLA-G dependence of aF HLA-G02-mediated lysis (concentration-response curve depicting the CDC of HLAG-β2m-Reh or Reh cells mediated by aF HLA-G02. Vertical axis: lysis (%) normalized to the minimum and maximum controls (N=1, 4-PL match); horizontal axis: antibody concentration [M]). Fig. 16C: aF HLA-G02-mediated CDC of HLAG-β2m-Reh cells (concentration-response curve depicting the CDC of HLAG-β2m-Reh cells mediated by aF HLA-G02.Vertical axis: lysis (%), based on minimum and maximum controls (N=3, mean ± SEM, 4-PL fit); horizontal axis: antibody concentration [M].

[0048] Fig. 17. Concentration-dependent effects of HLA-G02 and afucosylated HLA-G02 on phagocytosis of HLA-G-expressing target cells or mock-transfected cells compared with αCD47 antibody. Fig. 17A and B: representative data (mean ± SD) (Vertical axis: percentage of CTY+CD11b+ double positive cells; horizontal axis: antibody concentration in μg / mL). Fig. 17C and D: pooled data obtained using monocytes from three separate donors in duplicate in two independent experiments (Vertical axis: isotype control-corrected phagocytosis percentage = (antibody mean - isotype mean) ± SD; horizontal axis: antibody concentration in μg / mL).

[0049] Fig. 18. Target cell killing mediated by afucosylated HLA-G02. Percent depletion of HLA-G-expressing target cells by ADCP (vertical axis) at different concentrations of afucosylated HLA-G02 or αCD47 (horizontal axis, in μg / mL) after overnight incubation (from four individual donors, in duplicate, from two independent experiments). Mean and standard deviation relative to isotype control.

[0050] Fig. 19. Representative data of the concentration-dependent effect of afucosylated HLA-G02 and HLA-G02 IgG4P FALA on the phagocytosis of HLA-G-expressing cells alone (Fig. 19A) or in combination with anti-CD47 antibody (at a concentration of 1 μg / mL, Fig. 19B) compared with the corresponding isotype controls. Vertical axis: percentage of CTY+CD11b+ double-positive cells; horizontal axis: antibody concentration in μg / mL.

[0051] Fig. 20. Phagocytosis of HLA-G-expressing cells treated with afucosylated HLA-G02 alone or in combination with anti-CD47 antibody (at a concentration of 1 μg / mL). Fig. 20A shows data for afucosylated HLA-G02 (three donors, in duplicate, two independent experiments). Fig. 20B shows data with HLA-G02 IgG4P FALA (pooled data from four donors, each assayed in duplicate, three independent experiments). Phagocytosis values ​​(%, vertical axis) are corrected relative to the corresponding isotype control (mean Ab value - mean isotype value) and are presented as mean ± standard deviation. Horizontal axis: antibody concentration in μg / mL.

[0052] Fig. 21. Cytokine levels (vertical axis, pg / mL) (Fig. 21A: IFN-gamma; Fig. 21B: TNF-alpha; Fig. 21C: IL-2; Fig. 21D: IL-6; Fig. 21E: IL-8; Fig. 21F: IL-10) produced in PBMC cultures in the presence and absence of JEG3 cells over a range of afucosylated HLA-G02 concentrations (horizontal axis, μg / mL). Values ​​are presented as the mean + / - standard deviation for all 16 PBMC donors. The dotted lines indicate the mean level of each cytokine produced in the presence of 50 μg / ml afucosylated isotype control antibody, 50 μg / ml anti-CD3, or 100 ng / ml LPS.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention is described below with reference to specific non-limiting aspects and embodiments thereof and with reference to certain figures and examples.

[0055] Technical terms are used in accordance with common sense unless otherwise specified. Where specific meanings are assigned to certain terms, the definitions of these terms will be given in the context in which they are used.

[0056] When the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising."

[0057] When an indefinite or definite article is used to refer to a singular noun, such as "a," "an," or "the," the plural of that noun is also included unless otherwise stated.

[0058] The present invention relates to an anti-HLA-G antibody. In a first aspect, the present invention relates to an antibody that specifically binds to HLA-G, comprising:

[0059] a. a light chain variable region comprising:

[0060] CDR-L1 containing SEQ ID NO:1,

[0061] CDR-L2 containing SEQ ID NO:2, and

[0062] CDR-L3 containing SEQ ID NO:3; and

[0063] b. a heavy chain variable region comprising:

[0064] CDR-H1 containing SEQ ID NO:4,

[0065] CDR-H2 containing SEQ ID NO:5, and

[0066] CDR-H3 containing SEQ ID NO:6.

[0067] HLA-G

[0068] The term "HLA-G" or "human HLA-G" refers to human leukocyte antigen G, which is a classical HLA-I molecule, also known as the human major histocompatibility complex I (MHC) molecule. HLA-G typically forms an MHC-I complex with B2m.

[0069] Unless otherwise specified, the term "HLA-G" refers to any alternative splicing or naturally occurring variants or isoforms of human HLA-G that are naturally expressed by cells. A typical complete human HLA-G sequence comprises the sequence shown in SEQ ID NO: 107.

[0070] In some aspects, the antibody of the invention selectively binds to the extracellular domain (ECD) of HLA-G (or "HLA-G ECD"). A typical HLA-G ECD sequence comprises the sequence set forth in SEQ ID NO: 108.

[0071] The amino acid sequences and nucleic acid sequences of HLA-G and its isoforms are also well known in the art.

[0072] Seven HLA-G isoforms have been identified, four of which are membrane-bound (HLA-G1, HLA-G2, HLA-G3, HLA-G4) and three are soluble (HLA-G5, HLA-G6, and HLA-G7), which result from alternative splicing of the primary HLA-G transcript (Fig. 1). mRNA analysis of renal tumors suggests the presence of additional isoforms, some of which lack the α1 domain, but these have not been confirmed at the protein level (Tronik-Le Roux et al., Molecular Oncology, 11(2017), 1561–1578).

[0073] HLA-G1 and HLA-G5 have a structure similar to that of classical HLA-I molecules, i.e., heterodimeric molecules containing a heavy chain with three globular domains (α1, α2, and α3) linked to a light chain, namely, beta-2-microglobulin (abbreviated "β2m" or "B2m"). HLA-G1 and HLA-G5 can also exist as free alpha chains, i.e., not in a complex with B2m.

[0074] HLA-G2 and HLA-G6 contain only the α1 and α3 domains. HLA-G4 contains only the α1 and α2 domains. HLA-G3 and HLA-G7 contain only the α1 domain.

[0075] In one embodiment, the antibody of the invention binds to at least one of HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7. In one embodiment, the antibody of the invention binds to all HLA-G isoforms containing the alpha 3 domain, i.e., HLA-G1, HLA-G2, HLA-G5, and HLA-G6, including HLA-G1 and HLA-G5 complexed with B2m and expressed as molecules without B2m.

[0076] HLA-G1 and HLA-G5 can form homomultimers, such as dimers and trimers linked by a disulfide bond. In one embodiment, an antibody of the invention binds to monomeric HLA-G. In one embodiment, an antibody of the invention binds to dimeric HLA-G. In one embodiment, an antibody of the invention binds to trimeric HLA-G. In one embodiment, an antibody of the invention binds to monomeric, dimeric, and trimeric HLA-G.

[0077] Antibodies binding to HLA-G

[0078] Antibodies for use in the context of the present invention include whole antibodies and functionally active fragments thereof (i.e., molecules containing an antigen-binding domain that specifically binds to an antigen, also referred to as antigen-binding fragments). The features disclosed herein with respect to antibodies also apply to antibody fragments, unless the context otherwise requires. An antibody may be (or be derived from) monoclonal, multivalent, polyspecific, bispecific, fully human, humanized, or chimeric.

[0079] Whole antibodies, also known as "immunoglobulins (Ig)," generally refer to intact or full-length antibodies, i.e., containing elements of two heavy chains and two light chains linked by disulfide bonds, which, when assembled, form a characteristic Y-shaped three-dimensional structure. Classically, naturally occurring whole antibodies are monospecific, as they bind to a single type of antigen, and bivalent, as they have two independent antigen-binding domains. The terms "intact antibody," "full-length antibody," and "whole antibody" are used interchangeably to refer to a monospecific bivalent antibody with a structure similar to that of a native antibody, including the Fc region, as defined herein.

[0080] In whole antibodies, each light chain consists of a light chain variable region (abbreviated VL) and a light chain constant region (CL). Each heavy chain consists of a heavy chain variable region (abbreviated VH) and a heavy chain constant region (CH), which consists of three constant domains CH1, CH2, and CH3 or four constant domains CH1, CH2, CH3, and CH4, depending on the Ig class. The “class” of an Ig or antibody refers to the type of constant region and includes IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses, such as IgG1, IgG2, IgG3, and IgG4. Constant regions of antibodies can mediate the binding of immunoglobulin to tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0081] The VH and VL regions of the antibody of the present invention can be further subdivided into regions of hypervariability (or "hypervariable regions" or HVRs) that determine antigen recognition, called complementarity determining regions (CDRs), which are interspersed with structurally more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDRs and FRs together form a variable region. By convention, the CDRs in the variable region of the heavy chain of an antibody or its antigen-binding fragment are designated as CDR-H1, CDR-H2, and CDR-H3, and in the variable regions of the light chain as CDR-L1, CDR-L2, and CDR-L3. They are numbered sequentially from the N-terminus to the C-terminus of each chain.

[0082] CDRs are traditionally numbered according to the system developed by Kabat et al. This system is described by Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al. (see above)"). This numbering system is used in the present description unless otherwise noted.

[0083] Kabat residue designations do not always correspond directly to the linear amino acid residue numbering. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening or insertion into a structural component of the primary variable domain structure, whether the framework or complementarity-determining region (CDR). The correct Kabat residue numbering can be determined for a given antibody by aligning homologous residues in the antibody sequence with the "standard" Kabat-numbered sequence.

[0084] The CDRs of the variable domain of the heavy chain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise specified, the term “CDR-H1” used herein refers to residues 26-35 according to a combination of the Kabat numbering system and the definition of a topological loop according to Chothia.

[0085] The CDRs of the light chain variable domain according to the Kabat numbering system are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3).

[0086] In addition to the CDR loops, there is a fourth loop between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3), which is formed by framework 3 (FR3). According to the Kabat numbering system, framework 3 is defined by positions 66-94 in the heavy chain and positions 57-88 in the light chain.

[0087] Based on the alignment of sequences of different members of the immunoglobulin family, numbering schemes have been proposed, such as those described by Kabat et al., 1991 and Dondelinger et al., 2018, Frontiers in Immunology, Vol 9, Article 2278.

[0088] The antibody of the invention comprises a light chain variable region comprising CDR-L1 comprising SEQ ID NO:1, CDR-L2 comprising SEQ ID NO:2, and CDR-L3 comprising SEQ ID NO:3, and a heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:4, CDR-H2 comprising SEQ ID NO:5, and CDR-H3 comprising SEQ ID NO:6.

[0089] In one embodiment, the antibody of the invention comprises a light chain variable region comprising the CDRs of the light chain variable region of SEQ ID NO: 19 and a heavy chain variable region comprising the CDRs of the heavy chain variable region of SEQ ID NO: 93.

[0090] Antibodies containing such CDR sequences clearly possess an inventive step, as they provide an antibody with high affinity for HLA-G, high specificity for HLA-G (in particular, without cross-reactivity with other HLA-I molecules, despite their very high homology), a high degree of inhibition of HLA-G biological functions, and high stability, which is important for mass production. Moreover, when combined with an active Fc, they provide an antibody capable of directly killing HLA-G+ tumor cells.

[0091] The terms "constant domain(s)" and "constant region" are used interchangeably herein to refer to the domain(s) of an antibody that are(are) outside the variable regions. The constant domains are identical in all antibodies of the same isotype but differ between different isotypes. Typically, the constant region of a heavy chain is formed, from the N- to the C-terminus, by a CH1-hinge-CH2-CH3-optionally CH4 fragment containing three or four constant domains.

[0092] The constant region domains of the antibody molecule of the present invention, if present, can be selected taking into account the intended function of the antibody molecule and, in particular, the effector functions that may be required. For example, the constant region domains can be human IgA, IgD, IgE, IgG, or IgM domains. In particular, the constant region domains of human IgG, especially the IgG1 and IgG3 isotypes, can be used when the antibody molecule is intended for therapeutic use and the effector functions of the antibody are required. Alternatively, the IgG2 and IgG4 isotypes can be used when the antibody molecule is intended for therapeutic purposes and the effector functions of the antibody are not required. It should be understood that sequence variants of these constant region domains can also be used.For example, IgG4 molecules in which serine at position 241 (numbered according to the Kabat numbering system) is replaced by proline, as described by Angal et al. (Angal et al., 1993. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody as observed during SDS-PAGE analysis Mol Immunol 30, 105-108) and designated herein as IgG4P, can be used.

[0093] "Fc," "Fc fragment," and "Fc region" are used interchangeably to refer to the C-terminal region of an antibody, which contains the antibody constant region excluding the first constant region of the immunoglobulin domain. Thus, Fc refers to the last two constant domains of the C H2 and C H3IgA, IgD, and IgG, or the last three constant domains of IgE and IgM, as well as the flexible hinge N-terminus to these domains. The Fc region of the human IgG1 heavy chain is defined herein as comprising residues from C226 to the carboxyl terminus, with the numbering corresponding to the EU index according to Kabat. In the context of human IgG1, the lower hinge refers to positions 226-236, the CH2 domain refers to positions 237-340, and the CH3 domain refers to positions 341-447 according to the EU index according to Kabat. The corresponding Fc region of other immunoglobulins can be determined by sequence alignment.

[0094] In the context of the present invention, if a constant region or Fc region is present, it may be natural, as defined above, or may be modified in various ways, provided that it contains a functional FcR-binding domain and, preferably, a functional FcRn-binding domain. Preferably, the modified constant region or Fc region results in improved functionality and / or pharmacokinetics. Modifications may include the removal of certain portions of the Fc fragment. Modifications may further include various amino acid substitutions that can affect the biological properties of the antibody. Mutations that increase FcRn binding and, therefore, increase the half-life in vivo may also be present. Modifications may further include modification of the glycosylation profile of the antibody.The natural Fc fragment is glycosylated in the CH2 domain, and each of the two heavy chains contains an N-glycan linked to an asparagine residue at position 297 (Asn297). In the context of the present invention, an antibody can be glycomodified, i.e., engineered to have a specific glycosylation profile that, for example, leads to an improved property, such as improved effector function and / or improved serum half-life.

[0095] The antibodies disclosed herein are isolated. An "isolated" antibody is one that has been separated (e.g., by purification means) from a component of its natural environment.

[0096] The term "antibody" encompasses monovalent antibodies, i.e., antibodies containing only one antigen-binding domain (e.g., partial antibodies containing a full-length heavy chain and a full-length light chain linked together, also called "half-antibodies"), and multivalent antibodies, i.e., antibodies containing more than one antigen-binding domain.

[0097] The term "antibody" according to the invention also encompasses antigen-binding fragments of antibodies.

[0098] Antigen-binding fragments of antibodies include single-chain antibodies (e.g., scFv and dsscfv), Fab, Fab', F(ab')2, Fv, single-domain antibodies, or nanobodies (e.g., VH or VL, or VHH or VNAR). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International Patent Applications WO2011 / 117648, WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171.

[0099] Methods for the generation and production of these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0100] The term "Fab fragment" as used herein refers to an antibody fragment comprising a light chain fragment comprising a VL domain (light chain variable region) and a light chain constant domain (CL), as well as a VH domain (heavy chain variable region) and the first constant domain (CH1) of the heavy chain.

[0101] A typical "Fab' fragment" comprises a pair of heavy and light chains, in which the heavy chain comprises a variable region VH, a constant domain CH1, and a natural or modified hinge region, and the light chain comprises a variable region VL and a constant domain CL. Fab' dimers according to the present invention form F(ab')2, where, for example, dimerization can occur through the hinge.

[0102] The term "single-domain antibody" as used herein refers to an antibody fragment consisting of a single monomeric variable domain of an antibody. Examples of single-domain antibodies include VH or VL, or VHH, or VNAR.

[0103] “Fv” refers to two variable domains, such as cooperative variable domains such as a cognate pair or affinity-matured variable domains, i.e., a pair of VH and VL.

[0104] “Single chain variable fragment” or “scFv” as used herein refers to a single chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains.

[0105] “A disulfide bond-stabilized single-chain variable fragment” or “dsscFv” as used herein refers to a single-chain variable fragment that is stabilized by a peptide linker between the variable domains VH and VL and also includes an interdomain disulfide bond between VH and VL (see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012, WO2007109254).

[0106] In one embodiment, the disulfide bond between the VH and VL variable domains is between the two residues listed below (unless the context dictates otherwise; Kabat numbering is used in the list below). Wherever reference is made to Kabat numbering, the corresponding reference is Kabat et al., 1991 (5 th edition, Bethesda, Md), in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0107] In one embodiment, the disulfide bond is at a position selected from the group consisting of:

[0108] - VH37+VL95C, see, for example, Protein Science 6, 781-788 Zhu et al (1997);

[0109] - VH44+VL100, see, for example, Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012;

[0110] - VH44+VL105 see, e.g., J Biochem. 118, 825-831 Luo et al (1995);

[0111] - VH45+VL87 see, for example, Protein Science 6, 781-788 Zhu et al (1997);

[0112] - VH55+VL101, see, for example, FEBS Letters 377, 135-139 Young et al (1995);

[0113] - VH100+VL50, see, for example, Biochemistry 29, 1362-1367 Glockshuber et al (1990);

[0114] - VH100b+VL49, see, e.g., Biochemistry 29, 1362–1367, Glockshuber et al (1990);

[0115] - VH98 +VL46, see, for example, Protein Science 6, 781-788 Zhu et al (1997);

[0116] - VH101+VL46, see, for example, Protein Science 6, 781-788 Zhu et al (1997);

[0117] - VH105+VL43, see, for example, Proc. Natl. Acad. Sci. USA Vol. 90 pp. 7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung et al (1994),

[0118] - VH106+VL57, see, for example, FEBS Letters 377, 135-139 Young et al (1995),

[0119] and the position or positions corresponding to them in a pair of variable regions located in the molecule.

[0120] In one embodiment, a disulfide bond is formed between positions VH44 and VL100.

[0121] Polyspecific antibodies

[0122] The antibody of the invention may be a multispecific antibody. The term "multispecific or multi-specific antibody" as used herein refers to an antibody disclosed herein that has at least two binding domains, i.e., two or more binding domains, for example, two or three binding domains, wherein at least two binding domains independently bind two different antigens or two different epitopes on the same antigen. Multispecific antibodies are typically monovalent with respect to each specificity (antigen). The multispecific antibodies disclosed herein include monovalent and multivalent, for example, bivalent, trivalent, and tetravalent multispecific antibodies.

[0123] In one embodiment, the construct is a bispecific antibody. The term "bispecific or bi-specific antibody" as used herein refers to an antibody with two antigen-binding specificities. In one embodiment, the antibody comprises two antigen-binding domains, wherein one binding domain binds ANTIGEN 1 and the other binding domain binds ANTIGEN 2, i.e., each binding domain is monovalent for each antigen. In one embodiment, the antibody is a tetravalent bispecific antibody, i.e., the antibody comprises four antigen-binding domains, wherein, for example, two binding domains bind ANTIGEN 1 and the other two binding domains bind ANTIGEN 2. In one embodiment, the antibody is a trivalent bispecific antibody.

[0124] In one embodiment, the antibody construct is a trispecific antibody. The term "trispecific or tri-specific antibody," as used herein, refers to an antibody with three antigen-binding specificities. For example, the antibody is an antibody with three antigen-binding domains (trivalent), which independently bind three different antigens or three different epitopes on the same antigen, i.e., each binding domain is monovalent for each antigen.

[0125] A paratope is a region of an antibody that recognizes and binds to an antigen. The antibody of the invention may be a multiparatopic antibody. A "multiparatopic antibody," as used herein, refers to an antibody disclosed herein that comprises two or more different paratopes that interact with different epitopes of either the same antigen or two different antigens. Multiparatopic antibodies disclosed herein may be biparatopic, triparatopic, or tetraparatopic.

[0126] "Antigen-binding domain" as used herein refers to a portion of an antibody that comprises part or all of one or more variable domains, such as part or all of a pair of variable domains VH and VL, that specifically interact with a target antigen. A binding domain may comprise a single-domain antibody. In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises no more than one VH and one VL.

[0127] Many formats of polyspecific antibodies have already been created. Different classifications have been proposed, but polyspecific IgG antibody formats typically include bispecific IgG, complemented IgG, fragments of polyspecific (e.g., bispecific) antibodies, polyspecific (e.g., bispecific) fusion proteins, and conjugates of polyspecific (e.g., bispecific) antibodies, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95–106.

[0128] Methods for producing bispecific antibodies include, but are not limited to, the CrossMab method (Klein et al. Engineering therapeutic bispecific antibodies using CrossMab technology, Methods 154 (2019) 21-31), the knob-in-hole method (e.g., WO1996027011, WO1998050431), the DuoBody method (e.g., WO2011131746), and the Azymetric method (e.g., WO2012058768). Additional methods for producing bispecific antibodies are described, for example, in Godar et al., 2018, Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion on Therapeutic Patents, 28:3, 251-276. Bispecific antibodies include, but are not limited to, CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, common knob-in-hole LC, knob-in-hole assembly, charged pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ body, and orthogonal Fab.

[0129] A complemented IgG typically comprises a full-length IgG created by attaching an additional antigen-binding domain or antigen-binding fragment to the N- and / or C-terminus of the IgG heavy and / or light chain. Examples of such additional antigen-binding fragments include sdAb (e.g., VH or VL), Fv, scFv, dsscFv, Fab, and scFav antibodies. IgG fusion antibody formats include, but are not limited to, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgC(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-1g, Zybody, and DVI-IgG (four-in-one), such as described by Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0130] Multispecific antibody fragments include nanobody, nanobody-HAS, BiTE, diabody, DART, TandAb, sc-diabody, sc-diabody-CH3, diabody-CH3, ternary body, minibody; minibody, tri-bi-minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc-diabody-Fc, diabody-Fc, tandem scFv-Fc; and intrabody, as described, for example, by Spiess et al., Alternative Molecular Formats and Therapeutic Applications for Bispecific Antibodies. Mol Immunol. 67(2015):95-106.

[0131] Polyspecific fusion proteins include Dock and Lock, ImmTAC, HSA-body, sc-diabody-HAS, and tandem scFv-toxin.

[0132] The polyspecific antibody conjugates include IgG-IgG; Cov-X-body; and scFv1-PEG-scFv2.

[0133] Additional formats of polyspecific antibodies are described, for example, by Brinkmann and Kontermann, The making of bispecific antibodies, mAbs, 9:2, 182-212 (2017), in particular in Fig. 2, for example, tandem scFv, triple body, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, scFv4-IgG. Bibodies, tribodies and methods for producing them are disclosed, for example, in WO99 / 37791.

[0134] Examples of antibodies for use in the present invention include complemented IgG and complemented Fab, wherein the whole IgG or Fab fragment, respectively, is created by adding at least one additional antigen-binding domain (e.g., two, three, or four additional antigen-binding domains), for example, a single-domain antibody (such as VH or VL or VHH), scFv, dsscFv, dsFv to the N- and / or C-terminus of the heavy and / or light chain of said IgG or Fab, for example, as described in WO2009 / 040562, WO2010 / 035012, WO2011 / 030107, WO2011 / 061492, WO2011 / 061246 and WO2011 / 086091. In particular, the Fab-Fv format is described in WO2009 / 040562, and its disulfide-stabilized version Fab-dsFv is described in WO2010 / 035012. A single-linker Fab-dsFv, where the dsFv is connected to the Fab via a single linker between the VL or VH domain of the Fv and the C-terminus of the LC or HC of the Fab, is described in WO2014 / 096390.A complemented IgG comprising full-length IgG1 created by attaching a dsFv to the C-terminus of an IgG heavy or light chain is described in WO2015 / 197789.

[0135] Another example of an antibody for use in the present invention comprises a Fab linked to two scFvs or dsscFvs, wherein each scFv or dsscFv binds to the same or different targets (e.g., one scFv or dsscFv binds to a therapeutic target, and one scFv or dsscFv prolongs half-life by binding, for example, albumin). Such antibodies are described in WO2015 / 197772. Another example of an antibody for use in a fragment of the present invention comprises a Fab linked to only one scFv or dsscFv, as described, for example, in WO2013 / 068571 and Dave et al., Mabs, 8(7), 1319-1335, (2016).

[0136] Other well-known polyspecific antibody formats include:

[0137] In the context of the present description, a diabody refers to two Fv pairs, a first VH / VL pair and an additional VH / VL pair, which have two linkers between the Fv, where the VH of the first Fv is linked to the VL of the second Fv, and the VL of the first Fv is linked to the VH of the second Fv.

[0138] In the context of the present description, a triabody refers to a format similar to a diabody, containing three Fvs and three linkers between the Fvs.

[0139] In the context of the present description, a tetrabody refers to a format similar to a diabody, containing four Fvs and four linkers between the Fvs.

[0140] As used herein, a tandem scFv refers to at least two scFvs linked via a single linker, where one linker is between the Fvs.

[0141] In the context of the present description, a tandem scFv-Fc refers to at least two tandem scFvs, each of which is attached to the N-terminus of the CH2 domain of the -CH2CH3 fragment of the constant region, for example, via a hinge.

[0142] As used herein, Fab-Fv refers to an Fv fragment with a variable region attached to the C-terminus of each of the following components: CH1 of the heavy chain and CL of the light chain. This format can be represented as its PEGylated version.

[0143] For the purposes of this description, Fab'-Fv is analogous to FabFv in which the Fab portion is replaced by Fab'. This format can be represented as its PEGylated version.

[0144] As used herein, Fab-dsFv refers to FabFv in which a disulfide bond within the Fv stabilizes the attached C-terminal variable regions. This format can be represented as its PEGylated version.

[0145] As used herein, Fab-scFv is a Fab molecule with an scFv attached to the C-terminus of a light or heavy chain.

[0146] As used herein, Fab'-scFv is a Fab' molecule with scFv attached to the C-terminus of the light or heavy chain.

[0147] As used herein, DiFab refers to two Fab molecules linked via the C-terminus of the heavy chains.

[0148] As used herein, DiFab' refers to two Fab' molecules linked via one or more disulfide bonds in their hinge region.

[0149] In the context of the present description, an Sc-diabody is a diabody containing a linker between Fv, wherein the molecule contains three linkers and forms a normal scFv, in which the VH and VL ends are linked to one of the variable regions of an additional Fv pair.

[0150] In the context of the present description, Sc-diabody-Fc is two sc-diabodies, each of which is attached to the N-terminus of the CH2 domain of the -CH2CH3 fragment of the constant region, for example, via a hinge.

[0151] In the context of the present description, ScFv-Fc-scFv refers to four scFvs, each of which is attached to the N-terminus and C-terminus of both the heavy and light chains of the -CH2CH3 moiety.

[0152] As used herein, Sc-diabody-CH3 refers to two sc-diabody molecules, each of which is linked to a CH3 domain, such as through a hinge.

[0153] In the context of the present description, IgG-scFv is a full-length antibody with scFv at the C-terminus of each of the heavy chains or each of the light chains.

[0154] In the context of the present description, scFv-IgG is a full-length antibody with an scFv at the N-terminus of each of the heavy chains or each of the light chains.

[0155] In the context of the present description, V-IgG is a full-length antibody with a variable domain at the N-terminus of each of the heavy chains or each of the light chains.

[0156] In the context of the present description, IgG-V is a full-length antibody with a variable domain at the C-terminus of each of the heavy chains or each of the light chains.

[0157] DVD-Ig (also known as double V domain IgG) is a full-length antibody with four additional variable domains, one at the N-terminus of each heavy and light chain.

[0158] The present invention relates to a multispecific antibody comprising one binding domain that specifically binds to HLA-G, wherein said binding domain comprises:

[0159] a. a light chain variable region comprising:

[0160] CDR-L1 containing SEQ ID NO:1,

[0161] CDR-L2 containing SEQ ID NO:2, and

[0162] CDR-L3 containing SEQ ID NO:3; and

[0163] b. a heavy chain variable region comprising:

[0164] CDR-H1 containing SEQ ID NO:4,

[0165] CDR-H2 containing SEQ ID NO:5, and

[0166] CDR-H3 containing SEQ ID NO:6.

[0167] The antibodies of the invention specifically (or selectively) bind HLA-G. An antibody "specifically binds" a protein when it binds preferentially or with high affinity to the protein of interest (e.g., HLA-G), but exhibits little or no binding to other proteins. In other words, the antibody binds to the protein of interest without significant cross-reactivity with any other molecule. The specificity of the antibody can be further studied by determining whether the antibody binds to other related proteins, as discussed above, or whether it is able to differentiate between them.

[0168] In particular, an antibody that "specifically binds" HLA-G is not cross-reactive with another human protein, especially with another HLA-I molecule. In one embodiment, the antibody of the invention substantially does not bind to any of HLA-A, HLA-B, HLA-C, HLA-E, and HLA-F. In one embodiment, the antibody of the invention does not bind to any of HLA-A, HLA-B, HLA-C, HLA-E, and HLA-F. Exemplary sequences of HLA-A, HLA-B, HLA-C, HLA-E, and HLA-F are SEQ ID NOs: 132, 134, 136, 138, and 140, respectively. In one embodiment, the antibody of the invention does not bind to B2m.

[0169] The antibody of the invention can specifically bind the alpha-3 domain of HLA-G. By "specifically binding the alpha-3 domain of HLA-G" is meant that the antibody binds the alpha-3 domain of HLA-G without exhibiting cross-reactivity to another human protein and without cross-reactivity to another domain of HLA-G (e.g., the alpha-1 or alpha-2 domain).

[0170] Cross-reactivity can, for example, be assessed by any suitable method disclosed herein. The cross-reactivity of an antibody can be considered significant if the antibody binds to another molecule with a strength of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% of the strength with which it binds to the protein of interest. An antibody that is specific (or selective) can bind to another molecule with a strength that is less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the strength with which it binds to the protein of interest. The antibody can bind to another molecule with a strength that is less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the strength with which it binds to the protein of interest.

[0171] In one embodiment according to the present invention, the binding of an antibody to HLA-G is characterized by a dissociation constant (K D ) less than 20 nM, in particular less than 15 nM, in particular less than 10 nM, in particular less than 9 nM, in particular less than 8 nM, in particular less than 7 nM, in particular less than 6 nM, in particular less than 5 nM.

[0172] The term "K D "," as used herein, refers to the equilibrium dissociation constant, which is obtained from the ratio K d to K a (i.e. K d / K a ) and expressed as a molar concentration (M). K d and K a refer to the rate of dissociation and the rate of association, respectively, of a particular antigen-antibody (or its antigen-binding fragment) interaction. K values D for antibodies can be determined by methods well known in the art. The method for determining K Dantibodies is by using surface plasmon resonance (SPR), such as the Biacore® system, such as described in the examples provided herein, using recombinant HLA-G or a suitable fusion protein / polypeptide thereof. Typically, K values Dis determined by SPR at 25°C. In one example, affinity is measured using recombinant HLA-G extracellular domain (ECD) expressed in complex with B2m, as described in the Examples herein. For surface plasmon resonance, target molecules are immobilized on a solid phase and exposed to ligands in a mobile phase flowing along a flow cell. If ligand binds to the immobilized target, the local refractive index changes, resulting in a change in the SPR angle, which can be monitored in real time by recording the change in reflected light intensity. The rate of change of the SPR signal can be analyzed to obtain apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these quantities yields the apparent equilibrium constant (affinity) (see, e.g., Wolff et al. Cancer Res. 53:2560–65 (1993)).

[0173] The term "affinity" refers to the strength of interaction between an antibody and HLA-G. Binding affinity to HLA-G can be measured by HLA-G ECD or HLA-G bound or unbound to B2m. For example, binding to HLA-G can be assessed by measuring the binding affinity to a soluble HLA-G ECD comprising the sequence of SEQ ID NO: 108 or 110. In one example, binding to HLA-G is assessed by measuring the binding affinity to a soluble HLA-G ECD, for example, comprising the sequence of SEQ ID NO: 108 or 110, bound to B2m. In one embodiment, an antibody of the invention binds to HLA-G ECD with a dissociation constant (K D) less than 20 nM, in particular less than 15 nM, in particular less than 10 nM, in particular less than 9 nM, in particular less than 8 nM, in particular less than 7 nM, in particular less than 6 nM, in particular less than 5 nM. In one embodiment, the dissociation constant between the antibody of the invention expressed as a full-length antibody and a monomeric form of HLA-G (e.g., HLA-G ECD bound to B2m) is determined by the SPR method at a temperature of 25°C. In one embodiment, the dissociation constant is determined by the SPR method as described in Example 7.1.

[0174] In another example, binding to HLA-G can be assessed by measuring the binding affinity to cell membrane-expressed HLA-G comprising the sequence of SEQ ID NO: 107. Binding to cell membrane-expressed HLA-G can be analyzed by FACS. Typically, cell surface-expressed HLA-G is expressed in dimeric form. In one embodiment, an antibody of the invention binds to HLA-G on cells with a dissociation constant (K D ), determined by FACS, less than 2 nM, preferably less than 1 nM. In one embodiment, the antibody of the invention binds to JEG3 cells with a dissociation constant (K D ), determined by FACS, is less than 1 nM.

[0175] In one embodiment, the present invention relates to an antibody that specifically binds to HLA-G, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and wherein the heavy chain variable region comprises a CDR-H1 comprising SEQ ID NO: 4, a CDR-H2 comprising SEQ ID NO: 5, and a CDR-H3 comprising SEQ ID NO: 6; and wherein the antibody has a dissociation constant (K D ) less than 20 nM, in particular less than 15 nM, in particular less than 10 nM, in particular less than 9 nM, in particular less than 8 nM, in particular less than 7 nM, in particular less than 6 nM or, in particular, less than 5 nM. In one embodiment, the dissociation constant between the antibody of the invention expressed as a full-length antibody and the monomeric form of HLA-G is determined by the SPR method at a temperature of 25°C.

[0176] In one embodiment, the present invention relates to an antibody that specifically binds to HLA-G, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and wherein the heavy chain variable region comprises a CDR-H1 comprising SEQ ID NO: 4, a CDR-H2 comprising SEQ ID NO: 5, and a CDR-H3 comprising SEQ ID NO: 6; and wherein the antibody, expressed as a full-length antibody, binds to JEG3 cells with a dissociation constant (K D ), determined by FACS, less than 2 nM, preferably less than 1 nM.

[0177] In one embodiment, the antibody of the present invention is a blocking antibody. In the context of antibodies, the term "blocking" (or "blocks") describes an antibody that is capable of inhibiting or weakening the binding of a target (HLA-G) to its receptors. In one embodiment, the antibody of the present invention blocks the interaction between HLA-G and ILT2. In one embodiment, the antibody of the present invention blocks the interaction between HLA-G and ILT4. In one embodiment, the antibody of the present invention blocks the interaction between HLA-G and ILT2 and between HLA-G and ILT4. In one embodiment, the antibody of the present invention blocks the interaction between HLA-G and ILT2 and / or between HLA-G and ILT4, when HLA-G is expressed as a monomer and / or dimer and / or trimer.

[0178] Blocking of HLA-G binding to ILT2 and / or ILT4 can be assessed by measuring the blocking of the interaction between the extracellular domain (ECD) of HLA-G, associated or not with B2m expressed on the cell surface, and ILT2 and / or ILT4, for example, expressed as fusion proteins such as Fc fusion proteins (ILT2-Fc, ILT4-Fc). An ILT2-rabbit Fc fusion protein that comprises, for example, SEQ ID NO: 142 can be used. An ILT4-rabbit Fc fusion protein that comprises, for example, SEQ ID NO: 144 can be used. Blocking of HLA-G binding to ILT2 and / or ILT4 can be assessed as described in Example 8.

[0179] In some embodiments, the antibody of the invention does not block the association between HLA-G and B2m. In some embodiments, the antibody of the invention does not block the association between HLA-G and related peptides naturally expressed in complex with HLA-G.

[0180] In some embodiments, an antibody of the invention inhibits HLA-G multimerization. In some embodiments, an antibody of the invention inhibits HLA-G dimerization. In some embodiments, an antibody of the invention inhibits HLA-G trimerization.

[0181] In one embodiment of the IC 50 the antibodies of the present invention for blocking ILT2 binding to HLA-G is less than 50 pM, preferably IC 50 the antibodies of the present invention for blocking the binding of ILT2 to HLA-G, which is naturally expressed on the surface of JEG3 cells, is less than 40 pM, or less than 30 pM, or less than 20 pM, as determined, for example, by an in vitro assay using a large reaction volume, as described in Example 8. In a preferred embodiment, the IC 50the antibody of the present invention for blocking ILT2 binding to HLA-G, which is naturally expressed on the surface of JEG3 cells, is less than 20 pM. In one embodiment, ILT2 is expressed as an ILT2-rabbit Fc fusion protein comprising, for example, SEQ ID NO: 142. In one embodiment, the IC 50 the antibodies of the present invention for blocking ILT4 binding to HLA-G is less than 1800 pM, preferably IC 50 The antibody of the present invention for blocking ILT4 binding to HLA-G in an in vitro assay, as disclosed herein, is less than 1500 pM or less than 1400 pM. In one embodiment, ILT4 is expressed as an ILT4-rabbit Fc fusion protein comprising, for example, SEQ ID NO: 144. Blocking HLA-G binding to ILT4 can be assessed as described in Example 8.

[0182] In one embodiment, the present invention relates to an antibody that specifically binds to HLA-G, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and wherein the heavy chain variable region comprises a CDR-H1 comprising SEQ ID NO: 4, a CDR-H2 comprising SEQ ID NO: 5, and a CDR-H3 comprising SEQ ID NO: 6; and wherein the antibody has:

[0183] a. IC 50 to block the binding of ILT2 to HLA-G naturally expressed on the surface of JEG3 cells, which is less than 20 pM, as determined, for example, by an in vitro assay using a large reaction volume, as described in Example 8; and / or

[0184] b.IC 50 to block the binding of ILT4 to HLA-G, which is less than 1400 pM, as described, for example, in Example 8.

[0185] IC term 50, as used herein, refers to the half-maximal inhibitory concentration, which is a measure of the effectiveness of a substance, such as an antibody, in inhibiting a specific biological or biochemical function, which in the present invention is the binding activity of ILT2 or ILT4 to HLA-G. IC 50 is a quantitative measure indicating how much of a particular substance is needed to inhibit half of a given biological process, function, or activity.

[0186] In some embodiments, an antibody of the present invention inhibits HLA-G-mediated immunosuppressive function. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated immunosuppressive function by blocking the interaction between HLA-G and ILT2 and / or the interaction between HLA-G and ILT4. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated suppressive function of NK cells. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated suppressive function of cytotoxic T lymphocytes, such as CD8+ T lymphocytes and / or CD4+ T lymphocytes. In some embodiments, an antibody of the invention inhibits HLA-G-mediated suppressive function of regulatory T lymphocytes. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated B cell suppressive function.In some embodiments, an antibody of the present invention inhibits HLA-G-mediated suppressive function of monocytes. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated suppressive function of macrophages. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated suppressive function of dendritic cells. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated suppression of neutrophils. In some embodiments, an antibody of the present invention inhibits HLA-G-mediated suppression of phagocytosis.

[0187] In some embodiments, the antibody of the invention induces the production of proinflammatory cytokines such as TNF-alpha, IL-1, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 (also known as IL-12p70), IL-13, IL-15, IL-18, IFN-gamma, GM-CSF, CCL2, CCL3, CCL4, CCL5, TNF-alpha. In some embodiments, the antibody of the present invention promotes the recruitment of immune cells (monocytes, macrophages, dendritic cells, B cells, T cells or NK cells) to the tumor microenvironment. In some embodiments, the antibody of the present invention inhibits HLA-G function on tumor cells expressing HLA-G. In some embodiments, an antibody of the invention induces myeloid cell activation. In some embodiments, an antibody of the invention induces tumor cell killing, for example, through ADCC or CDC.In some embodiments, an antibody of the invention induces phagocytosis of tumor cells, such as ADCP. In some embodiments, an antibody of the invention inhibits angiogenesis. In some embodiments, an antibody of the invention inhibits tumor cell metastasis. In some embodiments, an antibody of the invention inhibits tumor cell proliferation.

[0188] In one embodiment, the invention relates to an antibody that specifically binds to HLA-G, comprising:

[0189] a. a light chain variable region comprising:

[0190] CDR-L1 containing SEQ ID NO:1,

[0191] CDR-L2 containing SEQ ID NO:2, and

[0192] CDR-L3 comprising SEQ ID NO:3; and

[0193] b. a heavy chain variable region comprising:

[0194] CDR-H1 containing SEQ ID NO:4,

[0195] CDR-H2 containing SEQ ID NO:5, and

[0196] CDR-H3 containing SEQ ID NO:6,

[0197] wherein the antibody blocks the binding of HLA-G to ILT2 and / or ILT4, preferably to ILT2 and ILT4, and wherein the antibody has a dissociation constant (K D ) with HLA-G less than 10 nM. In one embodiment, the K value D determined by the SPR method at 25°C using a full-length antibody of the invention. In one embodiment, the dissociation constant is determined for the monomeric form of HLA-G.

[0198] In one embodiment, the invention relates to an antibody that specifically binds to HLA-G, comprising:

[0199] a. a light chain variable region comprising:

[0200] CDR-L1 containing SEQ ID NO:1,

[0201] CDR-L2 containing SEQ ID NO:2, and

[0202] CDR-L3 containing SEQ ID NO:3; and

[0203] b. a heavy chain variable region comprising:

[0204] CDR-H1 containing SEQ ID NO:4,

[0205] CDR-H2 containing SEQ ID NO:5, and

[0206] CDR-H3 containing SEQ ID NO:6,

[0207] wherein the antibody inhibits at least one of the HLA-G-mediated immunosuppressive functions described above.

[0208] Antibodies for use in the present invention may be chimeric antibodies, humanized antibodies, or fully human antibodies.

[0209] In one embodiment, the antibody is chimeric. The term "chimeric" antibody refers to an antibody in which the variable domain (or at least a portion thereof) of the heavy and / or light chain is obtained from a specific source or species, such as a mouse, rat, rabbit, or similar species, while the remainder of the heavy and / or light chain (i.e., the constant region) is derived from another species, such as a human (Morrison; PNAS 81, 6851 (1984))). Chimeric antibodies are composed of elements obtained from two different species while retaining the characteristics of the species from which they are obtained. A subcategory of "chimeric antibodies" are "humanized antibodies".

[0210] Chimeric antibodies are typically produced using recombinant DNA techniques. The DNA can be modified by replacing the coding sequence of the constant regions of the human L and H chains with the corresponding constant regions of the H and L chains of non-human origin (e.g., mouse or rabbit).

[0211] Humanized antibodies (which include CDR-grafted antibodies) are antibody molecules that comprise one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e.g., U.S. Patent 5,585,089; WO91 / 09967). It should be understood that it may be necessary to transfer only the specificity-determining residues of the CDRs, rather than the entire CDR (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally comprise one or more framework region residues derived from the non-human species from which the CDRs are derived.

[0212] Fully human antibodies are antibodies in which the variable regions and constant regions (if present) of both the heavy and light chains are all of human origin or are substantially identical to sequences of human origin, but are not necessarily derived from the same antibody. Examples of fully human antibodies may include antibodies produced, for example, by the phage display methods described above, and antibodies produced by mice in which the variable region of a mouse immunoglobulin and, optionally, the constant region genes are replaced by human counterparts, for example as described generally in EP 0546073, US 5545806, US 5569825, US 5625126, US 5633425, US 5661016, US 5770429, EP 0438474 and EP 0463151.

[0213] In one embodiment, the antibody is human. Human antibodies comprise heavy or light chain variable regions or full-length heavy or light chains that are a "product" or "derived from" a specific germline sequence if the variable regions or full-length chains of the antibody are obtained from a system that utilizes human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest or screening a phage-displayed library of human immunoglobulin genes for the antigen of interest.A human antibody or antibody fragment that is a "product" of or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is most closely related (i.e., has the highest % identity) to the human antibody sequence. A human antibody that is a "product" of or "derived from" a particular human germline immunoglobulin sequence may have amino acid differences relative to the germline sequence caused, for example, by naturally occurring somatic mutations or the intentional introduction of a site-directed mutation.However, the amino acid sequence of the selected human antibody is typically at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared to the amino acid sequence of the germline immunoglobulin of other species (e.g., mouse germline sequences). In some cases, the amino acid sequence of the human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene.Typically, a human antibody derived from a specific human germline sequence will differ by no more than 10 amino acids from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody may differ by no more than 5 or even no more than 4, 3, 2, or 1 amino acid from the amino acid sequence encoded by the germline immunoglobulin gene.

[0214] Human antibodies can be produced by several methods known to those skilled in the art. Human antibodies can be produced by the hybridoma method using human myeloma cell lines or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Single Antibody Production Techniques and Applications, pp51-63, Marcel Dekker Inc., 1987). Alternative methods include the use of phage libraries or transgenic mice that utilize repertoires of human variable regions (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:11-23).

[0215] The antibodies of the present invention can be produced by any suitable method known in the art. To produce antibodies that specifically recognize HLA-G, HLA-G, including its fusion proteins, and cells (recombinantly or naturally) expressing HLA-G can be used. Various forms of HLA-G, as disclosed herein, can be used.

[0216] In another embodiment, the antigen used is HLA-G, preferably expressed on the surface of rabbit fibroblast cells, preferably produced as described in the examples below. In one embodiment, the antigen used is a complex of HLA-G, preferably expressed on the surface of rabbit fibroblast cells and preferably produced as described in the examples below, with B2m.

[0217] HLA-G or fragments thereof for use in host immunization can be obtained by methods well known in the art from genetically engineered host cells containing expression systems. In some cases, HLA-G or a fragment thereof may be part of a larger protein, such as a fusion protein, for example, fused with an affinity tag, etc.

[0218] Antibodies produced against HLA-G can be obtained, if immunization of an animal is required, by administering HLA-G or a portion thereof to an animal, preferably a non-human animal, using well-known and routine protocols, see, for example, Handbook of Experimental Immunology, DM Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many animals can be immunized, such as rabbits, mice, rats, sheep, cows, camels, or pigs. However, mice, rabbits, pigs, and rats are typically used. In one embodiment, the antibody of the invention is obtained by administering rat fibroblasts expressing HLA-G on their surface.

[0219] Monoclonal antibodies can be produced by various methods, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and methods using transgenic animals containing all or part of the human immunoglobulin loci. Several exemplary methods for producing monoclonal antibodies are disclosed herein.

[0220] For example, monoclonal antibodies can be produced by the hybridoma method (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma method, the human B-cell hybridoma method (Kozbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan RLiss, Inc., 1985).

[0221] Antibodies for use in the present invention can also be obtained by methods for producing an antibody from a single lymphocyte by cloning and expressing immunoglobulin variable region cDNA obtained from single lymphocytes selected for producing specific antibodies, for example, by the methods described in Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; in W092 / 02551; WO2004 / 051268 and International Patent Application WO2004 / 106377.

[0222] Monoclonal antibodies can also be produced by various phage display methods known in the art, including those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182:41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187:9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280). In some phage display methods, VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage, as described by Winter et al., Ann. Rev. Immunol, 12:433–455 (1994). Phage typically display antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need for hybridoma construction. Alternatively, a naive repertoire can be cloned (e.g., from humans) to generate a single source of antibodies to a wide range of foreign as well as self-antigens without any immunization, as described by Griffiths et al., EMBO J 12:725–734 (1993). Finally, naive libraries can also be created synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequence to encode highly variable CDR3 regions and rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381–388 (1992).Patent publications describing phage libraries of human antibodies include, for example: US 5750373 and US 2005 / 0079574, US2005 / 0119455, US2005 / 0266000, US2007 / 0117126, US2007 / 0160598, US2007 / 0237764, US2007 / 0292936 and US2009 / 0002360.

[0223] Antibody screening can be performed using assays that measure binding to HLA-G and / or assays that measure the ability to block HLA-G binding to one or more of its receptors. An example of a binding assay is an ELISA, for example, using a fusion protein of the target polypeptide immobilized on plates and a conjugated secondary antibody to detect antibody binding to the target. An example of a blocking assay is a flow cytometric assay that measures the blockade of protein ligand binding to the target polypeptide. A fluorescently labeled secondary antibody is used to determine the amount of such protein ligand binding to the target polypeptide.

[0224] Antibodies can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods for screening such libraries for antibodies possessing desired binding characteristics are known in the art.

[0225] The antibody of the present invention may comprise framework regions of an animal in which antibody formation has been initiated. For example, it will comprise the CDRs as defined above and the framework regions of a rabbit antibody, such as an antibody comprising the light chain variable region according to SEQ ID NO: 7 (the nucleotide sequence of which is shown in SEQ ID NO: 8) and the heavy chain variable region according to SEQ ID NO: 11 (the nucleotide sequence of which is shown in SEQ ID NO: 12).

[0226] In one preferred embodiment, the antibody of the present invention is humanized.

[0227] In one preferred embodiment, an antibody that binds to HLA-G, wherein the antibody is a humanized antibody, comprises a light chain variable region and a heavy chain variable region, wherein:

[0228] a. the light chain variable region comprises a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and

[0229] b. The heavy chain variable region comprises CDR-H1 comprising SEQ ID NO: 4, CDR-H2 comprising SEQ ID NO: 5, and CDR-H3 comprising SEQ ID NO: 6.

[0230] As used herein, the term "humanized" antibody refers to an antibody in which the heavy and / or light chain comprises one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g., a non-human antibody, such as a mouse or rabbit monoclonal antibody) grafted into the variable region framework of the heavy and / or light chain of an acceptor antibody (e.g., a human antibody). See review by Vaughan et al., Nature Biotechnology, 16:535-539, 1998. In one embodiment, instead of transferring the entire CDR to the human antibody framework, only one or more specificity-determining residues from any CDR described above are transferred (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity determining residues from one or more CDRs described above are transferred into the human antibody framework.In another embodiment, only the specificity determining residues from each CDR described above are transferred into the human antibody framework.

[0231] Any suitable acceptor variable region framework sequence can be used in CDR grafting, taking into account the class / type of the donor antibody from which the CDRs are derived, including mouse, primate, and human framework regions.

[0232] Preferably, the humanized antibody of the present invention has a variable domain comprising human acceptor framework regions and one or more CDRs specifically described herein. Thus, in one embodiment, a humanized antibody is provided that binds HLA-G, wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.

[0233] Examples of human scaffolds that can be used in the present invention include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY, and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences can be used; they are available at http: / / www.imgt.org / .

[0234] In the humanized antibody of the present invention, the acceptor heavy and light chains do not necessarily have to be derived from the same antibody and, if desired, may comprise composite chains having framework regions derived from different chains.

[0235] A suitable framework region of the light chain of the humanized antibody of the present invention is obtained from the human germline IGKV1D-13 IGKJ4 having SEQ ID NO: 103, the nucleotide sequence of which is shown in SEQ ID NO: 104.

[0236] A suitable heavy chain framework region of the humanized antibody of the present invention is obtained from the human germline IGHV3-66 IGHJ4 having the sequence shown in SEQ ID NO: 105, the nucleotide sequence of which is shown in SEQ ID NO: 106.

[0237] Accordingly, in one embodiment, a humanized antibody is provided that binds to HLA-G, wherein the antibody comprises a light chain variable region and a heavy chain variable region and wherein:

[0238] a. The variable region of the light chain comprises:

[0239] i. CDR-L1 comprising SEQ ID NO: 1; and

[0240] ii. CDR-L2 containing SEQ ID NO: 2; And

[0241] iii. CDR-L3 comprising SEQ ID NO: 3; and

[0242] b. variable region of the heavy chain, contains:

[0243] i. CDR-H1 comprising SEQ ID NO: 4; and

[0244] ii. CDR-H2 containing SEQ ID NO: 5 and

[0245] iii. CDR-H3 comprising SEQ ID NO: 6; and

[0246] wherein the light chain framework region is derived from the human germline IGKV1D-13 IGKJ4 comprising SEQ ID NO: 103; and the heavy chain framework region is derived from the human germline IGHV3-66 IGHJ4 comprising SEQ ID NO: 105.

[0247] In one embodiment, the antibody of the present invention comprises:

[0248] a. a light chain variable region comprising SEQ ID NO: 19 or 15 or 23; and / or

[0249] b. a heavy chain variable region comprising SEQ ID NO: 93, 27, 33, 57, 69, 75, 81 or 87.

[0250] In one embodiment, the antibody of the present invention comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 93.

[0251] In one embodiment, an antibody of the present invention comprises a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 27. In one embodiment, an antibody of the present invention comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 27. In one embodiment, an antibody of the present invention comprises a light chain variable region comprising SEQ ID NO: 23 and a heavy chain variable region comprising SEQ ID NO: 27. In one embodiment, an antibody of the present invention comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 33.In one embodiment, an antibody of the invention comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 57. In one embodiment, an antibody of the invention comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 69. In one embodiment, an antibody of the invention comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 75. In one embodiment, an antibody of the invention comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 81. In one embodiment, an antibody of the invention comprises a light chain variable region comprising SEQ ID NO: 19, and a heavy chain variable region comprising SEQ ID NO: 87.In one embodiment, an antibody of the invention comprises a light chain variable region comprising SEQ ID NO: 23 and a heavy chain variable region comprising SEQ ID NO: 93.

[0252] In one embodiment, the antibody of the invention is IgG1. In one embodiment, the antibody of the present invention is IgG1 and comprises:

[0253] a. a light chain comprising SEQ ID NO: 21 or 17 or 25; and / or

[0254] b. a heavy chain comprising SEQ ID NO: 95, 29, 35, 59, 71, 77, 83 or 89.

[0255] In one embodiment, the antibody of the present invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 95.

[0256] In one embodiment, an antibody of the present invention comprises a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 29. In one embodiment, an antibody of the present invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 29. In one embodiment, an antibody of the present invention comprises a light chain comprising SEQ ID NO: 25 and a heavy chain comprising SEQ ID NO: 29. In one embodiment, an antibody of the invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 35. In one embodiment, an antibody of the invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 59. In one embodiment, an antibody according to the present invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 71.In one embodiment, an antibody of the present invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 77. In one embodiment, an antibody of the present invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 83. In one embodiment, an antibody of the present invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 89. In one embodiment, an antibody of the present invention comprises a light chain comprising SEQ ID NO: 25 and a heavy chain comprising SEQ ID NO: 95.

[0257] Preferably, IgG1 contains an active Fc fragment, i.e., it has Fc-mediated effector functions. Thus, in one embodiment, the antibody of the invention has Fc-mediated effector functions.

[0258] The term "effector functions" refers to the biological activities inherent in the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP).

[0259] The term “complement-dependent cytotoxicity” or “CDC” refers to a mechanism of cell death induction in which the Fc effector domain of a target-bound antibody binds to and activates the complement component C1q, which in turn activates the complement cascade leading to target cell death.

[0260] The term "antibody-dependent cellular cytotoxicity" or "ADCC" is a cell death induction mechanism that depends on the interaction of antibody-coated target cells with effector cells possessing lytic activity, such as natural killer cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on effector cells.

[0261] The term "antibody-dependent cellular phagocytosis" or "ADCP" is a mechanism of phagocytosis induction that depends on the interaction of antibody-coated target cells or antibody-coated soluble targets with effector cells with phagocytic activity, such as macrophages and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells.

[0262] As described in the Examples, the expression pattern of HLA-G in normal, non-tumor tissues was examined, and it was found that HLA-G forms containing the epitope associated with the antibody of the invention are not expressed in healthy tissues, especially in pancreatic and pituitary tissues.

[0263] This contrasts with an earlier report by Cirulli et al. on HLA-G protein expression in pancreatic islets (Cirulli et al, DIABETES, Vol. 55, May 2006); they described the observed significant upregulation of HLA-G in islet cells cultured on an extracellular matrix that supports cell replication. For example, Boegel et al. also reported HLA-G gene expression in the pituitary gland, as well as in pancreatic islets and testes (Boegel et al, BMC Medical Genomics (2018) 11:36).

[0264] Thus, the results described in the examples of this disclosure are unexpected and demonstrate that, contrary to expectations based on the teachings of the prior art, an anti-HLA-G antibody capable of killing HLA-G-expressing cells, for example, via Fc-mediated effector functions, is a potential candidate for the treatment of solid tumors without the expected toxicity to patients due to binding to normal tissues. Furthermore, an antibody containing an active Fc can promote the recruitment and activation of effector cells into the tumor microenvironment (TME). Furthermore, direct killing of HLA-G+ tumor cells can induce the release of tumor antigens into the local environment, which will further stimulate the immune response.

[0265] The dual mechanism of such an antibody disclosed herein, capable of blocking the interaction between HLA-G and its inhibitory receptors and capable of killing cells, represents a significant advantage for the treatment of patients with upregulated HLA-G, such as in solid cancer.

[0266] Furthermore, tumor heterogeneity suggests that the importance of each mechanism (HLA-G blockade to stimulate immune cell activation and direct tumor cell killing via active Fc-dependent mechanisms) may vary among patients. Thus, multiple tumor cell killing mechanisms may benefit a broader patient population due to their ability to target different mechanisms in tumors with different characteristics, such as diverse HLA-G expression patterns.

[0267] Methods for selecting anti-HLA-G antibodies according to the invention

[0268] Due to the specific challenges associated with the production of anti-HLA-G antibodies (such as high homology to other HLA-I, identification of antibodies capable of blocking the interaction between HLA-G and its inhibitory receptors) and to identify antibodies that would be useful in therapy, there was a need to develop a dedicated detection, screening and testing strategy that includes measuring binding to HLA-G, assessing the binding affinity and specificity (absence of cross-reactivity with other HLA-I) and assessing the functional properties of the antibodies tested, as well as high-throughput measurement of structural aspects of binding (target epitope residues).

[0269] Therefore, a method for identifying an antibody according to the invention is provided, comprising:

[0270] a) immunizing a non-human mammal with an HLA-G immunogenic composition;

[0271] b) isolating B cells from said non-human mammal;

[0272] c) selection of antibodies produced by the said B cells that have the following properties:

[0273] i. bind to HLA-G with an affinity represented by the dissociation constant K D less than 20 nM; and

[0274] ii. not bind to HLA-I other than HLA-G; and

[0275] iii. Block the binding between HLA-G and ILT2 and / or between HLA-G and ILT4

[0276] Step a)

[0277] "Immunogenic composition" refers to a composition capable of eliciting an immune response in a non-human mammal upon administration of said composition. In a mammal to which the immunogenic composition is administered, such a composition typically provides expression of an immunogenic antigen of interest, to which antibodies can be produced as part of an immune response. "HLA-G immunogenic composition" refers to a composition capable of generating an immune response against HLA-G in a mammal to which said composition is administered.

[0278] "Immunization with protein" refers to a method of administering an immunogenic protein containing an antigen of interest, or an immunogenic portion of said protein containing said antigen of interest or an immunogenic portion thereof.

[0279] In one embodiment, the immunogenic composition comprises a full-length protein. In another embodiment, the immunogenic composition comprises an immunogenic portion of the protein. For example, in one embodiment, the immunogenic composition comprises full-length HLA-G in complex with B2m. In another embodiment, the immunogenic composition comprises full-length HLA-G in the absence of B2m. In another embodiment, the immunogenic composition comprises an immunogenic portion of HLA-G, with or without association with B2m. In another embodiment, the immunogenic composition comprises the extracellular domain of HLA-G in complex with B2m. In another embodiment, the immunogenic composition comprises the extracellular domain of HLA-G in the absence of B2m.

[0280] "DNA immunization" refers to a method of directly introducing into mammalian cells a genetically engineered nucleic acid molecule encoding a full-length protein or an immunogenic portion thereof containing an antigen of interest (also referred to herein as a nucleic acid-based vaccine or DNA immunization) to elicit an immunological response in said cells against said antigen of interest. DNA immunization utilizes the host's cellular machinery to express the peptide(s) corresponding to the introduced nucleic acid molecule and / or achieve the desired effect, in particular antigen expression at the cellular level, as well as immunotherapeutic effect(s) at the cellular level or within the host organism.

[0281] "Cellular immunization" refers to a method of administering cells that naturally express or that are transfected with an immunogenic protein containing an antigen of interest or an immunogenic portion of said protein containing said antigen of interest or an immunogenic portion thereof. In one embodiment, the immunization in step a) is carried out using cellular immunization with fibroblasts transfected with an immunogenic protein containing an antigen of interest or an immunogenic portion of said protein containing said antigen of interest or an immunogenic portion thereof.

[0282] By "immunogenic portion" is meant a portion of a protein or antigen of interest that retains the ability to induce an immune response in a non-human mammal when said portion of the protein or antigen of interest or DNA encoding it is administered to trigger the production of the antibodies of the invention.

[0283] HLA-G, including its fusion proteins, cells (recombinantly or naturally) expressing HLA-G, can be used to produce antibodies that specifically recognize HLA-G. Various forms of HLA-G can be used, as disclosed herein.

[0284] HLA-G or fragments thereof for use in host immunization can be obtained by methods well known in the art from genetically engineered host cells containing expression systems, or they can be isolated from natural biological sources. In some cases, HLA-G or a fragment thereof can be part of a larger protein, such as a fusion protein, for example, fused with an affinity tag, etc.

[0285] In one embodiment, the immunization step can be performed by protein immunization, DNA immunization, or cellular immunization, or any combination thereof.

[0286] In one embodiment, the non-human mammal is a mouse. In one embodiment, the non-human mammal is a rat. In one embodiment, the non-human mammal is a rabbit. In one embodiment, the rabbit is a New Zealand White rabbit. In one embodiment, the mammal is immunized by subcutaneous injection of an immunogenic composition. In one embodiment, the immunogenic composition comprises rabbit fibroblast cells transiently expressing HLA-G on the cell surface. In one example, the rabbit fibroblast cells are transfected with a DNA sequence encoding HLA-G that comprises SEQ ID NO: 111. In another embodiment, the immunogenic composition comprises rabbit fibroblast cells transiently co-expressing HLA-G and B2m on the cell surface.In one embodiment, the rabbit fibroblast cells are Rab9 rabbit fibroblast cells.

[0287] The immunization step can be performed according to a primary immunization protocol, implying a first administration (primary immunization or primary administration) of the immunogenic composition, and then at least one additional administration (booster immunization or booster administration), which is separated in time from the primary administration within the immunization protocol. Booster immunization includes one, two, three or more immunizations. In one embodiment, booster immunization includes two administrations of the immunogenic composition at 14-day intervals. In one embodiment, the immunization step includes a primary administration of rabbit fibroblasts transiently expressing HLA-G on the cell surface, followed by two booster immunizations at 14-day intervals. In one embodiment, the rabbit fibroblast cells are Rab9 rabbit fibroblast cells.

[0288] In one embodiment, primary immunization includes the administration of an adjuvant. In one embodiment, the adjuvant is administered at a site different from the injection site of the immunogenic composition. In one embodiment, the adjuvant is Freund's adjuvant. In one embodiment, both primary immunization and booster immunization include the administration of an adjuvant, such as Freund's adjuvant.

[0289] In one embodiment, the immunization step comprises a primary immunization in the presence of a first adjuvant followed by at least one booster immunization in the presence of a second adjuvant.

[0290] In one embodiment, the immunogenic composition is administered by subcutaneous injection, for example, into the shoulder.

[0291] "Adjuvant" refers to an immunostimulant. Adjuvants are substances well known in the art. Traditional adjuvants that act as immunostimulants or antigen delivery systems, or both, include, for example, alum, polysaccharides, liposomes, biodegradable polymer-based nanoparticles, and lipopolysaccharides. For example, an adjuvant can be Freund's adjuvant, Montanide's adjuvant, or Fama's adjuvant.

[0292] Step b)

[0293] Methods for isolating B cells are well known and typically involve isolating B cells from PBMCs (peripheral blood mononuclear cells), bone marrow, or secondary lymphoid organs, i.e., from a lymph node or spleen. In one embodiment, isolating antigen-specific memory B cells is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after immunization step a). In one embodiment, isolating B cells is performed 14 days after immunization step a). In one embodiment, step b) comprises sorting antigen-specific B cells by flow cytometry.

[0294] Stage c)

[0295] The screening steps in step c) can be performed in accordance with methods for measuring binding and blocking activity, such as those disclosed herein:

[0296] i. binds to HLA-G with an affinity represented by the dissociation constant (K D ) less than 20 nM.

[0297] Binding can be determined by soluble and / or membrane-bound HLA-G. Binding to HLA-G can be determined by surface plasmon resonance, such as using the Biacore® system, such as described in the examples. In one example, affinity for recombinant ECD HLA-G in complex with B2m is measured using Biacore, as disclosed in the examples of this specification, and antibodies that bind to K can be selected for further analysis. D less than 20 nM. In one example, the dissociation constant K D The difference between the antibody expressed in the form of full-length antibody and the monomeric form of HLA-G is determined by the SPR method at a temperature of 25°C.

[0298] Alternatively or additionally, binding to HLA-G can be determined by FACS in HLA-G-expressing cells, such as HEK293 cells transfected with HLA-G and B2m, or JEG3 cells naturally expressing HLA-G. Methods for measuring antibody affinity to HLA-G using FACS are presented in the examples described.

[0299] Advantageously, antibodies can be screened using binding assays to both soluble HLA-G and cell-expressed HLA-G.

[0300] ii. Does not bind to HLA-I except HLA-G

[0301] To assess binding specificity, i.e., the absence of cross-reactivity with other HLA-Is, a special screening strategy was developed. This strategy involved the creation of variant HLA-G constructs in which HLA-G-specific amino acids were replaced with consensus amino acids found in other HLA-Is ("HLA-G Null constructs"). The HLA-G Null constructs described in Example 1 may be particularly useful for screening HLA-G-specific antibodies.

[0302] "HLA-G Null 1,2,3" corresponds to an HLA-G variant in which the amino acids specifically expressed on HLA-G α1, α2 and α3 are replaced by consensus amino acids expressed on other HLA-I (20 amino acids are mutated).

[0303] In one embodiment, the method for identifying an antibody of the invention comprises screening antibodies to "HLA-G Null 1,2,3" obtained after step b) and selecting antibodies for which no binding is detected. Screening can be performed for the presence of "HLA-G Null 1,2,3" expressed on the cell surface (e.g., comprising SEQ ID NO: 115) or for the presence of soluble "HLA-G Null 1,2,3" (ECD) (e.g., comprising SEQ ID NO: 113).

[0304] Binding to other HLA-Is can be further assessed using the methods described in the examples. HEK293 cells can be transfected with DNA sequences encoding HLA-A, B, C, E, or F (e.g., DNA sequences containing SEQ ID NOs: 131, 133, 135, 137, and 139, respectively) and B2m (e.g., DNA sequences containing SEQ ID NO: 130). Binding to cells expressing HLA-Is can be assessed using FACS.

[0305] iii. Block the binding between HLA-G and ILT2 and / or between HLA-G and ILT4

[0306] Blocking the binding of HLA-G to ILT2 and / or ILT4 can be assessed by measuring the blocking of the interaction between HLA-G associated with B2m expressed on the surface of cells (e.g., naturally expressed on the surface of JEG3 cells or transiently expressed on the surface of HCT116 cells, as described in the Examples) and ILT2 and / or ILT4, for example, expressed as fusion proteins such as Fc-fusion proteins (ITT2-Fc, ILT4-Fc).

[0307] Antibodies having the desired properties and selected after step c) can be further characterized and differentiated based on additional assays, which include, for example, specificity assays, ADCC, ADCP, CDC, biophysical assays and stability assays, as well as based on the ability to modulate the immune environment and induce tumor cell killing in primary human tumors in culture ex vivo, for example, by the methods disclosed in the examples of the present description.

[0308] Epitope

[0309] In the present invention, the term "epitope" is used interchangeably for both conformational and linear epitopes. A conformational epitope consists of discontinuous regions of the primary amino acid sequence of an antigen, while a linear epitope is formed by a sequence of continuous amino acids.

[0310] In one embodiment, the antibody of the invention specifically binds to the alpha-3 domain of HLA-G. In one embodiment, the antibody of the present invention binds to an HLA-G epitope comprising residues F195 and Y197 according to SEQ ID NO: 107.

[0311] In some embodiments, an antibody of the invention binds to an HLA-G epitope, wherein said epitope comprises residues V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250 and Y257 (wherein the numbering corresponds to SEQ ID NO: 107).

[0312] In one embodiment, the antibody of the invention does not bind to B2m. Thus, the antibody of the invention preferentially binds to HLA-G in complex with B2m or in the absence of B2m. In one embodiment, the antibody of the invention does not bind to the same binding site on HLA-G as related HLA-G peptides naturally expressed in complex with HLA-G. Therefore, in one embodiment, the antibody of the invention does not block the association between HLA-G and related peptides naturally expressed in complex with HLA-G.

[0313] In one embodiment, the present invention relates to an anti-HLA-G antibody that binds to an epitope of HLA-G, wherein said epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all residues selected from the list consisting of V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250, and Y257 of HLA-G (SEQ ID NO: 107). In one embodiment, the present invention relates to a humanized IgG1 antibody that binds to an HLA-G epitope, wherein the epitope comprises residues V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250, and Y257 of human HLA-G (SEQ ID NO: 107). In one embodiment, the antibody is afucosylated IgG1.

[0314] In one embodiment, the present invention relates to an anti-HLA-G antibody that binds to an HLA-G epitope, wherein said epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all residues selected from the list consisting of V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250, and Y257 of HLA-G (SEQ ID NO: 107), determined at a contact distance of less than 4 Å. In one embodiment, the antibody is afucosylated IgG1.

[0315] In one embodiment, the present invention relates to a humanized IgG1 antibody that binds to an HLA-G epitope, wherein said epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all residues selected from the list consisting of V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250, and Y257 of HLA-G (SEQ ID NO: 107), determined at a contact distance of less than 4 Å. In one embodiment, the antibody is afucosylated IgG1.

[0316] In one embodiment, the present invention relates to a humanized IgG1 antibody that binds to an HLA-G epitope, wherein said epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all residues selected from the list consisting of V194, F195, Y197, E198, R219, Q224, Q226, D227, V248, V249, P250, E253, and Y257 of HLA-G (SEQ ID NO: 107), determined at a contact distance of less than 5 Å. In one embodiment, the antibody is afucosylated IgG1.

[0317] The epitope can be identified by any suitable epitope mapping method known in the art in combination with any of the antibodies provided by the present invention. Examples of such methods include screening peptides of various lengths derived from full-length HLA-G for binding to the antibody or a fragment thereof of the present invention and identifying the smallest fragment that can specifically bind to the antibody containing the sequence of the epitope recognized by the antibody. HLA-G peptides can be produced synthetically or by proteolytic cleavage of HLA-G. Antibody-binding peptides can be identified, for example, by mass spectrometric analysis. Methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), or hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS) can be used to identify the epitope bound by the antibody.Typically, when epitope identification is performed using X-ray crystallography, the amino acid residues comprising the epitope are considered to be those of the antigen located within 4 Å of the CDR. Once identified, the epitope can be used to generate fragments that bind to the antibody of the present invention and, if necessary, can be used as an immunogen to generate additional antibodies binding the same epitope.

[0318] The epitope specified in aspects and embodiments of the present invention is preferably an epitope characterized by X-ray crystallography. In one embodiment, the present invention relates to an anti-HLA-G antibody that binds to an HLA-G epitope, wherein said epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all residues selected from the list consisting of V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250, and Y257 of HLA-G (SEQ ID NO: 107), determined at a contact distance of less than 4 Å, wherein the epitope is characterized by X-ray crystallography.

[0319] In one embodiment, the present invention relates to a humanized IgG1 antibody that binds to an HLA-G epitope, wherein said epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all residues selected from the list consisting of V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250, and Y257 of HLA-G (SEQ ID NO: 107), determined at a contact distance of less than 4 Å, wherein the epitope is characterized by X-ray crystallography. In one embodiment, the antibody is afucosylated IgG1.

[0320] In one embodiment, the present invention relates to a humanized IgG1 antibody that binds to an HLA-G epitope, wherein said epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all residues selected from the list consisting of V194, F195, Y197, E198, R219, Q224, Q226, D227, V248, V249, P250, E253, and Y257 of HLA-G (SEQ ID NO: 107), determined at a contact distance of less than 5 Å by X-ray crystallography. In one embodiment, the antibody is afucosylated IgG1.

[0321] Furthermore, HDX-MS and NMR can be used to analyze interactions in solution, revealing allosteric or conformational changes not always apparent from crystallography. For example, HDX-MS data from a 30-second incubation with deuterium identified potential binding sites: 178-MLQRADPPKTHVTHHPVFD-196 and 214-ILTWQRDGEDQTQDVEL-230.

[0322] In one embodiment, the epitope determined by NMR using more stringent criteria that considers the excess of the average of all calculated shifts (>0.0764) comprises residues T200, L201, L215, W217, R219, D220, E229, A245, A246, V247, V249, S251, E253, Q255, T258, H260, V261, and W274.

[0323] In one embodiment, the epitope, as determined by NMR using more stringent criteria that considers the average of all calculated shifts (>0.1597), comprises residues H191, Y197, E198, R202, L230, V248, G252, C259, and K275.

[0324] Antibodies may compete for binding to HLA-G or bind to the same epitope as antibodies defined above in terms of light chain, heavy chain, light chain variable region, heavy chain variable region, or CDR sequences.

[0325] In particular, the present invention relates to an antibody that competes for binding to HLA-G or binds to the same epitope as an antibody comprising a combination of CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences of SEQ ID NOs: 1 / 2 / 3 / 4 / 5 / 6. The antibody can compete for binding to HLA-G or bind to the same epitope as an antibody that comprises a pair of VL and VH sequences of SEQ ID NOs: 19 and 93, respectively. The antibody may compete for binding to HLA-G or bind to the same epitope as IgG1 containing a combination of CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences with SEQ ID NO: 1 / 2 / 3 / 4 / 5 / 6. The antibody may compete for binding to HLA-G or bind to the same epitope as IgG1 containing a pair of VL and VH sequences with SEQ ID NO: 19 and 93, respectively.

[0326] In one embodiment, the invention relates to an antibody that cross-competes for binding to HLA-G with an antibody comprising a combination of CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences with SEQ ID NO: 1 / 2 / 3 / 4 / 5 / 6.

[0327] In the context of the invention, the antibodies provided herein that compete for binding to HLA-G or bind to the same epitope as the reference antibody of the present invention retain the beneficial properties of the reference antibody as described in the above sections, such as specificity for HLA-G, high affinity, and blocking activity of ILT2 and / or ILT4. In one example, an antibody that competes for binding to HLA-G or binds to the same epitope as the reference antibody of the invention has:

[0328] a. dissociation constant (K D) between said antibody expressed in the form of a full-length antibody and the monomeric form of HLA-G, which is less than 20 nM, in particular less than 15 nM, in particular less than 10 nM, in particular less than 9 nM, in particular less than 8 nM, in particular less than 7 nM, in particular less than 6 nM, or in particular less than 5 nM, as determined, for example, by the SPR method at a temperature of 25°C; and / or

[0329] b.IC 50 less than 20 pM to block the binding of ILT2 to HLA-G naturally expressed on the surface of JEG3 cells, as determined, for example, by an in vitro assay using a large reaction volume as described in Example 8; and / or

[0330] c.IC 50 less than 1400 pM to block ILT4 binding to HLA-G, as described, for example, in Example 8.

[0331] To determine whether an antibody competes for binding with a reference antibody, the binding assay described above is performed using two different experimental models. In the first model, the reference antibody is allowed to bind to the antigen under saturating conditions, followed by assessment of the binding of the test antibody to the antigen. In the second model, the test antibody is allowed to bind to the antigen under saturating conditions, followed by assessment of the binding of the reference antibody to the protein / peptide. If in both experimental models only the first (saturating) antibody is able to bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the antigen.As will be understood by one of skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, it may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope, or it may cause a conformational change resulting in a lack of binding.

[0332] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other to that antigen. Alternatively, two antibodies have the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0333] Further routine experiments (e.g., peptide mutation analysis and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is truly due to binding to the same portion of the antigen as the reference antibody, or whether the lack of observed binding is due to steric blocking (or another phenomenon). These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0334] Antibody variants

[0335] One skilled in the art will also appreciate that antibodies can undergo a variety of post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody, as well as the culture conditions. Such modifications can include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Accordingly, the C-terminal lysine of the antibody heavy chain may be absent.

[0336] In one embodiment, the C-terminal amino acid of the antibody is cleaved during post-translational modifications.

[0337] In one embodiment, the N-terminal amino acid of the antibody is cleaved during post-translational modifications.

[0338] In some embodiments, antibody variants are provided that have one or more amino acid substitutions, insertions, and / or deletions. Sites of interest for substitution mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest, and the products are screened for desired activity, such as maintaining / improving antigen binding, reducing immunogenicity, or improving ADCC, CDC, and / or ADCP.

[0339] In some embodiments, amino acid sequence variants of the antibodies disclosed herein are provided. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the anti-HLA-G antibody can be obtained by introducing appropriate modifications into the nucleotide sequence encoding the protein or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences (e.g., in one or more CDRs and / or framework sequences or in the VH and / or VL domain) of the anti-HLA-G antibody. Any combination of deletions, insertions, and substitutions can be used to obtain the final construct, provided that the final construct has the desired characteristics.

[0340] In some embodiments of the VH and VL sequence variants provided herein, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0341] It should be understood that one or more amino acid substitutions, additions, and / or deletions can be introduced into the CDRs provided by the present invention without significantly altering the ability of the antibody to bind to HLA-G and neutralize HLA-G activity. One skilled in the art can readily test the effect of any amino acid substitutions, additions, and / or deletions, for example, using the methods disclosed herein, especially those illustrated in the examples, to determine HLA-G binding and inhibition of HLA-G interactions with its natural ligands.

[0342] Therefore, in some embodiments of the VH and VL sequence variants, each CDR comprises no more than one, two, or three amino acid substitutions, wherein such amino acid substitutions are conservative, and wherein the antibody retains its HLA-G binding properties.

[0343] Accordingly, the present invention relates to an anti-HLA-G antibody comprising one or more CDRs selected from CDR-L1 (comprising SEQ ID NO: 1), CDR-L2 (comprising SEQ ID NO: 2), CDR-L3 (comprising SEQ ID NO: 2), CDR-L3 (comprising SEQ ID NO: 3), CDR-H1 (comprising SEQ ID NO: 4), CDR-H2 (comprising SEQ ID NO: 5) and CDR-H3 (comprising SEQ ID NO: 6), wherein one or more amino acids in the one or more CDRs are replaced with another amino acid, for example, an amino acid similar to that defined below.

[0344] In one embodiment, the present invention relates to an anti-HLA-G antibody comprising CDR-L1 (comprising SEQ ID NO: 1), CDR-L2 (comprising SEQ ID NO: 2), CDR-L3 (comprising SEQ ID NO: 3), CDR-H1 (comprising SEQ ID NO: 4), CDR-H2 (comprising SEQ ID NO: 5), and CDR-H3 (comprising SEQ ID NO: 6), for example, wherein one or more amino acids in one or more CDRs are replaced with another amino acid, for example, an amino acid similar to those defined below.

[0345] In one embodiment, the anti-HLA-G antibody of the present invention comprises a light chain variable region that comprises three CDRs, wherein CDR-L1 comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 1, and / or CDR-L2 comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 2, and / or CDR-L3 comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or similar to the sequence set forth in SEQ ID NO: 3.

[0346] In one embodiment, the anti-HLA-G antibody of the present invention comprises a heavy chain variable region that comprises three CDRs, wherein CDR-H1 comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 4, and / or CDR-H2 comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 5, and / or CDR-H3 comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or similar to the sequence set forth in SEQ ID NO: 6.

[0347] In one embodiment, the anti-HLA-G antibody of the present invention comprises a light chain variable region comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or similar to the sequence set forth in SEQ ID NO: 19, or SEQ ID NO: 15, or SEQ ID NO: 23.

[0348] In one embodiment, the anti-HLA-G antibody of the present invention comprises a heavy chain variable region comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or similar to the sequence set forth in SEQ ID NO: 93, 27, 33, 57, 69, 75, 81, or 87.

[0349] In one embodiment, the anti-HLA-G antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 19, and / or the heavy chain variable region comprises a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 93.

[0350] In one embodiment, the anti-HLA-G antibody of the present invention comprises CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences comprising SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, and the remaining portions of the variable regions of the light chain and the heavy chain are identical or similar by at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 93, respectively.

[0351] In one embodiment, the anti-HLA-G antibody of the present invention comprises a light chain comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or similar to the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 17, or SEQ ID NO: 25.

[0352] In one embodiment, the anti-HLA-G antibody of the present invention comprises a heavy chain comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or similar to the sequence set forth in SEQ ID NO: 95, 29, 35, 59, 71, 77, 83, or 89.

[0353] In one embodiment, the anti-HLA-G antibody of the present invention is IgG1 comprising a light chain comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 21, and a heavy chain comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 95.

[0354] In one embodiment, the anti-HLA-G antibody of the present invention is IgG1 comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, and the remaining portions of the light chain and the heavy chain are at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or similar to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 95, respectively.

[0355] The anti-HLA-G antibody variants provided herein retain the beneficial properties of the parent antibody (i.e., the unmodified antibody), i.e., the functional properties described above, such as high specificity, high affinity, and ILT2 and / or ILT4 blocking activity. In one example, the anti-HLA-G antibody variant of the present invention has:

[0356] a. dissociation constant (K D) between said antibody expressed as a full-length antibody and the monomeric form of HLA-G, which is less than 20 nM, in particular less than 15 nM, in particular less than 10 nM, in particular less than 9 nM, in particular less than 8 nM, in particular less than 7 nM, in particular less than 6 nM or in particular less than 5 nM, as determined, for example, by the SPR method at a temperature of 25°C; and / or

[0357] b.IC 50 less than 20 pM to block the binding of ILT2 to HLA-G naturally expressed on the surface of JEG3 cells, as determined, for example, by an in vitro assay using a large reaction volume as described in Example 8; and / or

[0358] s. IC 50 less than 1400 pM to block ILT4 binding to HLA-G, as described, for example, in Example 8.

[0359] Sequence identity and similarity

[0360] The degrees of identity and similarity between sequences can be easily calculated. "% sequence identity" (or "% sequence similarity") is calculated by: (1) comparing two optimally aligned sequences within a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a given window, etc.), (2) determining the number of positions containing identical (or similar) amino acids (e.g., identical amino acids that occur in both sequences, similar amino acids that occur in both sequences) to obtain the number of matching positions, (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., in the longer sequence, in the shorter sequence, in the given window), and (4) multiplying the result by 100 to obtain % sequence identity or percent sequence similarity.

[0361] Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology-based algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology region alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., ed. 1995, supplement)).

[0362] Preferred examples of algorithms that are suitable for determining percent sequence identity and similarity include the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al., Nuc. Acids Res. 25:3389–3402 (1977) and Altschul et al., J. Mol. Biol. 215:403–410 (1990). Polypeptide sequences can also be compared using the FASTA program using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides an alignment and percent sequence identity of the regions of best overlap between the query and target sequences.

[0363] In some embodiments, substitutions, insertions, or deletions may be made in one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind to a target.

[0364] For example, conservative substitutions can be introduced into CDRs that do not significantly reduce binding affinity. Such changes can be made outside the CDR residues that contact the antigen.

[0365] Conservative substitutions are shown in Table 1 along with more significant “typical substitutions.”

[0366] Table 1: Examples of amino acid substitutions

[0367] Initial balance Typical replacements Conservative replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu

[0368] Substantial modifications of the biological properties of an antibody variant can be achieved by selecting substitutions that differ significantly in their effect on the preservation of the structure of the polypeptide backbone in the region of the substitution, the charge or hydrophobicity of the molecule at the target site, or the main part of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (in A.L. Lehninger, Biochemistry second ed., pp. 73-75, Worth Publishers, New York (1975)).

[0369] One type of substitution variant involves substitution of one or more residues of the CDR region of a parent antibody (humanized or human antibody). Typically, the resulting variant(s) selected for further study will have altered certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. A typical substitution variant is an affinity-matured antibody, which can be conveniently produced, for example, by phage display-based affinity maturation methods. Briefly, one or more CDR residues are mutated, and the antibody variants are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0370] Changes (e.g., substitutions) can be made in the CDRs, for example, to improve the affinity of the antibody. Such changes can be made at HVR "hot spots," i.e., residues encoded by codons that undergo mutations at a high frequency during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179–196 (2008)), and / or residues that contact the antigen, with the resulting VH or VL variant being tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, e.g., by Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001) In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand rearrangement, or oligonucleotide-directed mutagenesis). A secondary library is then created.The library is then screened to identify any antibody variants with the desired affinity.

[0371] One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is alanine scanning mutagenesis (Cunningham and Wells (1989) Science, 244:1081–1085). In this method, a residue or several target residues are identified and replaced with alanine to determine whether this affects the interaction of the antibody with the antigen. Alternatively, or additionally, the X-ray structure of the antigen-antibody complex can be used to identify contact points between the antibody and its antigen. Variants can be screened to determine whether they possess the desired properties.

[0372] Constant Region Variants

[0373] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby creating a variant Fc region. The variant Fc region may comprise a human Fc region sequence (e.g., an Fc region of human IgG1, IgG2, IgG3, or IgG4) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0374] Certain antibody variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Patent 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0375] Antibodies with an increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described, for example, in US2005 / 0014934A1. These antibodies contain an Fc region with one or more substitutions therein that improve the binding of the Fc region to FcRn.

[0376] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU residue numbering).

[0377] Antibodies with reduced effector function include antibodies with a substitution of one or more residues of the Fc region 234, 235, 237, 238, 265, 269, 270, 297, 327 and 329 (see, for example, U.S. Patent 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297 and 327, where the amino acid residue is numbered in accordance with the EU numbering system.

[0378] To confirm reduction / depletion of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody does not bind to FcγR (therefore likely lacking ADCC activity) but retains the ability to bind FcRn. The primary cells that mediate ADCC, NK cells, express only FcγRIII, whereas monocytes express FcRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is described generally by Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patents 5,500,362; 5821337. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that described by Clynes et al. Proc. Nat 1Acad. Sci. USA 95:652-656 (1998).To confirm that the antibody is unable to bind C1q and therefore does not have CDC activity, C1q binding assays can also be performed. See, for example, the ELISA method for assessing binding of C1q and C3c in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MI Glennie, Blood 103:2738-2743 (2004)). FcRn binding and clearance / half-life determination in vivo can also be accomplished by methods known in the art (see, e.g., Petkova, SB et al, Int 1. Immunol. 18(12):1759-1769 (2006)).

[0379] The constant region domains of the antibody molecule of the present invention, if present, can be selected taking into account the intended function of the antibody molecule and, in particular, the effector functions that may be required. For example, the constant region domains can be domains of human IgA, IgD, IgE, IgG, or IgM. In particular, the constant region domains of human IgG, in particular the IgG1 and IgG3 isotypes, can be used when the antibody molecule is intended for therapeutic use and the effector functions of the antibody are required. Alternatively, the IgG2 and IgG4 isotypes can be used when the antibody molecule is intended for therapeutic purposes and the effector functions of the antibody are not required. It should be understood that sequence variants of these constant region domains can also be used.

[0380] In some embodiments, the antibody of the present invention is wild-type human IgG1 (referred to as IgG1).

[0381] In some embodiments, the antibody of the invention is IgG1 LALA, a mutant of the wild-type human IgG1 isoform, wherein the amino acid substitutions L234A / L235A (according to EU numbering) have been introduced into the constant region of IgG1. In one embodiment, the antibody of the invention comprises a light chain comprising the sequence of SEQ ID NO: 21 and a heavy chain comprising the sequence of SEQ ID NO: 97.

[0382] In some embodiments, the antibody of the present invention is IgG1 LALAGA, a mutant of the wild-type human IgG1 isoform, wherein the amino acid substitutions L234A / L235A / G237A (according to EU numbering) have been introduced into the constant region of IgG1.

[0383] In some embodiments, the antibody of the invention is IgG4P, a mutant of the wild-type human IgG4 isoform in which amino acid 228 (according to EU numbering) is replaced by proline, as described, for example, by Angal et al., Molecular Immunology, 1993, 30(1), 105-108. In one embodiment, the antibody of the present invention comprises a light chain comprising the sequence of SEQ ID NO: 21 and a heavy chain comprising the sequence of SEQ ID NO: 99.

[0384] In some embodiments, the antibody of the invention is IgG4 FALA, a mutant of the wild-type human IgG4 isoform, wherein F234A / L235A substitutions (according to EU numbering) have been introduced into the constant region of IgG4.

[0385] In some embodiments, the antibody of the invention is IgG4P FALA, a mutant of the wild-type human IgG4 isoform, wherein in the constant region of IgG4, amino acid 228 (according to EU numbering) is replaced by proline and the acidic substitutions F234A / L235A (according to EU numbering) are present. In one embodiment, the antibody of the present invention comprises a light chain comprising the sequence of SEQ ID NO: 21 and a heavy chain comprising the sequence of SEQ ID NO: 101.

[0386] Glycosylation variants

[0387] In some embodiments, the antibody described herein is altered to increase or decrease the degree of glycosylation of the antibody. Addition or removal of glycosylation sites in the antibody can be advantageously achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0388] In one embodiment, the antibody of the invention is glycomodified. In one embodiment, the antibody of the invention has a low fucose content or no fucose at all. By "fucose content" is meant the percentage of fucosylated forms in the N-glycans attached to the Asn297 residue of the Fc fragment of each heavy chain of each antibody. By "low fucose content" is meant a fucose content of less than or equal to 65 percent. Indeed, it is now known that low fucose content in an antibody composition plays a critical role in the ability of said composition to induce a strong ADCC response via Fc-gammaRIII. Preferably, the fucose content is less than or equal to 65 percent, preferably less than or equal to 60 percent, 55 percent or 50 percent, even less than or equal to 45 percent, 40 percent, 35 percent, 30 percent, 25 percent or 20 percent.However, the fucose content does not necessarily have to be zero, it can, for example, be less than or equal to 5 percent, 10 percent, 15 percent or 20 percent.

[0389] In one embodiment, the antibody of the invention is afucosylated IgG1. Methods for producing afucosylated IgG1 are well known and include obtaining cells genetically modified to produce afucosylated antibodies. In one embodiment, the afucosylated IgG1 of the invention is produced in CHO cells in which the gene encoding alpha-1,6-fucosyltransferase (FUT8) has been genetically knocked out using methods well known in the art. For example, KO FUT8 CHOSXE / DG44 cells can be used as described in the examples provided herein.

[0390] In one embodiment, the antibody of the invention has improved ADCC and / or ADCP function and / or has improved ability to deplete tumor cells expressing HLA-G. In one embodiment, the ADCC and / or ADCP function and / or the ability of the afucosylated antibody of the present invention to deplete tumor cells expressing HLA-G are improved compared to the corresponding conventional (i.e., fucosylated) antibody (i.e., an antibody comprising the same amino acid sequence but comprising fucose, for example, produced in CHO cells that have not been modified and express FUT8).In the context of the invention, "improved" activity (e.g., ADCC and / or ADCP, and / or tumor cell depletion) means that the activity (e.g., ADCC and / or ADCP, and / or tumor cell depletion) of the afucosylated antibody is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher than the same activity of the reference antibody (i.e., the corresponding normal, i.e., fucosylated antibody).

[0391] To confirm the increase in ADCC and / or ADCP activity, in vitro and / or in vivo cytotoxicity assays that are well known in the art can be performed, for example, as disclosed herein.

[0392] In one embodiment, the antibody of the present invention is an afucosylated IgG1 that comprises:

[0393] a. a light chain comprising SEQ ID NO: 21 or 17 or 25; and / or

[0394] b. a heavy chain comprising SEQ ID NO: 95, 29, 35, 59, 71, 77, 83 or 89.

[0395] In one embodiment, the antibody of the present invention is an afucosylated IgG1 that comprises:

[0396] a. a light chain that is at least 90% identical or similar to the sequence set forth in SEQ ID NO: 21 or 17 or 25; and / or

[0397] b. a heavy chain that is at least 90% identical or similar to the sequence set forth in SEQ ID NO: 95, 29, 35, 59, 71, 77, 83 or 89.

[0398] In one embodiment, the anti-HLA-G antibody of the present invention is an afucosylated IgG1 comprising a light chain comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 21, and a heavy chain comprising a sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or similar to the sequence set forth in SEQ ID NO: 95.

[0399] In one embodiment, the anti-HLA-G antibody of the present invention is an afucosylated IgG1 comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively, and the remaining portions of the light chain and the heavy chain are at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or similar to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 95, respectively.

[0400] In one embodiment, the anti-HLA-G antibody of the present invention is an afucosylated IgG1 that comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 93, and wherein the remaining portions of the light chain and heavy chain are at least 90% identical or similar to the sequences set forth in SEQ ID NO: 21 and 95, respectively.

[0401] In one embodiment, the antibody of the invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 95, and has an improved ADCC and / or ADCP function and / or has an improved ability to deplete tumor cells expressing HLA-G. In one embodiment, the ADCC and / or ADCP function and / or the ability to deplete tumor cells expressing HLA-G are improved compared to the corresponding conventional antibody (i.e., a fucosylated antibody that comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 95).

[0402] In one embodiment, the antibody of the invention comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 95, and has an improved CDC function and / or has an improved ability to deplete tumor cells expressing HLA-G. In one embodiment, the CDC function and / or the ability to deplete tumor cells expressing HLA-G are improved compared to the corresponding conventional antibody (i.e., a fucosylated antibody that comprises a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 95).

[0403] In one embodiment, the anti-HLA-G antibody of the present invention is an afucosylated IgG1 that comprises a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 93, or a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 95, and has an improved ADCC and / or ADCP and / or CDC function, and has an improved ability to deplete tumor cells expressing HLA-G.

[0404] Biological molecules, such as antibodies, contain acidic and / or basic functional groups that impart a net positive or negative charge to the molecule. The magnitude of the net "apparent" charge will depend on the absolute amino acid sequence of the target, the local environment of the charged groups in the three-dimensional structure, and the environmental conditions of the molecule. The isoelectric point (pI) is the pH at which a particular molecule or its solvent-accessible surface carries no net electrical charge. In one example, an HLA-G-binding antibody can be engineered to have a favorable isoelectric point. This can lead to antibodies with more robust properties, particularly those with favorable solubility and / or stability profiles and / or improved purification characteristics.

[0405] An antibody can, for example, be engineered by substituting an amino acid residue, such as replacing an acidic amino acid residue with one or more basic amino acid residues. Alternatively, basic amino acid residues can be introduced or acidic amino acid residues can be removed. Alternatively, if the molecule has an unacceptably high pI, acidic residues can be introduced to lower the pI, if necessary. It is important to note that manipulation of the pI must be performed carefully to maintain the desired activity of the antibody or fragment thereof. Thus, in one embodiment, the engineered antibody has the same or substantially the same activity as the "unmodified" antibody or fragment thereof.

[0406] To predict the isoelectric point of an antibody, programs such as ** ExPASY http: / / www.expasy.ch / tools / pi_tool.html and http: / / www.iut-arles.up.univ-mrs.fr / w3bb / d_abim / compo-p.html can be used.

[0407] Effector molecules

[0408] If desired, the antibody for use in the present invention may be conjugated to one or more effector molecules.

[0409] It should be understood that the effector molecule may comprise one effector molecule or two or more such molecules linked to form a single fragment that can be attached to the antibodies of the present invention. If it is desired to obtain an antibody fragment linked to an effector molecule, it can be obtained using standard chemical or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule either directly or through a linking agent. Methods for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical procedures include, for example, those described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 03031581.Alternatively, where the effector molecule is a protein or polypeptide, the linkage may be achieved using recombinant DNA procedures, for example as described in WO 86 / 01533 and EP 0392745.

[0410] The term "effector molecule" as used herein includes, for example, antitumor agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides, in particular radioiodides, radioisotopes, chelating metals, nanoparticles and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

[0411] Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that is harmful to cells (e.g., kills). Examples include combrestatins, dolastatins, epothilones, staurosporine, maytansinoids, spongistatins, rhizoxin, halichondrins, roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinatedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.

[0412] Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMCs, calicheamicins or duocarmycins) and antimitotic agents (eg, vincristine and vinblastine).

[0413] Other effector molecules may include chelated radionuclides such as 111 In and 90 Y, Lu 177 , bismuth 213 , Californian 252 , iridium 192 and tungsten 188 / rhenium 188; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids, and suramin.

[0414] Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, and transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin, a protein such as insulin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, a thrombotic agent or an antiangiogenic agent such as angiostatin or endostatin, or a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), nerve growth factor (NGF) or another growth factor and immunoglobulins.

[0415] Other effector molecules may include detectable substances useful, for example, in diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. See generally U.S. Patent 4,741,900 for metal ions that can be conjugated to antibodies for use as a diagnostic agent.Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include. 125 I, 131 I, 111 In and 99 Tc.

[0416] In another example, the effector molecule can increase the half-life of the antibody in vivo and / or reduce the immunogenicity of the antibody and / or enhance the delivery of the antibody to the immune system across the epithelial barrier. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins, or albumin-binding compounds, such as those described in WO 05 / 117984.

[0417] When the effector molecule is a polymer, it may typically be a synthetic or naturally occurring polymer, such as an optionally substituted straight-chain or branched-chain polyalkylene, polyalkenylene, or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

[0418] Specific optional substituents that may be present in the above-mentioned synthetic polymers include one or more hydroxyl, methyl or methoxy groups.

[0419] Specific examples of synthetic polymers include optionally substituted straight-chain or branched-chain poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, in particular optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof.

[0420] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.

[0421] The term "derivatives" as used herein includes reactive derivatives, such as thiol-selective reactive groups such as maleimides and the like. The reactive group may be linked to the polymer directly or via a linker segment. It is understood that the residue of such a group will in some cases form part of the product as a linking group between the antibody fragment and the polymer.

[0422] The polymer size can be varied as desired, but it is typically in the range of average molecular weight from 500 Da to 50,000 Da, such as 5,000 to 40,000 Da, such as 20,000 to 40,000 Da. The polymer size can be selected in particular based on the intended use of the product, such as the ability to localize to specific tissues such as tumors, or to increase the half-life in the blood (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). For example, if the product is intended to be removed from the bloodstream and penetrate into tissues, it may be advantageous to use a polymer with a small molecular weight, such as one with a molecular weight of approximately 5,000 Da. For applications where the product remains in the bloodstream, it may be advantageous to use a polymer with a higher molecular weight, such as one with a molecular weight in the range of 20,000 Da to 40,000 Da.

[0423] Suitable polymers include a polyalkylene polymer such as poly(ethylene glycol) or, in particular, methoxypoly(ethylene glycol) or a derivative thereof, and especially with a molecular weight in the range of from about 15,000 Da to about 40,000 Da.

[0424] In one example, antibodies for use in the present invention are attached to poly(ethylene glycol) (PEG) moieties. In one specific example, the antibody is an antibody fragment, and the PEG molecules can be attached via any available amino acid side chain functional group or amino acid terminal functional group located in the antibody fragment, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids can be naturally occurring in the antibody fragment or can be introduced into the fragment using recombinant DNA techniques (see, for example, U.S. Patent 5,219,996; U.S. Patent 5,667,425; WO 98 / 25971). In one example, an antibody molecule of the present invention is a modified Fab fragment, wherein the modification is the addition of one or more amino acids to the C-terminal end of its heavy chain for attachment of an effector molecule.Accordingly, the additional amino acids form a modified hinge region containing one or more cysteine ​​residues to which the effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

[0425] Similarly, PEG molecules can be covalently linked via the thiol group of at least one cysteine ​​residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment can be covalently linked to a sulfur atom of a cysteine ​​residue located in the fragment. The covalent bond is typically a disulfide bond or, in particular, a sulfur-carbon bond. If a thiol group is used as the attachment point, appropriately activated effector molecules can be used, for example, thiol-selective derivatives such as maleimides and cysteine ​​derivatives. The activated polymer can be used as a starting material for the preparation of polymer-modified antibody fragments as described above.The activated polymer can be any polymer containing a reactive thiol group, such as an α-halocarboxylic acid or ester thereof, e.g., iodoacetamide, an imide such as maleimide, a vinyl sulfone, or a disulfide. Such starting materials can be commercially purchased (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (purchased from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (purchased from Nektar, formerly Shearwater).

[0426] In one embodiment, the antibody is a modified Fab or di-Fab fragment that is PEGylated, i.e., has PEG (poly(ethyleneglycol)) covalently attached thereto, such as by the method disclosed in EP 0948544 or EP 1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example, PEG is attached to a cysteine ​​in the hinge region. In one example, a PEG-modified Fab fragment contains a maleimide group covalently linked to one thiol group in the modified hinge region.A lysine residue can be covalently linked to a maleimide group, and a methoxypoly(ethylene glycol) polymer with a molecular weight of approximately 20,000 Da can be attached to each of the amino groups on the lysine residue. Thus, the total molecular weight of PEG attached to the Fab fragment can be approximately 40,000 Da.

[0427] In one embodiment, the antibody is a modified Fab' fragment having at the C-terminus of its heavy chain a modified hinge region containing at least one cysteine ​​residue to which an effector molecule is attached. A suitable effector molecule is PEG attached by the method described in WO 98 / 25971 and WO 2004072116 or WO 2007 / 003898. Effector molecules can be attached to antibody fragments by the methods described in international patent applications WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171.

[0428] In one embodiment, the antibody is not attached to an effector molecule.

[0429] Polynucleotides and vectors

[0430] The present invention also relates to an isolated polynucleotide encoding an antibody of the present invention or a portion thereof (e.g., with the amino acid SEQ IDs listed in Table 2). As used herein, the term "isolated" means that the polynucleotide exists in a physical environment different from that in which it may be found in nature.

[0431] The isolated polynucleotide of the present invention may comprise synthetic DNA, such as that obtained by chemical treatment, cDNA, genomic DNA, or any combination thereof.

[0432] Table 2: Amino acid sequences of 12389 anti-HLA-G antibodies and their corresponding nucleic acid sequences

[0433] Antibody sequence SEQ ID NO amino acid SEQ ID NO of nucleic acid 12389gL2gH16, VL 19 20 12389gL2gH16, light chain 21 22 12389gL2gH16, VH 93 94 12389gL2gH16, IgG1 heavy chain 95 96 12389gL2gH16, IgG1 heavy chain LALA 97 98 12389gL2gH16, IgG4P heavy chain 99 100 12389gL2gH16, IgG4P heavy chain FALA 101 102

[0434] The present description provides examples of suitable sequences. Thus, in one embodiment, the present invention relates to an isolated polynucleotide encoding an antibody comprising a sequence shown in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102.

[0435] In one embodiment, the present invention relates to an isolated polynucleotide encoding an antibody of the present invention, wherein the polynucleotide encodes a light chain variable region, wherein the polynucleotide:

[0436] i. is at least 90% identical to SEQ ID NO: 20, 16, or 24; or

[0437] ii. comprises or consists of SEQ ID NO: 20, 16 or 24.

[0438] In one embodiment, the present invention relates to an isolated polynucleotide encoding an antibody of the present invention, wherein the polynucleotide encodes a heavy chain variable region, wherein the polynucleotide:

[0439] i. is at least 90% identical to SEQ ID NO: 94, 28, 34, 58, 70, 76, 82 or 88; or

[0440] ii. comprises or consists of SEQ ID NO: 94, 28, 34, 58, 70, 76, 82 or 88.

[0441] In one embodiment, the present invention relates to an isolated polynucleotide encoding an antibody of the present invention, wherein the polynucleotide encodes a light chain, wherein the polynucleotide:

[0442] i. is at least 90% identical to SEQ ID NO: 22, 18, or 26; or

[0443] ii. comprises or consists of SEQ ID NO: 22, 18 or 26.

[0444] In one embodiment, the present invention relates to an isolated polynucleotide encoding an antibody of the present invention, wherein the polynucleotide encodes a heavy chain, wherein the polynucleotide:

[0445] i. is at least 90% identical to SEQ ID NO: 96, 30, 36, 60, 72, 78, 84 or 90; or

[0446] ii. comprises or consists of SEQ ID NO: 96, 30, 36, 60, 72, 78, 84 or 90.

[0447] In one embodiment, the present invention relates to an isolated polynucleotide encoding a heavy chain of an IgG1 antibody of the present invention, which comprises a sequence set forth in SEQ ID NO: 96, 30, 36, 60, 72, 78, 84 or 90.

[0448] Also provided is an isolated polynucleotide encoding a light chain of an IgG1 antibody of the present invention, which comprises a sequence as set forth in SEQ ID NO: 22, 18 or 26.

[0449] In a preferred embodiment, the present invention relates to an isolated polynucleotide encoding a heavy chain and a light chain of an IgG1 antibody of the present invention, wherein the polynucleotide encoding the heavy chain comprises the sequence set forth in SEQ ID NO: 96, and the polynucleotide encoding the light chain comprises the sequence set forth in SEQ ID NO: 22.

[0450] The present invention also relates to a cloning or expression vector comprising one or more polynucleotides disclosed herein. In one example, the cloning or expression vector of the present invention comprises one or more isolated polynucleotides described above.

[0451] Standard molecular biology techniques can be used to obtain DNA sequences encoding the antibody of the present invention. The desired DNA sequences can be synthesized in whole or in part by oligonucleotide synthesis methods. Site-directed mutagenesis and polymerase chain reaction (PCR) methods can be used if necessary.

[0452] General methods for vector construction, transfection methods, and culture techniques are well known to those skilled in the art. See, for example, "Current Protocols in Molecular Biology," 1999, F.M. Ausubel (ed.), Wiley Interscience, New York, and the Maniatis Manual, published by Cold Spring Harbor Publishing.

[0453] Host cells for antibody production

[0454] Also provided is a host cell comprising one or more isolated polynucleotide sequences of the present invention or one or more cloning or expression vectors comprising one or more isolated polynucleotide sequences encoding an antibody of the present invention. Any suitable host cell / vector system can be used to express the polynucleotide sequences encoding the antibody of the present invention. Bacterial systems, such as those based on E. coli and other microbes, can be used, or eukaryotic host cell expression systems, such as mammalian cells, can also be used. Suitable mammalian host cells include CHO cells, myeloma cells, or hybridomas.

[0455] In a further embodiment, a host cell comprising such nucleic acid(s) or vector(s) is provided. In one such embodiment, the host cell comprises (e.g., is transformed): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an anti-HLA-G antibody and an amino acid sequence comprising a VH of an anti-HLA-G antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an anti-HLA-G antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an anti-HLA-G antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). In one embodiment, the host cell is a prokaryotic cell, such as an E. coli cell.In one embodiment, a method for producing an anti-HLA-G antibody is provided, comprising culturing a host cell containing a nucleic acid encoding the antibody as set forth above under conditions suitable for expression of the antibody, and optionally isolating the antibody from the host cell (or host cell culture medium).

[0456] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as disclosed herein. For example, antibodies can be produced in bacteria, particularly where glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003, pp. 245-254), which describes the expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0457] In addition to prokaryotes, suitable hosts for cloning or expression of antibody-encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, including fungi and yeast strains whose glycosylation pathways have been "humanized," allowing the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 2:1409–1414 (2004), and Li et al., Nat. Biotech. 24:210–215 (2006).

[0458] Suitable types of Chinese hamster ovary cells (CHO cells) for use in the present invention may include CHO and CHO-K1 cells, including dhfr-CHO cells such as CHO-DG44 cells and CHO-DXB11 cells, which can be used with a DHFR selectable marker, or CHOK1-SV cells, which can be used with a glutamine synthetase selectable marker. Other cell types used for antibody expression include lymphocyte cell lines, such as NS0 myeloma cells and SP2 cells, and COS cells. The host cell can be stably transformed or transfected with the isolated polynucleotide sequences or expression vectors of the present invention.

[0459] In one embodiment, the antibody of the invention is produced in a host cell genetically modified to reduce or abolish the function of alpha-1,6-fucosyltransferase (FUT8). In one embodiment, the genetically modified cell is a CHO cell with a knockout of the FUT8 gene (KO FUT8). In one embodiment, the host cell for producing the antibody of the invention is CHO-DG44. In one embodiment, the host cell for producing the antibody of the invention is KO FUT8 CHOSXE / DG44 cells, which can be obtained by the methods described in the examples provided herein.

[0460] Method for producing antibodies

[0461] The present invention also relates to a method for producing an antibody of the present invention, comprising culturing a host cell of the present invention under conditions suitable for producing the antibody of the present invention and isolating the antibody.

[0462] The present invention also relates to a method for producing a pharmaceutical composition containing the antibody of the present invention, comprising culturing a host cell of the present invention under conditions suitable for producing the antibody of the present invention, isolating the antibody, and preparing the antibody as a pharmaceutical composition.

[0463] An antibody may contain only a heavy or light chain polypeptide, in which case only the sequence encoding the heavy or light chain polypeptide need be used to transfect host cells. To produce antibodies containing both heavy and light chains, the cell line can be transfected with two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector can be used, where the vector contains sequences encoding both light and heavy chain polypeptides.

[0464] Thus, a method is provided for culturing a host cell and expressing an antibody, isolating the antibody, and, optionally, purifying the antibody to obtain an isolated antibody. In one embodiment, the method further comprises the step of conjugating an effector molecule to the isolated antibody.

[0465] The present invention also relates to a method for producing an antibody of the present invention, comprising culturing a host cell containing a vector of the present invention under conditions suitable for expressing a protein from DNA encoding an antibody molecule of the present invention, and isolating the antibody molecule.

[0466] The antibodies of the present invention are expressed at high levels by host cells. Thus, the antibody properties appear to be optimized for commercial processing.

[0467] In one embodiment, a purified antibody is provided, such as a humanized antibody, in particular an antibody of the invention, in a substantially purified form, in particular free of or substantially free of endotoxin and / or host cell protein or DNA.

[0468] The term “substantially free of endotoxin” generally refers to an endotoxin content of 1 IU per mg of antibody product or less, such as 0.5 or 0.1 IU per mg of product.

[0469] The term "substantially free of host cell protein or DNA" generally refers to a host cell protein and / or DNA content of 400 μg per mg of the antibody product or less, such as 100 μg or less per mg, in particular 20 μg per mg, as appropriate.

[0470] Pharmaceutical compositions, doses and dosage regimens

[0471] The antibody of the invention can be presented in a pharmaceutical composition or a diagnostic composition. Therefore, the present invention also relates to a pharmaceutical or diagnostic composition comprising the antibody of the present invention in combination with one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0472] Preferably, the pharmaceutical or diagnostic composition comprises an antibody that specifically binds HLA-G, wherein the antibody comprises:

[0473] a. a light chain variable region comprising:

[0474] i. CDR-L1 containing SEQ ID NO: 1;

[0475] ii. CDR-L2 containing SEQ ID NO: 2 and

[0476] iii. CDR-L3 comprising SEQ ID NO: 3; and

[0477] b. a heavy chain variable region comprising:

[0478] i. CDR-H1 containing SEQ ID NO: 4;

[0479] ii. CDR-H2 containing SEQ ID NO: 5 and

[0480] iii. CDR-H3 containing SEQ ID NO: 6.

[0481] In one embodiment, the antibody of the present invention is the sole active ingredient. In another embodiment, the antibody of the present invention is in combination with one or more additional active ingredients. In one embodiment, the antibody of the present invention is in combination with an antibody directed against CD47. Thus, in one embodiment, an antibody is provided that specifically binds to HLA-G, wherein the antibody comprises:

[0482] a. a light chain variable region comprising:

[0483] i. CDR-L1 containing SEQ ID NO: 1;

[0484] ii. CDR-L2 containing SEQ ID NO: 2 and

[0485] iii. CDR-L3 comprising SEQ ID NO: 3; and

[0486] b. a heavy chain variable region comprising:

[0487] i. CDR-H1 containing SEQ ID NO: 4;

[0488] ii. CDR-H2 containing SEQ ID NO: 5 and

[0489] iii. CDR-H3 containing SEQ ID NO: 6,

[0490] where the antibody is in combination with a second antibody that binds to CD47.

[0491] Alternatively, the pharmaceutical compositions comprise the antibody of the present invention, which is the only active ingredient, and it can be administered individually to a patient in combination (e.g., simultaneously, sequentially, or separately) with other therapeutic, diagnostic, or palliative agents.

[0492] The pharmaceutical compositions of the invention can be administered to a patient in a suitable manner to determine the required therapeutically effective amount. The term "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent required to treat, alleviate, or prevent a target disease or condition, or to exhibit a detectable therapeutic or prophylactic effect. The therapeutically effective amount of any antibody can first be estimated either by cell culture assays or in animal models, typically rodents, rabbits, dogs, pigs, or primates. An animal model can also be used to determine a suitable concentration range and route of administration. This information can then be used to determine useful doses and routes of administration in humans.

[0493] The precise therapeutically effective amount for a human will depend on the severity of the disease condition, the general health of the subject, the age, weight, and sex of the subject, the diet, the time and frequency of administration, the drug combination(s), the sensitivity of the reaction, and the tolerance / response to therapy. Typically, the therapeutically effective amount is from 0.01 mg / kg to 500 mg / kg, for example, from 0.1 mg / kg to 200 mg / kg, for example, 100 mg / kg. The pharmaceutical compositions can be presented as convenient unit dosage forms containing a predetermined amount of the active agent of the invention per dose.

[0494] Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol, and ethanol. Furthermore, such compositions may contain excipients such as wetting or emulsifying agents or pH buffering agents.

[0495] Suitable administration forms include those suitable for parenteral administration, such as by injection or infusion, such as bolus injection or continuous infusion, intravenous, inhalation, or subcutaneous. If the product is intended for injection or infusion, it may be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain auxiliary substances such as suspending, preservative, stabilizing, and / or dispersing agents. Alternatively, the antibody of the invention may be in dry form, requiring reconstitution with a suitable sterile liquid before use. Solid forms suitable for solution or suspension in liquid vehicles prior to injection may also be prepared.

[0496] After preparation, the composition of the present invention can be administered directly to a subject. Accordingly, the present invention provides the use of the antibody of the invention for the production of a medicament.

[0497] Preferably, the pharmaceutical composition of the present invention is adapted for administration to humans.

[0498] Therefore, in another aspect, the present invention relates to an antibody that specifically binds to HLA-G, wherein the antibody or a pharmaceutical composition containing the antibody is intended for use in therapy, wherein the antibody comprises:

[0499] a. a light chain variable region comprising:

[0500] i. CDR-L1 containing SEQ ID NO: 1;

[0501] ii. CDR-L2 containing SEQ ID NO: 2; And

[0502] iii. CDR-L3 comprising SEQ ID NO: 3; and

[0503] b. a heavy chain variable region comprising:

[0504] i. CDR-H1 containing SEQ ID NO: 4;

[0505] ii. CDR-H2 containing SEQ ID NO: 5 and

[0506] iii. CDR-H3 containing SEQ ID NO: 6.

[0507] Therapeutic indications

[0508] The terms "treatment," "treating," and the like, as used herein, refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the development of a disease or its symptom and / or may be therapeutic in terms of partially or completely curing the disease and / or an adverse effect associated with the disease. Thus, treatment encompasses any treatment of a disease in a mammal, in particular in a human, and includes: (a) preventing the onset of a disease in a subject who may be predisposed to the disease but who has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e. stopping its progression; and (c) alleviating the disease, i.e. initiating regression of the disease.

[0509] "Therapeutically effective amount" refers to the amount of an anti-HLA-G antibody that, when administered to a mammal or other subject for the treatment of a disease, is sufficient to provide such treatment. The therapeutically effective amount will vary depending on the anti-HLA-G antibody, the disease and its severity, as well as the age, weight, etc., of the subject being treated.

[0510] The antibodies of the invention, their formulations, or pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatment. For prophylactic purposes, the antibodies, formulations, or compositions are administered to a subject at risk of developing a disorder or condition disclosed herein in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. For therapeutic purposes, the antibodies are administered to a subject already suffering from a disorder or condition disclosed herein in an amount sufficient to treat, alleviate, or partially halt the progression of the condition or one or more of its symptoms. Such therapeutic treatment can result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods.

[0511] The present invention relates to a method for treating a disorder or condition disclosed herein in a subject in need thereof, wherein the method comprises administering to the subject an antibody or pharmaceutical composition of the present invention. Such an antibody is administered in a therapeutically effective amount.

[0512] The present invention also relates to an antibody or a pharmaceutical composition of the present invention for use in therapy, in particular for use in the treatment of a disorder or condition disclosed herein.

[0513] The present invention also relates to the use of an antibody or a pharmaceutical composition of the invention for the manufacture of a medicament, in particular for use in the treatment of a disorder or condition disclosed herein.

[0514] The antibodies of the present invention can be used to treat, prevent, or alleviate any condition associated with HLA-G activity; for example, any condition the development of which is due in whole or in part to signaling through the HLA-G receptor.

[0515] HLA-G-associated diseases or disorders include, but are not limited to, cancer (or tumors), infections, and autoimmune disorders.

[0516] In one embodiment, the invention relates to an antibody or pharmaceutical composition of the present invention for use in the treatment of a disease characterized by overexpression of HLA-G.

[0517] The antibodies of the present invention may be particularly useful for the treatment or prevention of cancer, including cancer characterized by overexpression of HLA-G. Thus, in one embodiment, the invention relates to an antibody or pharmaceutical composition of the invention for use in the treatment of cancer. In one embodiment, the invention relates to an antibody or pharmaceutical composition of the present invention for use in the treatment of cancer characterized by overexpression of HLA-G.

[0518] Cancer diseases in the context of the present invention include, for example, clear cell renal cell carcinoma (RCC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, breast cancer, head and neck carcinoma, gastric cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, and hematological cancer. The antibodies of the present invention can be useful for the treatment or prevention of hematological malignancies, such as hematological cancer.

[0519] The antibodies of the present invention may be particularly useful for the treatment or prevention of a solid tumor. Thus, in one embodiment, the invention relates to an antibody or pharmaceutical composition of the invention for use in the treatment of a solid tumor. In one embodiment, the invention relates to an antibody or pharmaceutical composition of the invention for use in the treatment of a solid tumor characterized by overexpression of HLA-G. In one embodiment, the invention relates to an antibody or pharmaceutical composition of the present invention for use in the treatment of clear cell renal cell carcinoma (RCC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, head and neck carcinoma, gastric cancer, or hepatocellular carcinoma. In one specific embodiment, the solid tumor is clear cell renal cell carcinoma (RCC).In another specific embodiment, the solid tumor is colorectal carcinoma (CRC).

[0520] In one embodiment, the present invention relates to the use of an antibody or a pharmaceutical composition of the invention for the manufacture of a medicament for use in the treatment of a solid tumor. In one embodiment, the present invention relates to the use of an antibody or a pharmaceutical composition of the invention for the manufacture of a medicament for use in the treatment of a solid tumor characterized by overexpression of HLA-G. In one embodiment, the present invention relates to the use of an antibody or a pharmaceutical composition of the present invention for the manufacture of a medicament for use in the treatment of clear cell renal cell carcinoma (RCC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, head and neck carcinoma, gastric cancer, or hepatocellular carcinoma.In one specific embodiment, the solid tumor is clear cell renal cell carcinoma (RCC). In another specific embodiment, the solid tumor is colorectal carcinoma (CRC).

[0521] In one embodiment, the invention relates to a method of treating a solid tumor in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or pharmaceutical composition of the invention. In one embodiment, the invention relates to a method of treating in a patient a solid tumor characterized by overexpression of HLA-G, comprising administering to said patient a therapeutically effective amount of an antibody or pharmaceutical composition of the invention.

[0522] In one embodiment, the solid tumor is selected from clear cell renal cell carcinoma (CRC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, head and neck carcinoma, gastric cancer, and hepatocellular carcinoma. In one specific embodiment, the solid tumor is clear cell renal cell carcinoma (RCC). In another specific embodiment, the solid tumor is colorectal carcinoma (CRC).

[0523] The present invention also relates to the use of the antibodies of the present invention as diagnostically active agents or in diagnostic assays, for example, for diagnosing a disease or its severity.

[0524] In one embodiment, the invention relates to a method for diagnosing a solid tumor using an antibody or pharmaceutical composition of the invention. In one embodiment, the invention relates to a method for diagnosing a solid tumor characterized by overexpression of HLA-G using an antibody or pharmaceutical composition of the invention.

[0525] In one embodiment, the invention relates to a method for diagnosing clear cell renal cell carcinoma (RCC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, head and neck carcinoma, gastric cancer, or hepatocellular carcinoma using an antibody or pharmaceutical composition of the invention. In one specific embodiment, the solid tumor is clear cell renal cell carcinoma (RCC). In another specific embodiment, the solid tumor is colorectal carcinoma (CRC).

[0526] Diagnostics can preferably be performed on biological samples. "Biological sample" includes various types of samples obtained from a human being and can be used in diagnostic or monitoring analysis. The definition encompasses cerebrospinal fluid, blood such as plasma and serum, and other liquid samples of biological origin such as urine and saliva, solid tissue samples such as biopsy or tissue culture specimens, or cells derived from them and their progeny. The definition also includes samples after any manipulation performed on them after their collection, such as treatment with reagents, solubilization, or enrichment with certain components such as polynucleotides.

[0527] Diagnostic testing can preferably be performed on biological samples that have not come into contact with the human or animal body. This diagnostic testing is also called in vitro testing. In vitro diagnostic testing may be based on an in vitro method for detecting HLA-G1 in a biological sample obtained from a subject.

[0528] In one embodiment, the invention relates to a method for diagnosing a solid tumor expressing HLA-G in a biological sample using an antibody or pharmaceutical composition of the invention. In one embodiment, the invention relates to a method for diagnosing clear cell renal cell carcinoma (RCC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, head and neck carcinoma, gastric cancer, or hepatocellular carcinoma in a biological sample using an antibody or pharmaceutical composition of the invention.

[0529] Examples

[0530] Example 1: Generation of HLA-G, HLA-I and ILT2, ILT4 proteins for use in screening assays

[0531] 1.1 HLA-G proteins: sequences

[0532] To generate HLA-G constructs for screening anti-HLA-G antibodies, the sequence of the most common HLA-G isoforms, HLA-G1 (membrane-bound) or HLA-G5 (soluble), containing the alpha-1, 2, and 3 domains of HLA-G, was used.

[0533] The extracellular domain, or ECD, sequence of HLA-G was determined based on crystal structure analysis (alpha-1 domain, in italics; alpha-2 domain; alpha-3 domain, underlined; terminal KQ residues, determined by crystallography). The 20 HLA-G-specific residues are shown in bold:

[0534] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEY WEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDY LALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA DPPKTHVTHHPV F D Y EATLRCWALGFYPAEI I LTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPL M LRWKQ (SEQ ID NO: 108)

[0535] Soluble HLA-G (ECD HLA-G):

[0536] Protein was expressed with an AVitev10His tag (signal peptide in bold, avidin affinity tag or AVI tag for biotinylation is underlined, Tev protease site is underlined and italicized, 10His tag is italicized):

[0537] MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDY LALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQ GLNDIFEAQKIEWHE LE ENLYFQG SGGSHHHHHHHHHH(SEQ ID NO: 109)

[0538] The purified final protein sequence used for screening assays contains the following sequence (containing the AVitev10His tag):

[0539] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVA EQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQGLNDIFEAQKIEWHELEENLYFQGSGGSHHHHHHHHH (SEQ ID NO: 110)

[0540] HLA-G DNA sequence for cell membrane-associated expression:

[0542] The corresponding membrane-bound protein contains the following sequence (signal peptide MVVMAPRTLFLLLSGALTLTETWA, cleaved after expression, transmembrane and cytoplasmic domain are shown in italics):

[0543] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQR RAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD(SEQ ID NO: 107)

[0544] "HLA-G Null 1,2,3"

[0545] "HLA-G Null 1,2,3" corresponds to HLA-G where the amino acids specifically expressed on α1, α2, and α3 of HLA-G are replaced by consensus amino acids expressed on other HLA-I (the mutated 20 amino acids in the sequences below are indicated in bold).

[0546] HLA-I consensus amino acids were derived from sequence information obtained from the immunopolymorphism database at EBI. Full-length HLA-I proteins were analyzed, and residue profile plots were constructed for each domain (alpha 1-3) across the entire HLA-I set, enabling the identification of HLA-G-specific residues (20 in total).

[0547] The sequence information was also used to generate an allelic consensus sequence for each HLA protein, in which positions in the canonical sequence were replaced by the most common residue found in all alleles.

[0548] To obtain the HLA-G Null 1,2,3 sequence, HLA-G-specific residues were replaced with consensus residues found in other HLA molecules at specific 20 positions.

[0549] Soluble protein (ECD HLA-G Null 1,2,3) was expressed with AVitev10His tag (signal peptide in bold, AVI tag underlined, Tev protease site underlined and italicized, 10His tag in italics):

[0550] MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSTAVSRPGRGEPRFIAVGYVDDTQFVR FDSDAASPRMEPRAPWVEQEGPEYWERETRNAKANAQTDRVNLRTLRGYYNQSEAGSHTLQWMYGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAAREAEQLRAYLEGT CVEWLHRYLENGKETLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKQ GLNDIFEAQKIEWHE LE ENLYFQG SGGSHHHHHHHHHH(SEQ ID NO: 112)

[0551] The peptide signal is cleaved after expression, and the purified final protein sequence used for screening assays includes the following sequence:

[0552] GSHSMRYFSTAVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMEPRAPWVEQEGPEYWERETRNAKANAQTDRVLRTLRGYYNQSEAGSHTLQWMYGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAAREAE QLRAYLEGTCVEWLHRYLENGKETLQRADPPKTHVTHHPVSDHEATLRCWALGYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKQGLNDIFEAQKIEWHELEENLYFQGSGGSHHHHHHHHHH (SEQ ID NO: 113)

[0553] HLA-G Null 1,2,3 DNA sequence for cell membrane-associated expression:

[0555] The corresponding membrane-bound protein contains the following sequence (signal peptide MVVMAPRTLFLLLSGALTLTETWA, cleaved after expression, transmembrane and cytoplasmic domain in italics):

[0556] GSHSMRYFSTAVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMEPRAPWVEQEGPEY WERETRNAKANAQTDRVNLRTLRGYYNQSEAGSHTLQWMYGCDLGSDGRLLRGYEQYAYDGKDY LALNEDLRSWTAADTAAQISKRKCEAAREAEQLRAYLEGTCVEWLHRYLENGKETLQRADPPKT HVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPS GEEQRYTCHVQHEGLPEPLTLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD(SEQ ID NO: 115)

[0557] "HLA-G Null 1.3"

[0558] "HLA-G Null 1,3" corresponds to HLA-G where the amino acids specifically expressed on α1 and α3 HLA-G are replaced by consensus amino acids expressed on other HLA-I (in SEQ ID NO: 117 below, the mutated amino acids are shown in bold).

[0559] The protein was expressed on the cell surface. The DNA sequence for cell membrane-associated expression includes:

[0561] The corresponding membrane-bound protein contains the following sequence (signal peptide MVVMAPRTLFLLLSGALTLTETWA, cleaved after expression, transmembrane and cytoplasmic domain in italics):

[0562] GSHSMRYFSTAVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMEPRAPWVEQEGPEY WERETRNAKANAQTDRVNLRTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDY LALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKT HVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPS GEEQRYTCHVQHEGLPEPLTLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD(SEQ ID NO: 117)

[0563] "HLA-G Null 3"

[0564] "HLA-G Null 3" corresponds to HLA-G in which the amino acids specifically expressed on α3 HLA-G are replaced by consensus amino acids expressed on other HLA-I (in SEQ ID NO: 119 below, the mutated 5 amino acids are shown in bold).

[0565] The protein was expressed on the cell surface. The DNA sequence for cell membrane-associated expression includes:

[0567] The corresponding membrane-bound protein contains the following sequence (signal peptide MVVMAPRTLFLLLSGALTLTETWA, cleaved after expression, transmembrane and cytoplasmic domain in italics):

[0568] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTA ADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKQ SSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD (SEQ ID NO: 119)

[0569] "HLA-G Null3 2AA"

[0570] "HLA-G Null3 2AA" corresponds to an HLA-G in which only 2 amino acids that are specifically expressed on α3 HLA-G and reported to interact with ILT2 / ILT4 (F195, Y197 HLA-G) are replaced by consensus amino acids expressed on other HLA-I (mutated amino acids are shown in bold).

[0571] The protein was expressed on the cell surface. The DNA sequence for cell membrane-associated expression includes:

[0573] The corresponding membrane-bound protein contains the following sequence (signal peptide MVVMAPRTLFLLLSGALTLTETWA, cleaved after expression, transmembrane and cytoplasmic domain in italics):

[0574] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQR RAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD(SEQ ID NO: 121)

[0575] Isolated HLA-G alpha 3 domain (wild type)

[0576] Soluble protein was expressed as a Tev-humanFc fusion protein (signal peptide in bold, Tev underlined, Fc fragment in italics) and was not co-expressed with B2m (“HLA-G alpha 3 domain isolated, lacking B2m”):

[0577] MSVPTQVLGLLLLWLTDARCDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPI LEENLYFQ GVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 122)

[0578] The peptide signal is cleaved after expression, and the purified final protein sequence used for screening assays includes the following sequence (Fc tag is cleaved):

[0579] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPILEENLYFQ (SEQ ID NO: 123)

[0580] DNA sequence for cell membrane-associated expression:

[0581] atgtccgtgccgacccaagtgctgggactgctcctgctctggctgactgacgctcgctgtgacccccctaagacccacgtcactcatcaccctgtgtccgaccatgaag ctaccctgagatgctgggccctgggtttctaccccgccgagattaccttgacctggcaaagggacggcgaagatcagacgcaagacaccgagctcgtggagactcggccagcgggg gatggaacattccagaaatgggccgcagtggtcgtgccgtccggagaagaacagcggtacacttgccacgtgcagcacgaaggcctgccggagcctctgacccttcgctggaagc agtcgagcctccccaccatcccgatcatggggattgtggccggccttgtggtgctggccgcagtcgtgaccggagcagctgtggcggctgtcctgtggcggaagaagtcaagcgat (SEQ ID NO: 124)

[0582] The corresponding membrane-bound protein contains the following sequence (signal peptide cleaved after expression, transmembrane and cytoplasmic domain are shown in italics):

[0583] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD(SEQ ID NO: 125)

[0584] HLA-G Null Alpha 3 Domain Highlighted (5 amino acids in bold)

[0585] The protein was expressed as a 10HistevAVI-tagged protein (Tag and GS linkers are in italics, signal peptide is in bold).

[0586] MSVPTQVLGLLLLWLTDARCGGSHHHHHHHHHGSGSENLYFQGLNDIFEAQKIEWHGGGSGSDPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKQSSLPTIPI (SEQ ID NO: 126)

[0587] After expression, the peptide signal is cleaved, and the 10His tag is removed during purification. The purified final protein sequence used for screening assays includes the following sequence (the N-terminal AVI tag and GS linker are in italics):

[0588] GLNDIFEAQKIEWHGGGSGSDPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKQSSLPTIPI (SEQ ID NO: 127)

[0589] B2m

[0590] All HLA-G constructs used in the screening assays described above (except for the B2m-free, wild-type, isolated alpha 3 domain of HLA-G) were coexpressed with B2m. For expression of soluble HLA-G constructs, B2m with the following sequence was coexpressed (signal peptide is shown in bold):

[0591] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 128)

[0592] After expression, the signal peptide is cleaved and the final purified protein used for screening assays contains the following sequence:

[0593] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 129)

[0594] All membrane-bound HLA-G constructs (including isolated alpha-3 domains expressed by cells) were coexpressed with B2m. The DNA sequence used for transfection is as follows:

[0595] atgtcacgctccgtggcactggctgtgctggccctgctctccctgtcgggtcttgaggccatccagaggactccgaagattcaagtctactcccgccatcctgccgaaaacggaaagtccaattttctgaactgctatgtgtcgggcttccacccctccgacatcgaagtggacc tcctgaagaacggggagcggattgaaaaggtcgagcacagcgacctgagcttctcgaaggactggtcattctacctcctgtactacactgaattcaccccaaccgaaaaggatgagtacgcgtgcagagtgaaccacgtgaccttgagccagccgaagatcgtgaaatgggaccgggatatg (SEQ ID NO: 130)

[0596] The corresponding B2m complex with membrane-bound HLA-G constructs contains the sequence SEQ ID NO: 129 (signal peptide MSRSVALAVLALLSLSGLEA, cleaved after expression).

[0597] Methods for the production and purification of HLA-G constructs expressed as soluble proteins

[0598] Protein expression and purification of isolated wild-type HLA-G α3 domain (TevHumanFc or TevHFc)

[0599] HLA-G α3 TevHFc was co-expressed with β2m using the Expi293 expression system ТМ (Life Technologies ТМ ), following the manufacturer's protocol. Cells were harvested 5 days after transfection, and the supernatants were immediately used for purification.

[0600] Supernatants containing HLA-G α3 TevHFc+β2m protein were applied to a Hitrap protein A column. Unbound protein and impurities were washed with PBS, and HLA-G α3 TevHFc+β2m protein was eluted with 0.1 M citric acid buffer pH 2, and the peak fractions were neutralized with 0.5 mL 2 M Tris pH 8. Fractions containing purified HLA-G α3 TevHFc+β2m protein were pooled, and the HFc tag was removed by incubating the protein with tev protease at a ratio of 1:100 for 2 hours at room temperature and for 2 hours at 4°C. The protein was then concentrated and purified by size-exclusion chromatography on an S75 26 / 60 column equilibrated with PBS. Fractions containing purified HLA-G α3 protein were pooled, concentrated, and aliquots were stored at -80°C until use.

[0601] Protein expression and purification of the isolated alpha 3 domain of Null HLA-G

[0602] 10histevAVI HLA-G α3 Null was co-expressed with β2m using the Expi293 expression system ТМ (Life Technologies ТМ), following the manufacturer's protocol. Cells were harvested 5 days after transfection, and the supernatants were immediately used for purification. Supernatants containing the 10histevAVI HLAG α3 Null+B2m protein were applied to a HisTrap Excel column (GE Healthcare) using the Äkta Purifier (GE Healthcare). Unbound protein and contaminants were washed with Cytiva HyClone phosphate-buffered saline. ТМ (PBS), 500 mM NaCl (pH 7.5). 10 mM imidazole and protein were eluted with Cytiva HyClone phosphate-buffered saline. ТМ (PBS), 500 mM NaCl (pH 7.5), 500 mM imidazole. Fractions containing purified 10histevAVI HLAG α3 Null+B2m protein were pooled, and the 10his tag was removed by incubating the protein with tev protease at a ratio of 1:100 for 2 hours at room temperature and for 2 hours at 4°C. The protein was then concentrated and purified by size-exclusion chromatography on an S75 26 / 60 column equilibrated with Cytiva HyClone phosphate-buffered saline. ТМ(PBS). Fractions containing purified AVI HLAG α3 Null+B2m protein were pooled, concentrated, and aliquots were stored at -80°C until use.

[0603] Protein expression and purification of HLA-G ECD (wild-type or Null variants) co-expressed with B2m

[0604] ECD HLA-G (WT or Null mutants) were co-expressed with β2m using the CHO-SXE expression system following the manufacturer's protocol. Briefly, CHO-SXE cells were grown in a shaking incubator at 37°C under 8% CO2 in serum-free CD CHO medium (Gibco) supplemented with Gibco® GlutaMAX. ТМ (1:1000) until a cell density of 6×10 is reached 6 / ml. The cells were then centrifuged at 1500 rpm and resuspended in fresh ExpiCHO medium. ТМ for expression (Gibco). Cells were transfected using 1 mg / L DNA in a 1:1 ratio of ECD HLA-G to β2m. Transfection was performed using the ExpiFectamine transfection kit. ТМ CHO and OptiPRO ТМSFM. Conditioned medium containing secreted proteins was collected 96 hours after transfection. The filtered cell culture supernatant was loaded onto a 5 ml HisTrap Excel column (GE Healthcare) using the Äkta Purifier (GE Healthcare). The column was washed with Cytiva HyClone phosphate-buffered saline. ТМ (PBS), 500 mM NaCl (pH 7.5), and the protein was eluted with the same buffer containing 500 mM imidazole. Fractions containing protein were analyzed by SDS-PAGE using NuPAGE 4-20% Tris-glycine (Thermo) and NuPAGE MES SDS running buffer (Thermo) and stained with Quick Coomassie Stain (VWR). Pure fractions were pooled before concentration in an Amicon® Ultra-15 centrifugal filter device (Millipore). Proteins were then further purified with a Superdex 200 16 / 600 column (GE Healthcare) using Cytiva HyClone phosphate-buffered saline. ТМ (PBS) as a working buffer. Protein purity was assessed using analytical size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were greater than 97% pure (typically at least 99%). Additionally, proteins were analyzed using liquid chromatography-mass spectrometry (LC-MS) to confirm that the molecular weight (MW) of the sequence matched the expected one.

[0606] Methods for producing cells expressing HLA-G constructs on the cell surface

[0607] Transient expression of HLA-G (including Null constructs) with B2m on the surface of ExpiHEK293

[0608] Co-transfection of Expi293 suspension cells TM HLA-G and B2m expression vectors in a 1:1 ratio were transfected using ExpiFectamine transfection reagent. TM 293 (ThermoFisher Scientific) and after 24 hours, cell surface protein expression was obtained.

[0609] Transient expression of HLA-G (including Null constructs) with B2m on the surface of CHO (PHAGE panning)

[0610] HLA-G or HLA-G Null 1,2,3 were co-transfected with B2m expression vector at a 1:1 ratio into proprietary CHO-SXE cells using ExpiFectamine Transfection Kit ТМ CHO (Gibco), following the manufacturer's recommendations. Cells expressing HLA-G on their surface were harvested after 48 hours.

[0611] HLA-G-expressing HCT116 cells

[0612] One day before transfection, HCT116 cells (ATCC CCL-247) were seeded at a density of 4×10 6 cells per 75 cm T-flask 2in 20 ml of complete RPMI growth medium and incubated for 24 hours at 37°C, 5% CO2. On the day of transfection, the growth medium was removed and replaced with 16 ml of complete growth medium. For each flask with cells to be transfected, 20 μg of HLA-G and β2m plasmids (1:1 ratio) were diluted in 4 ml of serum-free Opti-MEM® I Reduced Serum. 80 μl of Lipofectamine LTX® reagent were added to the Opti-MEM® DNA solution diluted above and incubated at room temperature for 30 minutes. After incubation, DNA-Lipofectamine LTX® complexes were added directly to each flask containing cells, and the flasks were placed in a CO2 incubator at 37°C for 22 ± 2 hours.

[0613] 1.2 HLA-I constructs

[0614] HLA-I constructs were co-expressed with β2m using the Expi293 expression system ТМ (Life Technologies), following the manufacturer's protocol.

[0615] HLA-I consensus sequences were derived from the amino acid sequences of HLA-I alleles known from publicly available sources. Corresponding sequence information for the analyzed alleles was obtained from the immunopolymorphism database at EBI. This information was used to generate an allelic consensus sequence for each HLA protein, in which positions in the canonical sequence were replaced with the most common residue found in all alleles.

[0616] The DNA sequences encoding the consensus amino acid sequences used for transfection / expression of cell membranes are shown below. The DNA sequence encoding B2m was as described above. The signal peptide in the secreted HLA-I proteins was removed after expression.

[0617] HLA-A

[0618] DNA sequence used for transfection / cell membrane expression:

[0620] The corresponding membrane-bound protein contains the following sequence:

[0621] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAHSQTDRVDLGTLRGYYNQSEAGSHTIQMMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHVAEQLRAYLEGTCVEW LRRYLENGKETLQRTDPPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV (SEQ ID NO: 132)

[0622] HLA-B

[0623] DNA sequence used for transfection / cell membrane expression:

[0625] The corresponding membrane-bound protein contains the following sequence:

[0626] GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQISKTNTQTYRESLRNLRGYYNQSEAGSHTLQRMYGCDVGPDGRLLRGHNQYAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVE WLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQSTIPIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 134)

[0627] HLA-C

[0628] DNA sequence used for transfection / cell membrane expression:

[0630] The corresponding membrane-bound protein contains the following sequence:

[0631] CSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNL RGYYNQSEAGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQLRAYLEGTCVEWL RRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEPSSQPTIPIVGIVAGLAVLAVLAVLGAVMAVVMCRRKSSGGKGGSCSQAASSNSAQGSDESLIACKA (SEQ ID NO: 136)

[0632] HLA-E

[0633] DNA sequence used for transfection / cell membrane expression:

[0635] The corresponding membrane-bound protein contains the following sequence:

[0636] GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCV EWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO: 138)

[0637] HLA-F

[0638] DNA sequence used for transfection / cell membrane expression:

[0640] The corresponding membrane-bound protein contains the following sequence:

[0641] GSHSLRYFSTAVSRPGRGEPRYIAVEYVDDTQFLRFDSDAAIPRMEPREPWVEQEGPQYWEWTTGYAKANAQTDRVALRNLLRRYNQSEAGSHTLQGMNGCDMGPDGRLLRGYHQHAYDGKDYISLNEDLRSWTAADTVAQITQRFYEAEEYAEEFRTY LEGECLELLRRYLENGKETLQRADPPKAHVAHHPISDHEATLRCWALGFYPAEITLTWQRDGEEQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPQPLILRWEQSPQPTIPIVGIVAGLVVLGAVVTGAVVAAVMWRKKSSDRNRGSYSQAAV (SEQ ID NO: 140)

[0642] 1.3. ILT2 / ILT4 constructs (Fc fusion products):

[0643] ILT2 was expressed as a soluble ILT2 ECD-rabbit Fc fusion protein (signal peptide in bold, rabbit Fc underlined):

[0644] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSES SDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPS LSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVT LLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLE KTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAP IEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 141)

[0645] The purified final protein sequence used for screening assays contains the following sequence:

[0646] GHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTL SAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAP EETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQ GWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLE KTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAP IEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 142)

[0647] ILT4 was expressed as a soluble ILT4 ECD-rabbit Fc fusion protein (signal peptide in bold, rabbit Fc underlined):

[0648] MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSD PLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLS VQPGPVVAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLC QSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLELE KTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAP IEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 143)

[0649] The purified final protein sequence used for screening assays contains the following sequence:

[0650] QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTL SAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPG ESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQ FHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLELE KTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAP IEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 144)

[0651] Expression and purification of ILT2rbFc and ILT4rbFc

[0652] Proteins were expressed by transient transfection using the Expi293 expression system ТМ HEK (Life Technologies ТМ ), following the manufacturer's protocol. Cells were harvested 5 days after transfection, and the supernatants were immediately used for purification.

[0653] Supernatants containing ILT2rbFc or ILT4rbFc protein were loaded onto a Hitrap protein A column. Unbound protein and impurities were washed with PBS, and ILT2rbFc or ILT4rbFc proteins were eluted with endonuclease-free 0.1 M citric acid buffer pH 2, and peak fractions were neutralized with 0.5 mL 2 M Tris pH 8. Next, fractions containing purified protein were pooled, concentrated, and purified by size exclusion chromatography on S200 26 / 60 using Cytiva HyClone phosphate-buffered saline (PBS). ТМ as a working buffer. Fractions containing purified ILT2rbFc or ILT4rbFc protein were pooled, concentrated, and aliquoted before storage at -80°C.

[0654] Example 2. Generation of antibodies by HLA-G immunization.

[0655] Due to the specific challenges associated with the production of anti-HLA-G antibodies (such as high homology with other HLA-I molecules, identification of antibodies capable of blocking the interaction between HLA-G and its inhibitory receptors) and to identify antibodies that would be useful in therapy, there was a need to develop a dedicated detection strategy, including a dedicated screening and testing strategy, which is described below.

[0656] Immunization and screening strategy

[0657] Several animals of different species (including mice and rabbits) were immunized with syngeneic cells expressing different forms of HLA-G with or without coexpression of B2m. After 3-5 injections, the animals were sacrificed, and PBMCs, spleen, bone marrow, and lymph nodes were collected. Serum binding was monitored using the immunogen.

[0658] Memory B cell cultures were generated, and supernatants were first screened for their ability to bind HLA-G at higher levels than an irrelevant control in a multiplex assay without washing using either the TTP Labtech Mirrorball system (plate reader) or Intellicyt iQue (flow cytometry). Cultures were screened for an irrelevant control protein, in-house developed HLA-G proteins, and / or HEK EXPI293 cells transiently expressing cell surface constructs of interest (HLA-G, HLA-G Null 1, 2, 3, HLA-G Null 3). Protein reagents were biotinylated to ensure streptavidin capture by the beads. A fluorescently labeled species-specific anti-Fc secondary antibody was used for antibody detection assays.

[0659] In the primary screening, approximately 3800 HLA-G-specific positive hits were identified from a total of 18 B cell culture experiments, each using 100-300 plates. Positive supernatants from the primary screening were then processed for further characterization in binding assays (soluble isolated alpha-3 Null domain and cell-expressed isolated alpha-3 domains, HLA-G Null 1,3, HLA-G Null3 2AA).

[0660] Wells with desired profiles were processed to detect the variable V region by detecting fluorescent spots and binding to the HLA-G ECD protein.

[0661] In parallel, plasma cells from bone marrow and lymph node were also directly screened for their ability to bind human HLA-G or, in particular, the α3 domain by detecting fluorescent spots. In this case, B cells secreting HLA-G-specific antibodies were collected on biotinylated human HLA-G ECD or isolated wild-type HLA-G α3 domain immobilized on streptavidin beads. Goat anti-species Fc-FITC conjugate detection reagent was used. Approximately 1,700 directly observable spots were collected.

[0662] Following reverse transcription (RT) and PCR of the collected cells, transcriptionally active PCR (TAP) products encoding antibody V regions were obtained and used for transient transfection of EXPI293 HEK cells. The resulting TAP supernatants containing recombinant antibodies were tested in the following assays:

[0663] - Cell binding to HLA-G and Null mutants described above to establish domain binding (multiplexed iQue).

[0664] - ILT2 blocking analysis

[0665] - Affinity measurement using Biacore.

[0666] The heavy and light chain variable region gene pairs from the TAP products of interest were then cloned as species corresponding to full-length IgG antibodies and re-expressed in a transient expression system. The recombinant cloned antibodies were then retested in the assays described above.

[0667] A total of 109 V (variable) regions were cloned and reported, and only 30 of them were specific to HLA-G and did not bind to other HLA-Is, among which only 9 showed blocking of HLA-G interaction with ILT2. All of these antibodies bound to the alpha-3 domain of HLA-G, and 5 of the 9 specific and blocking antibodies had different sequences and were sent for further testing (listed in Table 3 below).

[0668] Table 3: Antibodies produced by immunization selected for further analysis

[0669] ID antibody Derivative / immunogen HLA-G01 Directly observable spots, bone marrow / Rab9+hHLA-G cells HLA-G02 (VR12389 gL2gH16) B cell culture, spleen / Rab9+hHLA-G cells HLA-G03 B cell culture, lymph node / Rab9+hHLA-G cells HLA-G04 B cell culture, lymph node / Rab9+hHLA-G+B2m cells HLA-G05 Directly observable spots, bone marrow / Rab9 cells+hHLA-G

[0670] Humanization campaigns were conducted for several antibodies based on their properties, which were then evaluated in characterization assays, including HLA-G02 and HLA-G01, which emerged as the best performing antibodies. Humanization of VR12389 is described in Example 4. HLA-G03 was also humanized; however, the high affinity of the humanized antibody did not result in improved functional activity, as described in the examples below.

[0671] Additional analyses performed with purified antibodies included cell-based specificity assays, ILT4 blocking assay, and ADCC. Data obtained for purified IgG1 antibodies are described in the additional examples below.

[0672] In the present description, the antibody identifier HLA-G02 refers to the humanized IgG1 antibody VR12389gL2gH16.

[0673] The immunization method that led to the discovery of antibody 12389

[0674] Rab9 cells transiently expressing HLA-G

[0675] Rab9 fibroblast cells were cultured in RPMI medium + 10% FBS and 1% glutamine in 5-layer cell culture flasks. When the cells reached 90-100% confluency, the medium was removed, the cells were washed with 100 ml PBS, and removed from the cell culture flask by adding 100 ml Accutase and incubating for 10-15 minutes at room temperature. The collected cells were centrifuged and resuspended at a concentration of 5 × 10 7 cells / ml in Earles' balanced salt buffer. HLA-G DNA was added to the cells at a rate of 250 μg DNA / ml of cells. The Rab9+HLA-G DNA mixture was then transferred to electroporation cuvettes at a rate of 3 × 10 7cells / cuvette. The cuvettes were then exposed to a pulsed electrical voltage of 150-170 V (20 ms, 5.5 A) using a proprietary electroporator (Zapper) and a Gene Pulser Xcell ShockPod cuvette chamber (BIORAD). After the electrical pulse, the cells were quickly transferred to warm Rab9 medium and placed back into a fresh 5-layer cell culture flask.

[0676] After all cells were electroporated and transferred to a new culture flask, they were incubated for 24 hours at 370°C, 5% CO2. Cells were then harvested using Accutase (as described previously), counted, and frozen in a -80°C freezer in 2x10 cryovials. 7 cells per cryovial. After 24 hours, the frozen cells were transferred to a Dewar flask with liquid nitrogen for longer-term storage.

[0677] Before freezing 5×10 5Transfected cells were tested for HLA-G expression by staining for 1 h at 40°C with APC-conjugated anti-HLA-G antibody Sigma (clone MEM / G9) and running samples on a FACS Caliber.

[0678] On the day of immunization, for each injection, 1 vial of transfected cells was quickly thawed at 37°C and washed twice in 50 ml PBS before resuspending in 500 μl for administration to rabbits.

[0679] The DNA sequence encoding full-length HLA-G (SEQ ID NO: 111) was used for electroporation (HLA-G nucleic acid sequence optimized for expression in mammalian cells).

[0680] Immunization

[0681] One female New Zealand White rabbit was subcutaneously immunized with 2×10 7 Rabbit Rab9 fibroblasts transiently expressing HLA-G on their surface, obtained as described above, were immunized. An equal volume of complete Freund's adjuvant was injected subcutaneously into a separate area of ​​the body simultaneously with cell immunization.

[0682] The rabbit received two booster injections 14 days apart, during which Rab9 rabbit fibroblasts transiently expressed HLA-G on their surface. Before each immunization, heparinized blood (200 μl) was collected from the ear vein. Serum was collected from the blood samples after centrifugation at 10,000 rpm for 5 minutes in a tabletop centrifuge and frozen at -20°C. The experiment was terminated 14 days after the last booster vaccination using single-cell suspensions of spleen, lymph nodes, bone marrow, and peripheral blood mononuclear cells prepared and frozen in 10% DMSO / FCS at -80°C until used for B cell detection. At the end of immunization, blood was also collected and serum was prepared as described previously.

[0683] Detection and screening of B cells

[0684] VR12389 was detected in a spleen memory B cell culture. Spleen cells were cultured with a feeder cell line and supplements at 37°C for 5 days in 96-well plates. This culture was then subjected to screening using a wash-free multiplex flow cytometric assay (Intellicyt iQue). The culture supernatant containing the secreted antibody was mixed with screening reagents as described above (in-house developed HLA-G proteins and HLA-G constructs: HLA-G, HLA-G Null 1, 2, 3, HLA-G Null3, expressed in the cells). Screening cells used in the screening were stained differently, which allowed gating of different populations, and goat anti-rabbit antibody labeled with Dylight 405 was used as a secondary antibody to detect antibody binding.

[0685] Hits were defined as HLA-G-binding substances that were specific, i.e., did not bind to the HLA-G Null 1,2,3 mutant or irrelevant control transfection. B cells responsible for the hits were identified by tracking the original wells in which they were cultured, followed by fluorescent spot detection as described previously.

[0686] The harvested cells from both leading cultures with the best results after the spot detection step were processed using the same protocol. Reverse transcription (RT) and PCR of the harvested cells yielded transcriptionally active PCR (TAP) products encoding antibody V regions, which were used for transient transfection of EXPI293 HEK cells. The resulting TAP supernatants containing recombinant antibodies were characterized for cell binding, ILT2 blocking, and affinity before antibody cloning and expression on a larger scale.

[0687] As mentioned above, a number of animal species (including rats, mice, and rabbits) were immunized with syngeneic cells expressing various forms of HLA-G with or without co-expression of B2m, but not all immunization strategies were successful in producing anti-HLA-G antibodies, or, if anti-HLA-G antibody production was confirmed, these antibodies were not HLA-G-specific and / or did not block or were unable to bind the HLA-G protein expressed on the cell surface. It should be noted that immunization of rabbits with Rab9 cells expressing the isolated alpha-3 domain or expressing a rabbit-human HLA-G chimera did not induce the production of any anti-HLA-G antibodies.Thus, the present invention relates to a method of immunization that is particularly useful for detecting anti-HLA-G antibodies useful in therapy, wherein said method comprises immunizing a rabbit with Rab9 rabbit fibroblasts transiently expressing the full-length HLA-G sequence on the cell surface.

[0688] Example 3: Generation of antibodies using phage display

[0689] To identify antibodies that could be useful in therapy, a second approach was developed in parallel with immunization campaigns to attempt to identify antibodies that specifically bind to HLA-G and that could be useful in therapy from phage display libraries. In this case, due to the unique challenges associated with producing anti-HLA-G antibodies useful in therapy, a dedicated screening and testing strategy was developed.

[0690] Phage Display Libraries

[0691] To generate HLA-G-binding antibodies, three naive combinatorial human scFv phage libraries were used, using different constructs to generate HLA-G-selective binders that do not bind or bind poorly to other HLA-Is. Libraries were biopanned in three or four rounds of selection using recombinant HLA-G expressed only on the cell surface or using the extracellular domain of the recombinant HLA-G protein in the final round. To enrich for HLA-G-specific binders, an optional subtraction step of HLA-G Null 1,2,3 (soluble protein or cell-expressed) was included in the final round.

[0692] Briefly, cell biopanning consisted of co-transfecting DNA constructs encoding human HLA-G and β2m into ExpiCHO in the first round or into Expi293 HEK in subsequent rounds and incubating these cells with blocked phage virions previously depleted on untransfected cells or cells transfected with HLA-G Null 1, 2, 3. Protein biopanning was performed by incubating blocked phage particles with either plate-coated HLA-G or biotinylated HLA-G in solution, followed by capture with magnetic beads coated with streptavidin or neutravidin. After several washes with PBS Tween buffer, target-bound phages were eluted and reamplified by infection with E. coli TG1.

[0693] Phage screening

[0694] After the final round of selection, 1,692 monoclonal isolated phages were screened by ELISA against biotinylated HLA-G ECD captured on streptavidin-coated plates. Binding was detected using an HRP-conjugated antibody to the M13 pVIII coat protein. Biotinylated HLA-G Null 1, 2, and 3 were used to assess the specificity of these monoclonal phage clones. 359 binders of interest were sequenced, and diversity was analyzed based on the CDR3 sequence motif of the heavy chain variable region. Eighty-one unique clones were then reformatted into rabbit IgG scFv-Fc fusion proteins in a mammalian expression vector for further characterization.

[0695] Further characterization of HLA-G-selective binders

[0696] scFv-Fc was expressed in Expi293 HEK cells. Their binding and specificity were verified by flow cytometry using an IntelliCyt iQue Screener Plus. Diluted supernatants containing antibodies were added to ExpiHEK cells cotransfected with human HLA-G or HLA-G and β2-microglobulin. Binding was detected using a fluorescent antibody specific for the Fc fragment.

[0697] 21 cellular HLA-G binders that did not bind HLA-G Null 1,2,3+B2m were further evaluated as scFv-Fc and / or after reformatting into full-length human IgG1 by SPR, using ILT2 blocking assay, binding to HLA-A, -B, -C, -E, -F expressed on HEK, and binding to JEG3 cells by flow cytometry.

[0698] 14 antibodies were confirmed to have high specificity for HLA-G with no or minimal binding to other HLA-I molecules, among which only 6 antibodies blocked the interaction between HLA-G and ILT2.

[0699] Among the 6 specific and blocking antibodies, 3 antibodies had different sequences and were subjected to further testing (HLA-G06, HLA-G07, HLA-G08).

[0700] Additional analyses performed with purified antibodies included cell-based specificity assays, ILT4 blocking assay, and ADCC. The data obtained for purified antibodies are described in the additional examples below.

[0701] Example 4: Humanization of antibody 12389

[0702] Antibody 12389 was humanized by grafting CDRs from the rabbit V region onto the frameworks of the human germline antibody V region. To detect antibody activity, several framework residues from the rabbit V region were also retained in the humanized sequence. These residues were selected following the protocol described by Adair et al. (1991) (WO91 / 09967). The alignment of the rabbit antibody V region (donor) sequences with the human germline V region (acceptor) sequences is shown in Figs. 2 and 3, along with the designed humanized sequences. CDRs grafted from the donor to the acceptor sequence follow the Kabat definition (Kabat et al., 1987), with the exception of CDR-H1, where the combined Chothia / Kabat definition is used (see Adair et al., WO91 / 09967).

[0703] For antibody 12389, the human V region of IGKV1D-13 plus the J region of IGKJ4 (IMGT, http: / / www.imgt.org / ) were selected as the light chain CDR acceptor. All light chain framework residues in the humanized graft variants were from the human germline gene, except for one or two residues from the group including residues 3 and 70, where the donor residues of Valine (V3) and Glutamine (Q70) are preserved relative to SEQ ID NO: 7 (rabbit VL), respectively.

[0704] The human V region IGHV3-66 plus the J region IGHJ4 (IMGT, http: / / www.imgt.org / ) were selected as the heavy chain CDR acceptor of antibody 12389. Like many rabbit antibodies, the VH gene of antibody 12389 is shorter than the selected human acceptor. When aligned with the human acceptor sequence, framework 1 of the VH region of antibody 12389 lacks the N-terminal residue, which is retained in the humanized antibody (Fig. 3). Framework 3 of the rabbit 12389 VH region also lacks two residues (75 and 76, relative to SEQ ID NO:11, rabbit VH) in the loop between strands D and E of the beta sheet: in the humanized graft variants, the gap is filled by the corresponding residues (lysine 75, K75; asparagine 76, N76) from the selected human acceptor sequence.All heavy chain framework residues in the humanized graft variants are derived from a human germline gene, with the exception of one or more residues from the group consisting of residues 24, 48, 49, 71, 73, 78, and 96, where the donor residues valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), valine (V78), and glycine (G96) are retained with reference to SEQ ID NO: 11, respectively.

[0705] Humanized antibody chain variants and combinations thereof were expressed and assessed for binding affinity to human HLA-G compared to the parent antibody.

[0706] Expression in Expi293 cells

[0707] Genes encoding variant heavy and light chain V region sequences were designed and constructed using the automated synthesis approach at ATUM (Newark, CA). For transient expression in mammalian cells, the humanized light chain V region genes were cloned into the pMhCK light chain expression vector, which contained DNA encoding the human kappa chain constant region (allotype Km3). The humanized heavy chain V region genes were cloned into the human gamma-4 heavy chain expression vector pMhg4PFL, which contained DNA encoding the human gamma-4 heavy chain constant region with the S228P hinge-stabilizing mutation (Angal S., King DJ, Bodmer MW, Turner A., ​​Lawson ADG, Roberts G., Pedley B., and Adair JR. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody. Mol. Immunol.1993, 30 (1):105-8), or into the gamma-1 heavy chain expression vector pMhg1FL, which contained DNA encoding the constant region of the human gamma-1 heavy chain (G1m17, allotype 1). Cotransfection of the resulting heavy and light chain vectors into Expi293 suspension cells. TM was carried out using the ExpiFectamine transfection reagent TM 293 (A14525, ThermoFisher Scientific), which resulted in the expression of humanized recombinant IgG4P and IgG1 antibodies.

[0708] Affinity Measurement by SPR

[0709] As described below, the assay format consisted of capturing anti-HLA-G IgG with an immobilized IgG antibody specific for human Fc, followed by titration of HLA-G on the captured surface.

[0710] The HLA-G binding affinity of anti-HLA-G IgG antibody was determined by surface plasmon resonance using a Biacore T200 (GE Healthcare Biosciences AB). Assays were performed at 25°C. Affinity-purified goat F(ab')2 fragment to human IgG (Jackson ImmunoResearch) was immobilized on a Series S CM5 sensor chip (GE Healthcare Bio-Sciences AB) by amine coupling to a level of approximately 6,000 response units (RU). HBS-EP buffer + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, GE Healthcare Bio-Sciences AB) was used as the running buffer at a flow rate of 10 μl / min. The reference surface was prepared by activating and deactivating the corresponding flow cell.

[0711] An injection of 10 μL of anti-HLA-G IgG at concentrations ranging from 0.15 to 0.7 μg / mL was used to capture immobilized anti-human IgG, Fc. Human ECD HLA-G+B2m was titrated against the captured anti-HLA-G IgG at 50 nM and a flow rate of 30 μL / min for 60 seconds, followed by dissociation for 150 seconds. The surface was regenerated at a flow rate of 10 μL / min by injection of 10 μL of 40 mM HCl followed by injection of 5 μL of 5 mM NaOH.

[0712] Background-subtracted binding curves were analyzed with Biacore T200 Evaluation software (version 3.0) using a 1:1 binding model fitted to the local Rmax.

[0713] Antibodies were analyzed at the beginning and end of the assay, and the results showed good accuracy. High-quality data were obtained for all samples, which are summarized in Tables 4 and 5.

[0714] Table 4: Affinity data for 12389 (chimeric rabbit V regions / human Fc) and humanized grafts expressed as hIgG4P

[0715] Antibody 12389 Residues of the donor light chain Residues of the donor heavy chain ka (M -1 s -1 ) kd (s -1 ) Affinity (KD) nM 12389 (chimeric rabbit V regions / human Fc) - - 9,49E+05 9,20E-03 9,69 12389gL1gH1 V3, Q70 V24, I48, G49, K71, S73, V78, G93 8,48E+05 6,78E-03 7,99 12389gL2gH1 V3 V24, I48, G49, K71, S73, V78, G93 9,28E+05 7,08E-03 7,63 12389gL3gH1 - V24, I48, G49, K71, S73, V78, G93 8,45E+05 6,97E-03 8,25 12389gL2gH4 V3 I48, G49, K71, S73, V78, G93 8,88E+05 7,18E-03 8,09 12389gL2gH5 V3 V24, G49, K71, S73, V78, G93 8,17E+05 1,01E-02 12,4 12389gL2gH6 V3 V24, I48, K71, S73, V78, G93 8,39E+05 1,04E-02 12,5 12389gL2gH8 V3 V24, I48, G49, S73, V78, G93 7,87E+05 1,21E-02 15,4 12389gL2gH9 V3 V24, I48, G49, K71, V78, G93 8,50E+05 6,03E-03 7,09 12389gL2gH11 V3 V24, I48, G49, K71, S73, G93 8,50E+05 9,07E-03 10,7 12389gL2gH12 V3 V24, I48, G49, K71, S73, V78 8,44E+05 6,24E-03 7,39 12389gL2gH13 V3 I48, G49, K71, V78, G93 9,56E+05 6,32E-03 6,61 12389gL2gH14 V3 I48, G49, K71, S73, V78 8,75E+05 6,64E-03 7,58 12389gL2gH15 V3 V24, I48, G49, K71, V78 8,96E+05 5,67E-03 6,33 12389gL2gH16 V3 I48, G49, K71, V78 9,02E+05 5,46E-03 6,06 12389gL3gH16 - I48, G49, K71, V78 8,33E+05 5,60E-03 6,72

[0716] Table 5: Affinity data for 12389 (chimeric rabbit V regions / human Fc) and 12389gL2gH16 expressed as hIgG1 in two experiments

[0717] Antibody 12389 Residues of the donor light chain Residues of the donor heavy chain ka (M -1 s -1 ) kd (s -1 ) Affinity (KD) nM 12389 (chimeric rabbit V regions / human Fc) - - 8,41E+05 9,10E-03 10,80 12389gL2gH16 IgG1 V3 I48, G49, K71, V78 7,29E+05 3,48E-03 4,78 12389 (chimeric rabbit V regions / human Fc) - - 7,82E+05 9,18E-03 11,70 12389gL2gH16 IgG1 V3 I48, G49, K71, V78 6,72E+05 3,64E-03 5,42

[0718] As shown in Table 4, all grafts except 12389gL2gH5, 12389gL2gH6, 12389gL2gH8, 12389gL2gH11 had KD less than 10 nM and lower than the KD measured for parental 12389 (chimeric rabbit V region / human Fc).

[0719] Graft 12389gL2gH16, which retained the VH donor framework residues I48, G49, K71, and V78, as well as the V3 residue of the VL donor framework, had the highest binding affinity to human HLA-G measured by surface plasmon resonance and retained functionality when expressed in different formats such as IgG4P and IgG1 (Tables 4-5), so it was selected for further characterization.

[0720] Example 5. Expression and purification of HLA-G01-HLA-G08.

[0721] The antibodies were transiently expressed as IgG1 in CHO cells transfected with a DNA vector encoding LC and HC of HLA-G01-HLA-G08 antibodies (LC:HC ratio 1:1) and purified by protein A affinity chromatography according to known methods for further testing.

[0722] Protein concentration was determined by reading the absorbance at 280 nm in nanodrop format, and purity was determined by analytical size-exclusion HPLC. Monomer content was determined by analytical size-exclusion chromatography and SDS-PAGE. Endotoxin levels were determined using Charles River Endosafe® LAL reagent cartridge technology and the Endosafe® nexgen-PTS reader, where a level of <1 IU / mL was considered acceptable.

[0723] The final purified sample had high purity, with a monomer content of >98%. The final purified sample was analyzed by intact mass spectrometry to confirm the mass of the heavy and light chains, expected modifications, and identity.

[0724] Example 6. Production of afucosylated HLA-G02 antibody

[0725] Method for obtaining KO FUT8 CHOSXE / DG44 cells

[0726] Guide RNAs (gRNAs) were designed to knock out two exons containing the active site encoding sequence for alpha1,6-fucosyltransferase (FUT8). gRNAs 2-8 (7 total) were designed using Benchling software to create multiple deletions in both the forward and reverse strands, with the maximum possible deletion being 4 kb. Guide RNAs were used as a pool to maximize potential knockouts. The gRNA sequence is presented in Table 6 below.

[0727] Table 6: gRNA sequences

[0728] Name Subsequence FUT8-gRNA-2 GAUGGAGGCUGUCUACAAUG FUT8-gRNA-3 GUCAGGGCUGUAGCACACUG FUT8-gRNA-4 GAAGUGGUAGUAACUUUACA FUT8-gRNA-5 AUUAGUAUCCCUAGUCAUGG FUT8-gRNA-6 UGGUACACCUAGUACUACUG FUT8-gRNA-7 UGACUAUACAAAUUUCUGGG FUT8-gRNA-8 AGUCAACAAUGUCUUAGACA

[0729] gRNA pools were prepared at final concentrations in pmol as shown in Table 7. Cas09 (ThermoFisher) was prepared at the required concentration as shown in Table 7.

[0730] 3×10 6CHO SXE / DG44 cells were prepared for nucleofection: after centrifugation at x100g for 8 min, the cells were washed in PBS, centrifuged again at x100g for 8 min, and resuspended in 100 μl of nucleofection solution to obtain 3×10 4 / µl cells. Pre-transfection mixtures were prepared as shown in Table 7 below and left without cells for 10-60 minutes at room temperature to allow formation of Cas09 / gRNA complexes.

[0731] Table 7: Pre-transfection mixes

[0732] Ratio 9:1 Ratio 6:1 Ratio 3:1 Negative control Simulation control Nucleofection solution 60 µl 60µl 60 µl 60 µl 60 µl gRNA 20 µl (90 pmol) 20 µl (60 pmol) 20 µl (30 pmol) - - Cas09 (3.22 mg / ml) 3.33 µl 3.33 µl 3.33 µl 3.33 µl -

[0733] Before nucleofection (Nucleofector 4D, Lonza), 16.67 µl of cells (3×10 4cells / µl) according to the manufacturer's instructions. Cells were recovered by adding 900 µl of pre-warmed CD CHO medium to small, upright T25 flasks. After incubation for 24 h at 37°C, 5% CO2, the medium was replaced with fresh, pre-warmed CD CHO (the antibiotics penicillin, streptomycin, and amphotericin B were added to reduce the risk of contamination), and once the cells had recovered and were dividing, they were transferred to a 125 ml shaker (after 96-120 h).

[0734] Ten days after nucleofection, the cells were ready for FACS sorting. Cells were stained with LCA (Lens Culinaris agglutinin conjugated to fluorescein). Cells were prepared for FACS by centrifugation at 100 g for 8 minutes, washing with PBS, centrifugation again at 100 g for 8 minutes, and resuspension in prewarmed CD CHO medium; LCA dye was then added at a concentration of 20 μg / mL and left for 45 minutes. Cells were then washed twice with PBS (centrifugation at 100 g for 8 minutes, resuspension in PBS) to remove any unbound dye. LCA bound to fucose on the cell surface of FUT8-positive cells; FUT8-negative (knockout) cells were not stained. Cells were collected in prewarmed CD CHO medium. Cells were maintained at 37°C, 5% CO2 for recovery and passage until the desired cell density was reached.

[0735] Production of afucosylated HLA-G antibodies

[0736] Anti-HLA-G02 antibody constructs were expressed in the engineered CHO-SXE cell line (Cain et al 2012), which was further modified to knockdown the α-1,6-fucosyltransferase (FUT8) enzyme as described above. The ExpiCHO transfection system (Thermo Fisher Scientific) was used to transiently generate afucosylated antibodies from these cells. Following a high-yield protocol, cells were seeded at a density of 6 × 10 6cells / ml in ExpiCHO expression medium. For a 200 ml culture, 200 μg DNA was diluted in 8 ml Opti-PRO serum-free medium (SFM) and mixed with 7.4 ml Opti-PRO SFM containing 640 μl ExpiFectamine transfection reagent before addition to the cells. Cells were transferred to an 8% CO2, 37°C incubator with a shaking platform at 190 rpm. On the first day after transfection, 48 ml of growth medium and 1200 μl of enhancer were added to the cells and they were returned to the incubator, with the temperature reduced to 32°C. Cultures were harvested on day 10 by centrifugation for 1 hour at 4000 rpm. Cell culture supernatants were clarified by filtration through a 0.22 μM Stericup filter.

[0737] The product titer was determined by loading 100 μl of the supernatant onto a protein G column attached to an Infinity high-performance liquid chromatography (HPLC) system. The product was eluted from the column with 150 mM sodium chloride, pH 2.1, and signal A 280compared with a purified Fab standard. The clarified supernatant was loaded onto a MabSelect Sure column (GE Healthcare) at 5 ml / min and washed with 3 column volumes (CV) of PBS, pH 7.4. The captured protein was eluted from the column with 0.1 M sodium citrate buffer, pH 3.6. The eluate was neutralized with 2 M Tris-HCl, pH 8.5. To remove high molecular weight species, the affinity-purified protein was loaded onto a Superdex 16 / 60 gel filtration chromatography column equilibrated with 10 mM phosphate-buffered saline, pH 7.4, at 1 ml / min, and the eluted fractions were pre-analyzed by analytical SE-UPLC to pool the relevant fractions. The pooled fractions were subjected to SDS-PAGE and SE-UPLC to determine the quality and purity of the protein.

[0738] In the present description, unless otherwise specified, “HLA-G02” refers to unmodified normal (i.e., fucosylated) VR12389gL2gH16 IgG1. “Afucosylated HLA-02” refers to the corresponding afucosylated IgG1 antibody produced according to the methods described herein.

[0739] Example 7: Binding, affinity, and specificity of HLA-G antibodies

[0740] 7.1 Affinity to HLA-G WT measured by SPR

[0741] The binding affinity of anti-HLA-G antibodies (hIgG1 format) to HLA-G was determined by surface plasmon resonance using a Biacore T200 (GE Healthcare Biosciences AB). Assays were performed at 25°C. Affinity-purified goat F(ab')2 fragment to human IgG (Jackson ImmunoResearch) was immobilized on a Series S CM5 sensor chip (GE Healthcare Bio-Sciences AB) by amine coupling to a level of approximately 6,000 response units (RU). HBS-EP buffer +(10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, GE Healthcare Bio-Sciences AB) was used as the running buffer with a flow rate of 10 μl / min. The reference surface was prepared by activating and deactivating the corresponding flow cell.

[0742] 10 μL injection of anti-HLA-G antibodies at concentrations ranging from 0.6 to 0.9 μg / mL was used for capture by immobilized anti-human IgG, Fc. Human ECD HLA-G+B2m was titrated against the captured anti-HLA-G hIgG1 at different maximum concentrations of 4000, 400, 100, and 50 nM at a flow rate of 30 μL / min for 60, 90, or 120 seconds, followed by dissociation for 120, 180, or 240 seconds (Table 8).

[0743] Table 8:

[0744] Capture Sample Concentration (nM) Contact (c) Dissociation (c) Both normal HLA-G02 and afucosylated HLA-G02 HLA-G 50 90 180 HLA-G01, HLA-G03, HLA-G05 HLA-G 100 90 180 HLA-G04 HLA-G 400 120 240 HLA-G06, HLA-G07, HLA-G08 HLA-G 4000 60 120

[0745] The results are presented in Table 9.

[0746] Table 9: Affinity of anti-HLA-G antibodies determined by SPR method

[0747] Antibody ID ka (M -1 s -1 ) kd (s -1 ) KD (nM) HLA-G01 5,85E+05 3,35E-03 5,72 HLA-G02 (VR12389gL2gH16) 6,18E+05 3,15E-03 5,09 HLA-G06 3,09E+05 7,91E-02 256 HLA-G03 4,04E+05 2,34E-04 0,58 HLA-G07 4,39E+05 2,00E-01 456 HLA-G04 5,61E+04 4,33E-04 7,72 HLA-G05 2,00E+05 1,40E-03 6,98 HLA-G08 2,19E+05 7,66E-02 349

[0748] HLA-G06, HLA-G07, and HLA-G08 had the lowest affinity (higher KD value) for HLA-G, as determined by the SPR method. HLA-G01, HLA-G02, and HLA-G03 had the highest affinity for HLA-G.

[0749] Another assay also assessed the affinity of afucosylated HLA-G02 and found similarity to the regular (fucosylated) HLA-G02 counterpart. The results are presented in Table 10 below.

[0750] Table 10: SPR affinity of conventional and afucosylated anti-HLA-G02 antibodies

[0751] Antibody ID ka (M -1 s -1 ) kd (s -1 ) KD (nM) HLA-G02 (normal, i.e. fucosylated) 8,24E+05 3,54E-03 4.30 Afucosylated HLA-G02 8,07E+05 3,55E-03 4.40

[0752] 7.2 Affinity to HLA-G Null 1,2,3 measured by SPR to assess specificity

[0753] The binding affinity of anti-HLA-G IgG1 antibodies to HLA-G Null 1, 2, and 3 was determined by surface plasmon resonance using a Biacore T200 (GE Healthcare Biosciences AB). Assays were performed at 25°C. Affinity-purified goat F(ab')2 fragment to human IgG (Jackson ImmunoResearch) was immobilized on a Series S CM5 sensor chip (GE Healthcare Bio-Sciences AB) by amine coupling to a level of approximately 6,000 response units (RU). HBS-EP buffer + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, GE Healthcare Bio-Sciences AB) was used as the running buffer with a flow rate of 10 μl / min. The reference surface was prepared by activating and deactivating the corresponding flow cell.

[0754] An injection of 10 μL of anti-HLA-G antibodies at concentrations ranging from 0.6 to 0.9 μg / mL was used for capture by the immobilized Fc-specific antibody to human IgG. The human “HLA-G Null 1,2,3” mutant AVItev10HisTag+B2m was titrated over the captured anti-HLA-G IgG with an initial concentration of 20 μM at a flow rate of 30 μL / min for 60 s, followed by dissociation for 150 s. The surface was regenerated at a flow rate of 10 μL / min by injection of 10 μL of 40 mM HCl followed by injection of 5 μL of 5 mM NaOH.

[0755] Background-subtracted binding curves were analyzed using Biacore T200 Evaluation software (version 3.0) using equilibrium analysis. The results are presented in Table 11.

[0756] Table 11: Affinity to HLA-G Null 1,2,3 determined by SPR

[0757] Antibody ID KD (μM) HLA-G01 No binding HLA-G02 (normal, i.e. fucosylated) No binding Afucosylated HLA-G02 No binding HLA-G06 No binding HLA-G03 No binding HLA-G07 No binding HLA-G04 No binding HLA-G05 No binding HLA-G08 7,8

[0758] HLA-G01 - HLA-G07 binding was not detected. HLA-G08 showed some binding to HLA-G Null 1,2,3 and is therefore less specific for HLA-G.

[0759] 7.3 Binding to wild-type HLA-G expressed by HEK compared to HLA-G Null 1,2,3 to assess antibody specificity

[0760] Binding to wild-type HLA-G expressed on human embryonic kidney (HEK293) cells was measured and compared with binding to HLA-G Null 1,2,3 to determine binding specificity. Binding to dimeric HLA-G can be assessed primarily using cell-based assays, whereas the SPR assay described above only measures binding to monomeric HLA-G.

[0761] HEK293 cells transfected with either HLA-G / β2m or HLA-G Null 1,2,3 / β2m were incubated with anti-HLA-G IgG1 for two hours at 4°C in 384-well V-bottom plates (Greiner). IgG concentrations ranging from 100 nM to 0.05 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. After incubation, the cells were washed three times in assay buffer and then incubated for 20 minutes at 4°C with 20 μl of staining solution (3.75 μg / ml R-phycoerythrin-conjugated goat F(ab')2 fragment affinity purified to human IgG (H+L) (Jackson ImmunoResearch) and Viability Dye e780 (Life Technologies)). Following the washing step, the cells were incubated for 10 minutes in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. The cells were then washed and resuspended in 40 μl of PBS. Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of PE-positive cells.EC50 and Emax were calculated from the mean fluorescence intensity using FlowJo analysis software. The results are presented in Table 12.

[0762] Table 12: Affinity to HLA-G and HLA-G Null 1,2,3 expressed by HEK determined by FACS

[0763] Antibody ID Binding to HEK cells HLA-G WT EC50 (nM) HLA-G WT Emax (MFI) HLA-G null 1,2,3 EC50 (nM) HLA-G null 1,2,3 Emax (MFI) HLA-G01 1,54 129298 ND 61 HLA-G02 (VR12389 gL2gH16) 1,65 141300 ND 67 Fucosylated HLA-02 0,92 89198 ND 63 HLA-G06 1,26 115689 ND 53 HLA-G03 2,02 189443 ND 101 HLA-G07 1,03 104296 ND 37 HLA-G04 2,73 178887 ND 30 HLA-G05 2,96 165437 ND 143 HLA-G08 1,57 137718 35,90 2858

[0764] HLA-G03 had the lowest affinity for HLA-G expressed on HEK293 compared to HLA-G01 and HLA-G02. Thus, HLA-G01 and HLA-G02 were more preferable than HLA-G03. The affinity of afucosylated HLA-G02 for HLA-G expressed on HEK was similar to that of its conventional (fucosylated) counterpart. HLA-G08 was found to bind to HLA-G Null 1, 2, and 3, confirming that this antibody is less specific.

[0765] 7.4. Binding affinity to JEG3 cells determined by FACS analysis

[0766] The binding affinity of anti-HLA-G IgG was measured using a cell-based flow cytometry assay using human trophoblastic choriocarcinoma (JEG3) cells naturally expressing HLA-G. This assay may be preferable for measuring binding to cells naturally expressing HLA-G, including binding to dimeric HLA-G on the cells, whereas the SPR assay described above only measures binding to monomeric HLA-G.

[0767] JEG3 cells were incubated with 1.5 ml of anti-HLA-G IgG1 solution for two hours at 4°C in microcentrifuge tubes (Eppendorf). IgG concentrations ranging from 10 nM to 0.0005 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. Cells were transferred to 384-well V-bottom plates (Greiner) and washed three times in assay buffer, incubated with 20 μl of staining solution for 20 min at 4°C (7.5 μg / ml R-phycoerythrin-conjugated goat F(ab')₂ fragment affinity purified to human IgG (H+L) (Jackson ImmunoResearch) and Viability Dye e780 (Life Technologies)). After the washing step, cells were incubated for 10 min in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. Cells were then washed and resuspended in 20 μl of PBS. Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of PE-positive cells.KD was calculated from the mean fluorescence intensity using FlowJo analysis software. The results are presented in Table 13.

[0768] Table 13: Affinity to JEG3 cells determined by FACS

[0769] ID antibodies Affinity by FACS KD (nM) HLA-G01 0,045 HLA-G02 (VR12389 gL2gH16) 0,020 HLA-G06 - HLA-G03 0,068 HLA-G07 - HLA-G04 - HLA-G05 0,174 HLA-G08 -

[0770] HLA-G01 and HLA-G02 showed higher affinity in FACS analysis. The results confirmed that HLA-G03 has lower affinity for HLA-G when expressed on the surface of cells naturally expressing HLA-G, especially in the form of dimers, compared to HLA-G01 and HLA-G02.

[0771] 7.5. Binding to HLA-I and HLA-G of wild-type (WT) / subjected knockdown HLA-G in JEG3

[0772] To further investigate the binding specificity of anti-HLA-G IgG, binding to consensus HLA-A / B / C / E / F molecules expressed on HEK293 cells was measured. In the same assay, binding to HLA-G expressed on JEG3 cells was also measured and compared with knockdown (KD) HLA-G in JEG3 cells.

[0773] HEK293 cells transfected with the HLA-A, B, C, E, or F / β2m consensus sequence and JEG3 WT and JEG3 KD were incubated with anti-HLA-G IgG1 for two hours at 4°C in a 384-well V-bottom plate (Greiner). IgG concentrations ranging from 100 nM to 0.05 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. After incubation, cells were washed three times in assay buffer and then incubated with 20 μl of staining solution for 20 minutes at 4°C (3.75 μg / ml R-phycoerythrin-conjugated goat F(ab')2 fragment, affinity-purified to human IgG (H+L) (Jackson ImmunoResearch) and Viability Dye e780 (Life Technologies). After the washing step, cells were incubated for 10 minutes in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. Cells were then washed and resuspended in 40 μl of PBS.Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of PE-positive cells. EC50 and Emax were calculated from the mean fluorescence intensity using FlowJo analysis software. The results are presented in Tables 14 and 15.

[0774] Table 14: Binding to HLA-A / B / C / E / F

[0775] ID antibodies The EC50 (nM) of binding to HLA HLA-A BINDING Emax (MFI) HLA-B binding EC50 (nM) HLA-B BINDING Emax (MFI) HLA-C binding EC50 (nM) HLA-C BINDING Emax (MFI) HLA-G01 ND 373,3 ND 221,6 ND 119 HLA-G02 ND 57,3 ND 142,6 ND 65 Fucosylated HLA-G02 ND 191 ND 91 ND 117 ID antibodies HLA-E binding EC50 (nM) HLA-E BINDING Emax (MFI) HLA-F binding EC50 (nM) HLA-F Emax (MFI) HLA-G01 ND 120,5 ND 54 - - HLA-G02 ND 45 ND 67,6 - Fucosylated HLA-G02 ND 54 ND 73

[0776] No binding to HLA-I consensus molecules was detected ("ND"). The results confirmed that HLA-G01 and HLA-G02 antibodies are highly specific for HLA-G.

[0777] Table 15: JEG3 WT Binding

[0778] Antibody ID BINDING JEG3 WT EC50 (nM) JEG3 WT Emax CONNECTION (MFI) HLA-G01 0,38 58094 HLA-G02 0,43 63979 Fucosylated HLA-G02 0,2 44416 HLA-G06 4,10 6966 HLA-G03 0,41 38189 HLA-G07 3,42 4900 HLA-G04 3,65 31373 HLA-G05 1,26 44413

[0779] Furthermore, no binding to JEG3 KD was detected. HLA-G03 had a similar EC50 but a much lower Emax compared to HLA-G01 and HLA-G02 (or afucosylated HLA-G02), and therefore was not as good as HLA-G01 or HLA-G02.

[0780] 7.6. Identification of the anti-HLA-G antibody-binding domain in HLA-G proteins

[0781] Binding to HLA-G Null3, HLA-G Null1,3, and HLA-G Null3 2AA expressed on human embryonic kidney (HEK293) cells was measured to characterize the binding domain of anti-HLA-G IgG.

[0782] HEK293 cells transfected with HLA-G Null3 / β2m, HLA-G Null1.3 / β2M, and HLA-G Null3 2AA / β2M were incubated with anti-HLA-G IgG1 for two hours at 4°C in a 384-well V-bottom plate (Greiner). IgG concentrations ranging from 100 nM to 0.05 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. After the incubation period, the cells were washed three times in assay buffer and then incubated with 20 μl of staining solution for 20 min at 4°C (3.75 μg / ml R-phycoerythrin-conjugated goat F(ab')2 fragment affinity purified to human IgG (H+L) (Jackson ImmunoResearch) and Viability Dye e780 (Life Technologies). After the washing step, the cells were incubated for 10 min in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. The cells were then washed and resuspended in 40 μl of PBS.Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of PE-positive cells. EC50 and Emax were calculated from the mean fluorescence intensity using FlowJo analysis software. The results are presented in Table 16.

[0783] Table 16: Binding to HLA-G Null3, HLA-G Null 1.3, and HLA-G Null3 2AA expressed on HEK293

[0784] ID antibodies HLA-G Null3 EC50 (nM) HLA-G Null3 Emax (MFI) HLA-G Null 1.3 EC50 (nM) HLA-G Null 1.3 Emax (MFI) HLA-G Null3 2AA EC50 (nM) HLA-G Null3 2AA Emax (MFI) HLA-G01 ND 214 ND 182 ND 281 HLA-G02 ND 97 ND 87 ND 44 HLA-G06 ND 348 ND 318 ND 281 HLA-G03 ND 166 ND 247 ND 186 HLA-G07 0.09 124 ND 206 ND 97 HLA-G04 ND 204 ND 278 ND 317 HLA-G05 ND 17 ND 111 ND 178 HLA-G08 13,38 4893 17,02 11716 15,50 2318

[0785] The data showed that antibodies including HLA-G02 were specific to the alpha-3 domain of HLA-G. The data confirmed that HLA-G08 had the lowest specificity.

[0786] 7.7. HLA-G02 specificity was assessed in an experiment with PBMCs

[0787] The specificity of HLA-G02 was further confirmed using PBMCs obtained from 50 different donors representing a variety of HLA-I alleles. The goal was to confirm that the anti-HLA-G antibody is specific and does not cross-react with other HLA-I molecules expressed on PBMCs, particularly CD4+ T cells.

[0788] PBMCs were purified from peripheral venous blood and stored in liquid nitrogen in freezing medium (90% FBS + 10% DMSO). PBMCs from 50 different donors were thawed and resuspended in 1 ml of complete RPMI medium (RPMI 1640 medium plus 10% fetal bovine serum, 2 mM glutamax, and 1% penicillin / streptomycin). The cells were centrifuged at 300 rpm for 10 min and washed twice with PBS. The cell pellets were resuspended in 1 ml of Facs buffer (PBS, 0.5% BSA, and 2 mM EDTA), and the cells were seeded in a 96-well plate at a concentration of 50 μl of cell suspension per well.

[0789] Cells were stained with anti-human CD4-APC (Biolegend, 2.5 μl / well) and anti-HLA-G antibodies (HLA-G02 or the control “pan-HLA,” IgG1, which binds to HLA-I and is not specific for HLA-G) or an isotype control (50 μl of a solution with a concentration of 20 μg / ml per well). Cells were incubated at room temperature in the dark for 20 min, then washed twice with Facs buffer and resuspended in 50 μl of Facs buffer containing FITC-conjugated goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 1 / 100 dilution). Cells were incubated for another 20 min at room temperature in the dark, then washed twice with Facs buffer. Cells were resuspended in 100 µl / well of Facs buffer, and samples were processed on a Canto II instrument (HTS 1), collecting 10,000 events per sample. Analysis was performed using FlowJo v10.6.0 software by measuring the mean fluorescence intensity (MFI) of each CD4+ cell population from each donor.The graphs were constructed using Graphpad Prism software.

[0790] The results are shown in Fig. 4. Fig. 4 shows that 50 different donors of the specific anti-HLA-G antibody HLA-G02 lack binding to CD4 T cells compared with the pan-HLA antibody. The data are expressed as the mean fluorescence intensity (MFI) for each donor.

[0791] 7.8 HLA-G02 binding to cell membrane-associated HLA-G isoforms: HLA-G1, HLA-G2, HLA-G3, or HLA-G4

[0792] Human Expi293F cells were transfected with a plasmid encoding HLA-G1, HLA-G2, HLA-G3, or HLA-G4 using the ExpiFectamine Transfection Kit ТМ293 cells were transfected according to the manufacturer's protocol (ThermoFisher Scientific, part number A14524). Twenty-four hours after transfection, cells were harvested, washed with PBS (300 rpm, 10 min), and resuspended in PBS. Cells were seeded in 96 wells / plate and stained with anti-HLA-G antibodies HLA-G02 (human IgG1) or commercial 4H84 antibodies (Abcam, mouse IgG1) at a final concentration of 10 μg / ml and incubated in the dark for 20 min. The cells were then washed twice with Facs buffer (PBS, 0.5% BSA, and 2 mM EDTA) and resuspended in 50 μl of Facs buffer containing PE-conjugated goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 1 / 100 dilution) or PE-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, 1 / 100 dilution). The cells were incubated for another 20 min at room temperature in the dark and then washed twice with Facs buffer. The cells were resuspended in 100 μl of Facs buffer, and the samples were processed on a Canto II instrument (HTS 1).

[0793] The results are shown in Fig. 5. Fig. 5A shows that the HLA-G alpha-3 domain-specific antibody HLA-G02 recognized both HLA-G1 and HLA-G2 (68 and 17% of positive cells, respectively), but not HLA-G3 or HLA-G4, compared with the negative control (neg CTRL, an irrelevant antibody). As a positive control, a commercial mouse anti-HLA-G antibody 4H84, specific for the α1 domain, recognized all isoforms of HLA-G1, HLA-G2, HLA-G3, or HLA-G4 (staining in 81, 34, 67, and 16% of cells, respectively).

[0794] Figure 5B shows more specifically that the HLA-G02 antibody, specific for the alpha-3 domain of HLA-G, recognizes HLA-G2 (17% of positive cells) compared with the negative control (irrelevant antibody). The commercial mouse anti-HLA-G antibody 4H84, specific for the α1 domain, was used as a positive control for HLA-G2 (34% of positive cells).

[0795] Example 8: Evaluation of the ability of anti-HLA-G antibodies to block the interaction between HLA-G and ILT2 or ILT4.

[0796] 8.1 Blocking the interaction of JEG3 and ILT2

[0797] Anti-HLA-G IgG was incubated with HLA-G-expressing human trophoblastic choriocarcinoma (JEG3) and rabbit FcILT2 to measure their ability to block ILT2 binding to HLA-G.

[0798] JEG3 cells were incubated with anti-HLA-G IgG1 for one hour at 4°C in 384-well V-bottom plates (Greiner). IgG concentrations ranging from 100 nM to 0.05 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. After the incubation period, the cells were washed with assay buffer and then incubated with 20 μl of ILT2rbFc solution at a concentration of 3 μg / ml for one hour at 4°C. After incubation for 20 min at 4°C, 5 μl of staining solution (7.5 μg / ml fluorescein (FITC)-conjugated Fc-specific goat F(ab')2 fragment, affinity-purified to rabbit IgG (Jackson ImmunoResearch) and Viability Dye e780 (Life Technologies)) were added to each well. After the washing step, the cells were incubated for 10 min in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. The cells were then washed and resuspended in 40 μl PBS.Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of FITC-positive cells. IC50 and percent inhibition were calculated from the mean fluorescence intensity using FACSDiva analysis software. The results are presented in Table 17.

[0799] Blocking the interaction between JEG3 and ILT2: a large reaction volume

[0800] In the previous ILT2 blocking assay, some antibodies showed very low IC50. At such low concentrations, ligand depletion likely occurs due to the small reaction volume, which may lead to an overestimation of the IC50 value. To overcome ligand depletion, a large volume of anti-HLA-G IgG solutions (the best blocking antibodies identified in the previous assay) was incubated with JEG3 expressing HLA-G and rabbit Fc-ILT2 to improve the measurement of their ability to block ILT2 binding to HLA-G.

[0801] JEG3 cells were incubated with 1.5 mL of anti-HLA-G IgG1 solution for two hours at 4°C in microcentrifuge tubes (Eppendorf). IgG concentrations ranging from 10 nM to 0.005 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. The cells were transferred to 384-well V-bottom plates (Greiner) and washed three times with assay buffer. Then, they were incubated with 20 μL of ILT2rbFc solution at a concentration of 3 μg / mL for one hour at 4°C. After incubation, the cells were washed and incubated with 20 μl of staining solution (7.5 μg / ml fluorescein (FITC)-conjugated Fc-specific goat fragment F(ab')2 affinity-purified to rabbit IgG and Viability Dye e780 (Life Technologies)) for 20 min at 4°C. After the washing step, the cells were incubated for 10 min in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. The cells were then washed and resuspended in 20 μl of PBS.Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of FITC-positive cells. IC50 and percent inhibition were calculated from the mean fluorescence intensity using FlowJo analysis software. The results are presented in Table 17.

[0802] 8.2 Blocking the interaction between HLA-G expressed on HEK and ILT2.

[0803] Anti-HLA-G IgG was incubated with HLA-G-transfected HEK293 cells and rabbit FcILT2 to measure their ability to block ILT2 binding to HLA-G.

[0804] HEK293 cells transfected with HLA-G / β2M were incubated with anti-HLA-G IgG1 for one hour at 4°C in 384-well V-bottom plates (Greiner). IgG concentrations ranging from 100 nM to 0.05 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. After incubation, the cells were washed with assay buffer and then incubated with 20 μl of ILT2rbFc solution at a concentration of 1 μg / ml for one hour at 4°C. After incubation for 20 min at 4°C, 5 μl of staining solution (3 μg / ml fluorescein (FITC)-conjugated Fc-specific goat F(ab')2 fragment, affinity-purified to rabbit IgG (Jackson ImmunoResearch) and Viability Dye e780 (Life Technologies)) were added to each well. After the washing step, the cells were incubated for 10 min in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. The cells were then washed and resuspended in 40 μl PBS.Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of FITC-positive cells. IC50 and percent inhibition were calculated from the mean fluorescence intensity using FACSDiva analysis software. The results are presented in Table 17.

[0805] 8.3 Blocking the interaction between HLA-G expressed on HCT116 and ILT4

[0806] Anti-HLA-G IgG was incubated with HLA-G-transfected human colon cancer cells (HCT116) and rabbit FcILT4 to measure their ability to block ILT4 binding to HLA-G.

[0807] HCT116 cells transfected with HLA-G / β2M were incubated with anti-HLA-G IgG for one hour at 4°C in 384-well V-bottom plates (Greiner). IgG1 concentrations ranging from 100 nM to 0.05 nM were obtained by dilution in PBS, 1% FBS, and 0.1% sodium azide. After incubation, the cells were washed with assay buffer and then incubated with 20 μl of ILT4rbFc solution at a concentration of 0.4 μg / ml for one hour at 4°C. After incubation, 5 μl of staining solution (1.5 μg / ml fluorescein (FITC)-conjugated Fc-specific goat F(ab')2 fragment, affinity-purified to rabbit IgG (Jackson ImmunoResearch) and Viability Dye e780 (Life Technologies)) were added to the solution in each well for 20 min at 4°C. After the washing step, the cells were incubated for 10 min in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, protected from light. The cells were then washed and resuspended in 40 μl PBS.Samples were processed on a FACS Canto II instrument in HTS mode to determine the percentage of FITC-positive cells. IC50 and percent inhibition were calculated from the mean fluorescence intensity using FACSDiva analysis software. The results are presented in Table 17.

[0808] Table 17: Blocking activity of anti-HLA-G antibodies (IC50 values)

[0809] Antibody ID ILT2rbFc ILT2rbFc ILT2rbFc ILT4rbFc JEG3 Flow Average IC50 (nM) Large volume JEG3, average IC50 (nM) HEK-HLAG, average IC50 value (nM) HCT116-HLAG, mean IC50 value (nM) HLA-G01 0,59 0,039 1,58 1,78 HLA-G02 (VR12389 gL2gH16) 0,53 0,017 1,37 1,31 afucosylated HLA-G02 0,51 0,021 0,83 1,38 HLA-G06 7,40 - 10,54 4,58 HLA-G03 0,47 0,038 1,59 1,86 HLA-G07 1,82 - 2,33 2,82 HLA-G04 1,10 - 2,05 3,32 HLA-G05 0,74 - 1,51 2,59 HLA-G08 1,58 - 9,84 2,75

[0810] HLA-G01 and HLA-G02 were identified as the best blockers of HLA-G association with ILT2 and HLA-G association with ILT4. The blocking properties of afucosylated HLA-G02 were similar to those of its conventional (i.e., fucosylated) counterpart.

[0811] Example 9: Efficacy and activity of HLA-G01-HLA-G08 antibodies in ADCC of HLA-G-expressing cells.

[0812] Antibody-dependent cellular cytotoxicity (ADCC) is an immune mechanism by which cells expressing Fc receptors, such as NK cells, can recognize and kill antibody-coated cells. This process is critical for anti-cancer responses and is a key mechanism underlying the efficacy of many anti-cancer therapies. The ability of a subset of anti-HLA-G IgG1 antibodies to induce ADCC in vitro was determined using two different types of HLA-G-expressing target cells. These cells were either transfected with HLA-G and human beta-2-microglobulin (HCT116 colorectal carcinoma cells) or endogenously expressed the target on their cell surface (JEG3 cells).

[0813] Methods:

[0814] Transfection of the colorectal carcinoma cell line HCT116

[0815] HCT116 cells were transfected with an HLA-G construct that also encoded a green fluorescent protein (GFP) tag. Thus, cells successfully transfected with HLA-G also expressed GFP and could be easily identified and reliably tracked using flow cytometry. For transfection, 4×10 6 A total of 40 μg of DNA (20 μg each of the human HLA-G ECD GFP and β2M plasmids) and 80 μl of Lipofectamine LTX were used for culture of HCT116 cells in a T75 tissue culture flask. After 24 hours, the cells were detached from the flask and used as target cells as described below.

[0816] In vitro ADCC assay

[0817] HLA-G transfected JEG3 or HCT116 target cells were plated (2×10 4Cells / well (50 µl) were added to a round-bottomed polypropylene plate in the appropriate culture medium. Anti-HLA-G or control antibodies were prepared as 4X concentrated stock solutions in the same medium and added at 50 µl / well to the appropriate wells. All antibodies were tested in either duplicate or triplicate depending on the number of available donor NK cells. Some target cells were left without any antibodies and used as untreated controls.

[0818] Primary human NK cells were isolated from whole blood by negative selection using a magnetic bead kit (MilteNY Biotech). Purified NK cells were resuspended in RPMI + 10% FBS, 2 mM L-glutamine in the minimum volume required for the assay. NK cells (100 μl / well) were added to the appropriate wells of the assay plate on top of target cells and antibodies. The effector:target ratio ranged from 10:1 to 3:1 depending on the number of donor NK cells.

[0819] The assay plate was incubated at 37°C and 5% CO2 for ~3 hours. After 3 hours, the number of viable target cells was measured by flow cytometry. The assay plate was centrifuged at 300g for 3 minutes to pellet the cells, and each well was stained for the epithelial cell marker Epcam and the NK cell marker CD56. Staining antibodies (anti-Epcam PE and anti-CD56 BV421) were diluted to 1 / 100 in cell staining buffer, and 100 μl / well were added to each well. The plate was incubated at room temperature for 15 minutes. After staining, the cells were washed twice with PBS at 150 μl / well, and between each wash, the plate was centrifuged at 300g for 3 minutes. At the end of staining, the cells in each well were resuspended in a final volume of 100 µl / well of PBS containing 50 nM TO-PRO viable cell dye. ТМ -3.

[0820] After 10 minutes, exactly 70 μl of sample was taken from each well using a BD FACS Canto II Instrument, and the data were analyzed using FlowJoV10.60 software. The total number of viable target cells was determined for each well. Viable target cells were identified first as TO-PRO- ТМ 3-negative cells, and then, secondly, as CD56-negative and having high levels of SSC. Cells were then selected for Epcam (JEG3) or Epcam and GFP (HCT116) expression. For each tested sample, the percentage of depletion was calculated compared to untreated cells or cells treated with an isotype control, and the data were transferred to GraphPad Prism 8.1.1 software for analysis.

[0821] Results:

[0822] Figure 6 shows the percentage of Epcam+ GFP+ depleted HCT116 target cells after treatment with different anti-HLA-G antibodies or an IgG1 isotype control antibody. Each antibody was tested at two different concentrations: 1 μg (white bars) or 0.01 μg / mL (striped bars). The E:T ratio was 3.5:1. Each bar represents the mean (and range) of three data points, and each dot / square represents an individual sample. The data are from a single representative donor.

[0823] Table 18 shows the average percentage of Epcam+ GFP+ depletion of HCT116 cells by each antibody shown in Fig. 6 (ND: not detected).

[0824] Table 18: Average percentage of Epcam+ GFP+ depletion in HCT116 cells

[0825] Antibody ID Average % depletion of GFP+ cells (1 μg / ml) Average % depletion of GFP+ cells (0.01 μg / ml) HLA-G01 67,15 69,41 HLA-G02 73,11 70,51 HLA-G06 75,79 55,90 HLA-G03 75,01 61,45 HLA-G07 57,20 2,46 HLA-G04 66,48 43,83 HLA-G05 73,68 51,78 HLA-G08 71,20 39,00 Isotype IgG1 0,93 ND

[0826] Several antibodies showed similar mean % depletion of GFP+ cells at the highest antibody concentration (1 μg / mL). The highest mean % depletion of GFP+ cells, observed at the lowest concentration (0.01 μg / mL), was observed for HLA-G01 and HLA-G02 antibodies. These antibodies were further characterized in ADCC assays.

[0827] Figure 7 shows the percentage of Epcam+ GFP+ depleted HCT116 target cells after treatment with anti-HLA-G antibodies HLA-G01 and HLA-G02 or an isotype control antibody IgG1 from three separate experiments (3 different donors). Antibodies were tested at a concentration of 1 μg / mL (Figure 7A) or 0.01 μg / mL (Figure 7B). The E:T ratio ranged from 2.5 to 3:1. Each bar represents the mean (and range) of data from a single experiment, and each dot, square, or triangle represents an individual replicate.

[0828] Table 19 shows the average depletion percentage of Epcam+ GFP+ HCT116 cells by each antibody at a concentration of 1 μg / mL, as shown in Fig. 7A.

[0829] Table 19: Average percentage of depletion of Epcam+ GFP+ HCT116 (1 μg / mL antibody)

[0830] Human IgG1 antibody Average % attrition, experiment 1 Average % attrition, experiment 2 Average % attrition, experiment 3 HLA-G01 81,61 70,21 67,15 HLA-G02 87,71 79,25 73,11 isotype -34,56 -44,76 0,93

[0831] Table 20 shows the average percentage of Epcam+ GFP+ depletion of HCT116 cells by each antibody at a concentration of 0.01 μg / mL, as shown in Fig. 7B.

[0832] Table 20: Average percentage of depletion of Epcam+ GFP+ HCT116 (0.01 μg / mL antibody)

[0833] Human IgG1 antibody Average % attrition, experiment 1 Average % attrition, experiment 2 Average % attrition, experiment 3 HLA-G01 83,83 70,79 69,41 HLA-G02 85,09 75,92 69,42 isotype ND ND ND

[0834] According to the ADCC assay results, at both antibody concentrations, HLA-G02 antibody had better cell depletion activity than HLA-G01.

[0835] Figure 8 shows the percentage of Epcam+ GFP+ depletion in HCT116 cells after titration with anti-HLA-G antibodies HLA-G01 and HLA-G02 compared to the isotype control. The E:T ratio was 4:1. Each point represents the mean (and range) of three replicates. The data shown are from one representative donor.

[0836] Based on these results, the HLA-G02 antibody was selected for further characterization and development, specifically, an afucosylated version of HLA-G02 was generated for comparison with conventional IgG1 as described above.

[0837] Afucosylation of antibodies has been shown to increase FcγRIII:Fc binding affinity and reportedly enhances the ADCC potential of IgG1 molecules. Therefore, an afucosylated version of HLA-G02 was tested for its ability to induce ADCC in transfected HCT116 or JEG3 cells. The afucosylated antibodies were compared with the same antibody V regions produced in the conventional IgG1 format.

[0838] Figure 9A shows the percentage depletion of JEG3 cells after treatment with conventional HLA-G02 IgG1 (solid line) or afucosylated HLA-G02 IgG1 (“aF HLA-G02”, dotted line). The E:T ratio was 10:1. Each point represents the mean (and range) of two replicates. Data obtained for one representative donor are shown.

[0839] Figure 9B shows the percentage of Epcam+ GFP+ depletion in HCT116 cells after treatment with conventional HLA-G02 IgG1 (solid line) or afucosylated HLA-G02 IgG1 (“aF HLA-G02”, dashed line). The E:T ratio was 5:1. Each point represents the mean (and range) of three replicates. Data obtained for one representative donor are shown.

[0840] The HLA-G02 antibody demonstrated superior activity and efficacy in cell killing assays and was therefore selected for further development as a therapeutic candidate. The afucosylated version of HLA-G02 demonstrated improved ADCC compared to its conventional (i.e., fucosylated) counterpart.

[0841] Example 10: Efficacy and activity of the HLA-G antibody HLA-G02 in phagocytosis of HLA-G-expressing cells.

[0842] HLA-negative K562 cells were used as a target and transfected to express HLA-G. Pre-labeled target cells (CTY+) were incubated with monocytic-derived macrophages (CD11b+) with HLA-G02 antibodies in hIgG1 format. Phagocytosis was analyzed by measuring the percentage of CTY+CD11b+ cells.

[0843] Methods:

[0844] CD14+ monocytes were purified from human peripheral venous blood using the Pan Monocyte Isolation kit (MilteNY), an indirect magnetic labeling system for isolating intact monocytes. The cells were differentiated into macrophages using 50 ng / mL recombinant MCSF in complete RPMI medium (RPMI 1640 medium plus 10% fetal bovine serum, 2 mM glutamax, and 1% penicillin / streptomycin) for 7 days at 37°C, 5% CO2.

[0845] HLA-negative K562 erythroleukemia cells were either mock-transfected or transfected with HLA-G and B2m using the 4D-Nucleofector System and the L SF Cell Line 4D-Nucleofector Kit TMX (Lonza, ref# V4XC-2024) and cultured in complete RPMI medium for 24 h at 37°C, 5% CO2. The next day, the cells were harvested, washed, and labeled with Cell Trace Yellow (Thermofisher), washed again, and seeded at 25,000 cells per well in 100 μl of complete RPMI medium in a 96-well round-bottom ultra-low-adherence plate (Corning Costar). Cells were then incubated with either anti-CD47 antibody, anti-HLA-G antibodies, or isotype control at a concentration of 10 μg / ml for 1 h at 37°C, 5% CO2. After washing, cells were combined with monocyte-derived macrophages (50,000 macrophages per well) at a macrophage:target cell ratio of 2:1. The mixed cells were incubated for 2 hours at 37°C, 5% CO2. The cells were then washed and resuspended in PBS plus 10% purified human Fc-gammaR binding inhibitor (Thermofisher) for 20 minutes at 4°C, and then stained with anti-CD11b-APC (Biolegend) for 20 minutes at 4°C.Cells were washed and resuspended in PBS plus 2 mM EDTA plus 0.5% BSA in the presence of DAPI (500 ng / ml) to eliminate dead cells. Samples were obtained by flow cytometry on a BD FACSCanto. Analysis was performed using FlowJo v10.6.0 software by measuring the percentage of CTY+CD11b+ double-positive cells. Anti-CD47 was used as a positive control. Studies have shown that inhibiting CD47 on target cells inhibits the interaction of CD47 with the SIRPa receptor expressed on macrophages, leading to increased phagocytosis. CD47 expression has been shown to be upregulated in tumor cells, and anti-CD47 antibodies are currently being tested in clinical trials. The phagocytosis assay is a good control for assessing the phagocytic activity of monocyte-derived macrophages and demonstrates that both mock-transfected and HLA-G-transfected cells are capable of phagocytosis.

[0847] Results:

[0848] Table 21 shows the HLA-G-specific phagocytosis activity of anti-HLA-G antibodies on mock-transfected K562 compared with HLA-G-expressing K562 target cells (HLA-G / B2m K562). Data are expressed as percentages of CTY+CD11b+ double-positive cells and are representative of three independent experiments in duplicate.

[0849] Table 21: Phagocytic activity of anti-HLA-G02

[0850] IgG1 antibody % CTY+CD11b+ cells (mean value) false transf. K562 HLA-G / B2m K562 CTL isotype 26,8 24,6 anti-CD47 53,6 49,6 HLA-G02 25,6 39,6

[0851] Table 22 shows the statistically significant HLA-G-specific phagocytosis activity of HLA-G02 compared to the IgG1 isotype control. Data represent pooled data from 6 donors (in duplicate). Data were exported to Excel and normalized to the mean percentage of phagocytosis of mock-transfected cells.

[0852] Table 22: Phagocytic activity of anti-HLA-G02 (6 donors)

[0853] IgG1 antibody % CTY+CD11b+ cells relative to the control mean mock-transfected K562 HLA-G / B2m K562 Donor A Donor B Donor C Donor D Donor E Donor F Donor A Donor B Donor C Donor D Donor E Donor F CTL isotype 100,0 100,0 100,0 100,0 100,0 100,0 104,8 92,5 110,5 115,9 92,2 105,4 anti-CD47 100,0 100,0 100,0 100,0 100,0 100,0 98,6 99,4 109,8 88,6 86,5 71,6 HLA-G02 100,0 100,0 100,0 100,0 100,0 100,0 123,6 109,8 215,9 2561 202,4 184,8

[0854] Figure 10 shows the titration of HLA-G-specific phagocytosis activity of human anti-HLA-G IgG1 antibody HLA-G02 on mock-transfected (Figure 10A) and HLA-G / B2m-transfected K562 target cells (Figure 10B) compared with anti-CD47 antibody and isotype control. Data are expressed as percentage of CTY+CD11b+ double-positive cells and are representative of 1 of 2 donors (Table 23).

[0855] Table 23: Titration of HLA-G-specific phagocytosis activity of HLA-G02

[0856] Concentration (mcg / ml) % CTY+CD11b+ cells (mean value) mock-transfected K562 HLA-G / B2m K562 IgG1 isotype HLA-G02 Neg. CTL1 Neg. CTL2 aCD47 IgG1 isotype HLA-G02 neg. CTL1 neg. CTL2 aCD47 10 17,4 15,5 17,6 17,4 24,9 20,4 44,2 22,1 19,9 27,6 1 17,3 15,8 18,7 17,4 31,2 18,9 46,4 21,1 20,4 29,4 0,1 17,0 17,0 18,8 18,5 22,7 17,3 43,8 19,9 18,1 22,9 0,01 16,2 17,6 19,7 18,2 17,1 17,4 32,4 21,4 18,7 19,3 0,001 15,1 15,1 15,1 15,1 15,1 19,4 19,4 19,4 19,4 19,4

[0857] Figure 11 shows the titration of HLA-G-specific phagocytosis activity of conventional and afucosylated (aF) HLA-G02 formats on mock-transfected (Figure 11A) and HLA-G-expressing K562 target cells (Figure 11B) compared with anti-CD47 antibody and isotype control. Data are expressed as percentage of CTY+CD11b+ double-positive cells and are representative of 1 of 3 donors in 2 independent experiments (Table 24).

[0858] Table 24: Titration of HLA-G-specific phagocytic activity of HLA-G02 and afucosylated HLA-G02

[0859] Antibody concentration (μg / ml) % CTY+CD11b+ cells (mean value) Mock-transfected K562 HLA-G / B2m K562 isotype HuIgG1 HLA-G02 aF HLA-G02 aCD47 isotype HuIgG1 HLA-02 aF HLA-G02 aCD47 10 17,0 24,3 26,6 22,5 43,3 48,0 1 18,6 21,5 24,1 43,1 22,8 43,0 47,8 45,5 0,1 17,6 20,6 21,0 33,0 22,2 43,7 45,3 37,6 0,01 18,3 19,3 18,9 23,8 23,7 36,2 36,0 26,5 0,001 18,5 25,3 25,1 20,6 21,4 28,8 30,9 25,2 0,0001 19,6 21,4 23,3 21,4 28,7 29,2 0,00001 17,8 21,0 24,0 21,5 28,0 28,0 0,000001 18,5 18,6 21,3 23,5 25,3 26,1

[0860] Afucosylated HLA-G02 showed improved ADCP compared to its normal (i.e., fucosylated) HLA-G02 counterpart.

[0861] Example 11: Epitope mapping of antibody VR12389 by X-ray crystallography.

[0862] Production of HLA-G fusion protein

[0863] Peptide B2mHLAG C42S mut tev10his-HLA-G (C42S), in which the cysteine ​​required for homodimerization is mutated to serine (signal peptide in bold (cleaved after expression), peptide underlined, GS linker in italics, B2m sequence, GS linker in italics, HLA-G sequence with C42S highlighted as a gray shadow, tev cleavage site in bold and italics, GS linker in italics, 10his tag),

[0864] MSVPTQVLGLLLLWLTDARC RIIPRHLQL GCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIV KWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSASPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLR GYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVT HHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQLEENLYFQGSGGSHHHHHHHHHH (SEQ ID NO: 145)

[0865] PeptideB2mHLAG C42S mut tev10his protein was expressed by transient transfection using the Expi293 expression system ТМ (Life Technologies ТМ), following the manufacturer's protocol. Cells were harvested 5 days after transfection, and the supernatants were immediately used for purification. Supernatants containing the PeptideB2mHLAG C42S mut tev10his protein were loaded onto a Histrap NiExcel column. Unbound protein and contaminants were washed with PBS, 500 mM NaCl, 20 mM imidazole, pH 7.4, and the PeptideB2mHLAG C42S mut tev10his protein was eluted with PBS, 500 mM NaCl, 500 mM imidazole, pH 7.4. Fractions containing purified PeptideB2mHLAG C42S mut tev10his protein were pooled, and the his-tag was removed by incubating the protein with tev protease at a ratio of 1:100 for 2 h at room temperature and 2 h at 4°C. The protein was concentrated and further purified by size-exclusion chromatography on an S200 26 / 60 column equilibrated with 20 mM Tris, 50 mM NaCl, pH 7.4 buffer. Fractions containing purified PeptideB2mHLAG C42S mut protein were pooled, concentrated to 2.94 mg / ml, and stored in 1 mg aliquots at -80°C.

[0866] PeptideB2mHLAG C42S mut protein was detected by SDS-PAGE, which migrated to a position on the gel corresponding to the expected molecular weight (MW) of the glycosylated protein.

[0867] The amino acid sequence of the obtained PeptideB2mHLAG C42S mut protein used for complexation and in the crystal structure was as follows:

[0868] RIIPRHLQLGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEY ACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSASPRMEPRAPWVEQEGPEYWEEETRNT KAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENG KEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQLEENLYFQ (SEQ ID NO: 146)

[0869] Fab cleaning VR12389

[0870] Rabbit Fab (VR12389) (the light chain sequence shown in SEQ ID NO:9 and the heavy chain sequence shown in SEQ ID NO:13) was expressed as secreted proteins in ExpiCHO cells. The light chain and heavy chain expression constructs were cotransformed at a 1:1 molar ratio. Secreted proteins were purified by passing the conditioned medium through protein G beads and eluted with 0.1 M glycine, pH 2.7. Fractions were neutralized by adding 2 M Tris-HCl, pH 8.5. The protein was dialyzed in PBS, pH 7.2, then concentrated to 5.62 mg / mL and stored at 4°C.

[0871] Protein peptide B2mHLAG C42S mut with RbFab VR12389

[0872] The fusion peptide_β2m_HLA-G was incubated at a molar ratio of 1:1.1 with RbFab VR12389 for 1 hour. The complex was then purified on a Superdex 200 16 / 600 column (GE Healthcare) using 10 mM Tris-HCl, 150 mM NaCl (pH 7.5) as a running buffer. Fractions were analyzed by SDS-PAGE using NuPAGE 4-20% Tris-glycine gels (Thermo), and then the purest complex fractions were concentrated to 10.4 mg / mL using an Amicon® Ultra-15 centrifugal filtration device (Millipore).

[0873] Crystallography of the peptideB2mHLAG C42S mut protein with RbFab VR12389

[0874] Crystallization media for the complex were determined using several commercially available crystallization screens. These determinations were performed in a sessile drop format using MRC Swissci 96-well 2-drop crystallization plates (purchased from Molecular Dimensions, cat. #MD11-00-100). First, the reservoirs were filled with 75 µL of each crystallization media on the screens using a Microlab STAR liquid handling system (Hamilton). Then, 300 nL of the HLA-G / VR12389 complex and 300 nL of reservoir solutions were dispensed into the wells of the crystallization plates using a Mosquito liquid handler (TTP LabTech). Crystals were identified in ProPlex-HT96 screen G4 medium (Molecular Dimensions) containing 2 M ammonium sulfate and 0.1 M Tris at pH 8.0. The crystals were frozen using a reservoir solution containing 25% glycerol as a cryoprotectant. Diffraction data were collected using a Diamond light source.The structure was obtained using molecular replacement in Phaser. Phenix.refine and Coot were used in alternating cycles of automatic and manual fitting.

[0875] By superimposing the crystal structure of HLA-G in complex with VR12389 with the crystal structures of HLA-G in complex with ILT2 and ILT4 (obtained from the literature, see, e.g., Q Wang et al., Cellular & Molecular Immunology, 2019 and Shiroishi, PNAS vol 103, No 44, P 16412-16417, 2006), it was found that VR12389 prevents the interaction of HLA-G with ILT2 and ILT4 receptors through steric hindrance (Fig. 12).

[0876] At a contact distance of <4 Å, the HLA-G epitope recognized by the VR12389 antibody contains HLA-G residues V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250, and Y257.

[0877] At a contact distance of <5 Å, the HLA-G epitope recognized by the VR12389 antibody contains HLA-G residues V194, F195, Y197, E198, R219, Q224, Q226, D227, V248, V249, P250, E253, and Y257.

[0878] Example 12: Epitope mapping of antibody 12389 using HDX-MS and NMR.

[0879] Unlike crystallography, which is performed under static conditions, HDX-MS and NMR are techniques that allow the analysis of interactions in solution and can demonstrate allosteric or conformational changes that are not always obvious in crystallography.

[0880] HDX-MS Materials and Methods

[0881] Sample Preparation and Data Collection

[0882] For HDX-MS analysis, 12 μM human HLA-G ECD (SEQ ID NO: 110) was complexed with 36 μM antibody 12389 (expressed in either IgG1 or Fab format) and incubated for 1 hour at 4°C.

[0883] 4 μL of HLA-G or HLA-G complex were diluted in 57 μL of 10 mM phosphate in H2O (pH 7.0) or 10 mM phosphate in D2O (pD 7.0) at 25°C. The deuterated samples were then incubated for 0.5, 2, 15, and 60 min at 25°C. After the reaction, all samples were quenched by mixing at a 1:1 ratio with quench buffer (4 M guanadine hydrochloride, 250 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 100 mM phosphate) at 1°C. The mixed solution had a final pH of 2.5. The mixture was immediately injected into the nanoAcquity HDX module (Waters Corp.) for peptic digestion. Peptide digestion was then performed online on a Waters enzymatic digestion column in 0.2% formic acid in water at 20°C and a flow rate of 100 µl / min. All time points for deuterated samples and non-deuterated controls were obtained in triplicate, with blank runs between individual data points.

[0884] The peptide fragments were then captured using a chilled Acquity BEH C18 1.7 μM VANGUARD precolumn for 3 min. The peptides were then eluted into a chilled Acquity UPLC BEH C18 1.7 μM 1.0×100 system using the following gradient: 0 min, 5% B; 6 min, 35% B; 7 min, 40% B; 8 min, 95% B, 11 min, 5% B; 12 min, 95% B; 13 min, 5% B; 14 min, 95% B; 15 min, 5% B (A: 0.2% HCOOH in H2O, B: 0.2% HCOOH in acetonitrile). Peptide fragments were ionized by electrospray ionization in positive mode on a Synapt G2-Si mass spectrometer (Waters). Data were collected in ToF mode only in the m / z range of 50–2000 Th using the MSe method (low collision energy, 4 V; high collision energy: smooth ramp from 18 V to 40 V). Glu-1-fibrinopeptide B peptide was used for internal lock mass correction.

[0885] HDX-MS Data Processing

[0886] MS Data ENon-deuterated HLA-G control samples were used for sequence identification using Waters Protein Lynx Global Server 2.5.1 (PLGS). Only the HLA-G sequence database was used for peptide searches, with a precursor intensity threshold of 500 counts and three matching product ions set for protein identification. The ion count files for the three control samples were combined into a peptide list imported into Dynamx v3.0 software.

[0887] The peptides were further filtered in DynamX. The filtering parameters used were a minimum and maximum peptide sequence length of 4 and 25, respectively, a minimum intensity of 1000, minimum MS / MS products of 2, minimum products per amino acid of 0.2, and a maximum MH+ error threshold of 10 ppm. DynamX v3.0 was used to quantify the isotopic envelopes resulting from deuterium uptake for each peptide at each time point. In addition, all spectra were examined and inspected visually to ensure correct identification of m / z peaks, and only peptides with a high signal-to-noise ratio were used for HDX-MS analysis.

[0888] After manual filtering in Dynamx, statistical analysis and filtering were performed using Deuteros, using the statistical analysis published by Houde et al. (2011). Deuteros creates a Woods plot, which displays peptide length, initial and final residues, global coverage, and a metric on the Y axis, which represents absolute absorbance (in Da). This is the difference in absorbance in the presence of the ligand (bound) and the apo form. In Woods plots, confidence interval filtering was first applied to all peptides at each time point. Peptides with differential deuteration outside the selected confidence limits were considered insignificant.

[0889] Results:

[0890] In the presence of VR12389, a total of twelve peptides showed a statistically significant reduction in deuterium incorporation upon binding of the anti-HLA-G antibody, nine of which demonstrated significant protection. The major protection spanned residues 178–196 (MLQRADPPKTHVTHHPVFD) and 214–230 (ILTWQRDGEDQTQDVEL). Both regions are within the α3 domain (and the five terminal α2 residues). The region showing moderate protection upon antibody binding spanned residues 234–249 (RPAGDGTFQKWAAVVV) and is likely due to a conformational change. Peptides showing similar exchange patterns in the presence and absence of antibody contain minor deuterium incorporation.

[0891] Table 25: Peptides exhibiting reduced deuterium incorporation upon binding of VR12389 to HLA-G as determined by HDX-MS

[0892] Start End Peptide sequence Deuterium inclusion 234 249 RPAGDGTFQKWAAVVV Medium protected 184 195 RADPPKTHVTHHPVF Medium protected 180 196 QRADPPKTHVTHHPVFD Medium protected 181 195 RADPPKTHVTHHPVF Heavily protected 217 229 WQRDGEDQTQDVE Heavily protected 214 230 ILTWQRDGEDQTQDVEL Heavily protected 178 196 MLQRADPPKTHVTHHPVFD Heavily protected 216 229 TWQRDGEDQTQDVE Heavily protected 180 195 QRADPPKTHVTHHPVF Heavily protected 178 195 MLQRADPPKTHVTHHPVF Heavily protected 181 196 RADPPKTHVTHHPVFD Heavily protected 216 227 TWQRDGEDQTQD Heavily protected

[0893] In conclusion, according to HDX-MS analysis with 30 s deuterium incubation, the potential binding domains for VR12389 are 178-MLQRADPPKTHVTHHPVFD-196 and 214-ILTWQRDGEDQTQDVEL-230.

[0894] Nuclear magnetic resonance (NMR) spectroscopy

[0895] Epitope mapping of the VR12389 antibody was determined by NMR spectroscopy using Fab fragments of the antibody.

[0896] Materials

[0897] 2 H / 13 C / 15 N-tagged expression of the α3 domain of HLA-G

[0898] Competent E. coli BL21(DE3) (New England BioLabs #C2527H) was transformed with 1 μg of short N-His α3 HLA-G ATUM #393044 (HLA-G residues 207-300) using a standard heat shock method. Transformed cells were plated on LB agar plates containing 100 μg / ml carbenicillin and incubated overnight at 37°C. The next day, one colony was used to inoculate 10 ml of LBroth containing 100 μg / ml carbenicillin (Merck #C1389) and grown with shaking at 37°C and 200 rpm for 5 hours (New Brunswick Excella E25). Then to inoculate 500 ml of minimal medium labeled 2 H / 13 C / 15 N (described below), 1 ml of the starter culture was used and grown overnight in disposable 2-liter Erlenmeyer flasks (VWR #734-1904) at 37°C with shaking at 200 rpm. The optical density (OD) was recorded the following day. 600) overnight culture (Amersham Biosciences Ultrospec 3100 pro). Expression cultures were then inoculated with the overnight culture to a final OD 600 , equal to 0.1.

[0899] Cultures for expression in labeled minimal medium 2 H / 13 C / 15 N, were grown in 500 ml batches in disposable 2-liter Erlenmeyer flasks with shaking at 37°C and 200 rpm until OD 600 , equal to 0.9. Then, α3 HLA-G expression was induced using 500 μM IPTG (Sigma # I6758). Afterwards, the induced cultures were left at 37°C for another 4 hours and then collected by centrifugation at 7000 g for 30 minutes (Beckman Coulter J6-MI). The collected pellets were then frozen at -20°C before cell lysis.

[0900] Untagged expression of human β2m

[0901] Competent E. coli BL21(DE3) was transformed with 1 μg of human β2m (residues: 21-119) ATUM#358573 and grown as above. The next day, one colony was used to inoculate 10 ml of LBroth (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 1 mM NaH2O) containing 100 μg / ml carbenicillin and grown with shaking at 37°C and 200 rpm for 5 hours. Then, 1 ml of the starter culture was used to inoculate 500 ml of LBroth containing 100 μg / ml carbenicillin and grown overnight in a 2 L disposable Erlenmeyer flask at 37°C with shaking at 200 rpm. The OD was recorded the following day. 600 overnight culture. After this, the expression cultures were inoculated with the overnight culture to a final OD 600 , equal to 0.1.

[0902] 2x TY (tryptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L) were again grown in 500 mL batches in disposable 2 L Erlenmeyer flasks with shaking at 37°C at 200 rpm until OD 600 3.0. The incubator temperature was then reduced to 17°C. After 30 min, the cultures were supplemented with 20× nutrient solution (1 M MOPS, pH 7.2, 20 mM MgCl2, 20 mM MgSO4, 20% glycerol), and expression was induced with 150 μM IPTG. The induced cultures were then left at 17°C for 16 h and then collected by centrifugation (7000 g for 30 min). The collected pellets were then frozen at -20°C before cell lysis.

[0903] Lysis of bacterial cells

[0904] Adapted the purification and refolding protocol described by Craig S. Clements et al. The production, purification and crystallization of a soluble heterodimeric form of a highly selected T-cell receptor in its unliganded and liganded state. Biological Crystallography, 2002.

[0905] The cell pellets were lysed in lysis buffer: 50 mM Tris, pH 8.0, 1% (vol / vol) Triton X-100, 1% (vol / vol) sodium deoxycholate, 100 mM NaCl, 10 mM DTT, 1 mg DNase I (Biomedicals), 5 mM MgCl2, complete protease inhibitor cocktail (Roche). After 10 minutes of continuous mixing at room temperature, 10 mM EDTA was added. The resuspended cell pellet was then passed through a CF Cell Disrupter (Constant Systems) three times at 4°C and 1.36 atm (20 psi). The lysate was then clarified by centrifugation at 48,000 g for 1 h at 4°C (Beckman Coulter Avanti JXN-26), and the undissolved pellet was washed twice with wash buffer containing 1:50 mM Tris, pH 8.0, 0.5% (v / v) Triton X-100, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.2 mM complete protease inhibitor cocktail. Between washes, resuspended inclusion bodies were centrifuged at 48,000 × g for 30 min.For the final wash, which followed the second wash, inclusion bodies were washed one last time in buffer: 50 mM Tris, pH 8.0, 1 mM EDTA, 1 mM DTT, complete protease inhibitor cocktail. The purified inclusion bodies were then redissolved in 20 mM Tris, pH 8.0, 8 M urea (Sigma #U5378), 0.5 mM EDTA, 1 mM DTT. Purification fractions were analyzed by SDS-PAGE with NuPAGE 4-12% Bis-Tris gel (Thermo #NP0322) and NuPAGE MES SDS running buffer (Thermo #NP0002) and stained with Quick Coomassie Stain.

[0906] Refolding 2 H / 13 C / 15 N α3 HLA-G and unlabeled human β2m

[0907] Then, before refolding, the resolubilized inclusion bodies were diluted with 1.5 M guanidine HCl, 5 mM sodium acetate, 5 mM EDTA to a concentration of approximately 1 mg / mL.

[0908] First, human β2m was added dropwise into the refolding buffer: Tris, pH 8.5, 0.4 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 2 mM EDTA. This was followed by the addition of the α3 domain of HLA-G labeled 2 H / 13 C / 15 N, in a 1:1 molar ratio. To carry out the refolding reaction, the solution was left at room temperature for 16 hours with gentle stirring. A Spectra / Por dialysis membrane with a molecular weight of 3000 MWCO was then used to dialyze the refolding reaction product in dialysis buffer: 5 mM Tris, pH 8.5, at a dilution ratio of 1:20. The dialysis buffer was changed once during the 24-hour dialysis at room temperature.

[0909] Cleaning of the collapsed complex

[0910] Then, the folded HLA-G α3 / β2m complex was purified using the AKTA Pure system (GE Healthcare) and a HiTrap Q column (Cytiva Life Sciences) using the following buffers and purification sequence: Buffer A: 10 mM Tris, 10 mM NaCl, pH 8.5. Buffer B: 10 mM Tris, 500 mM NaCl, pH 8.5. Purification sequence: run at 5 ml / min, equilibrate with 5 CV buffer A, load dialyzable refolding reaction product, wash with 10 CV buffer A, eluting with 0-40% B in 10 CV, hold at 40% for 10 CV, 40-100% B in 20 CV, hold at 100% B for 10 CV.

[0911] Fractions were analyzed by SDS-PAGE using NuPAGE 4-12% Bis-Tris gel (Thermo) and NuPAGE MES SDS running buffer (Thermo) and stained with Quick Coomassie Stain (VWR #SERA35081.01). Pure fractions were pooled before concentration in a 10,000 MWCO Amicon Ultra (Millipore) and loaded onto an S75 300 / 10 Increase gel filtration column (Cytiva Life Sciences) with 150 mM NaCl, 10 mM Tris, pH 7.4, 0.02% NaN3 as running buffer. Fractions were again analyzed by SDS-PAGE, and pure fractions were pooled. The final purified sample was analyzed again by SDS-PAGE and then concentrated to ~350 µM. Protein concentration was determined using a Thermo Scientific Nanodrop 2000 spectrophotometer.

[0912] Purification of Fab reagents:

[0913] Fab reagents were purified using the AKTA Pure system (GE Healthcare) and a Gammabind Plus Sepharose column (Cytiva Life Sciences). Before capture, the supernatants were concentrated using the AKTA Flux system (GE Healthcare) to concentrations exceeding 300 mg / L. The following buffers and purification sequence were used: Buffer A: 10 mM PBS, pH 7.4. Buffer B: 0.1 M glycine HCl, pH 2.7. 5 CV of buffer A were used for equilibration. The supernatant was loaded at a rate that ensured a contact time of at least 20 minutes. 5 CV of buffer A were used for w...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to HLA-G, wherein the antibody or antigen-binding fragment thereof comprises: a. a light chain variable region comprising: i. CDR-L1 containing SEQ ID NO:1; ii. CDR-L2 containing SEQ ID NO:2 and iii. CDR-L3 comprising SEQ ID NO:3; and b. a heavy chain variable region comprising: i. CDR-H1 containing SEQ ID NO:4; ii. CDR-H2 containing SEQ ID NO:5 and iii. CDR-H3 containing SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof blocks the binding of HLA-G to ILT2 and ILT4 and / or inhibits HLA-G-mediated immunosuppressive function.

3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (K D ) for HLA-G less than 10 nM.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1-3, which specifically binds to the alpha 3 domain of HLA-G.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof binds to an HLA-G epitope comprising residues F195 and Y197 with reference to SEQ ID NO:

107.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof binds to an epitope of HLA-G, wherein the epitope comprises V194, F195, Y197, E198, Q224, Q226, D227, V248, V249, P250 and Y257 of HLA-G (SEQ ID NO:107).

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the epitope is characterized by X-ray crystallography.

8. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

9. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises: a. a light chain variable region comprising SEQ ID NO: 19 or 15 or 23; and / or b. a heavy chain variable region comprising SEQ ID NO:93, 27, 33, 57, 69, 75, 81 or 87.

10. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO:19 and a heavy chain variable region comprising SEQ ID NO:

93.

11. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody is a full-length antibody.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the full-length antibody is IgG1, IgG1 LALA, IgG1LALAGA, IgG4, IgG4P or IgG4P FALA.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the antibody is IgG1.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody is afucosylated IgG1.

15. An antibody or antigen-binding fragment thereof according to claim 13 or 14, wherein the antibody comprises: a. a light chain comprising SEQ ID NO:21 or 17 or 25; and / or b. a heavy chain comprising SEQ ID NO:95, 29, 35, 59, 71, 77, 83 or 89.

16. An antibody or antigen-binding fragment thereof according to claim 13 or 14, wherein the antibody comprises: a. a light chain that is at least 90% identical or similar to SEQ ID NO:21 or 17 or 25; and / or b. a heavy chain that is at least 90% identical or similar to SEQ ID NO:95, 29, 35, 59, 71, 77, 83 or 89.

17. The antibody or antigen-binding fragment thereof according to claim 13 or 14, wherein the antibody comprises a light chain variable region comprising SEQ ID NO:19 and a heavy chain variable region comprising SEQ ID NO:93, and wherein the remaining portions of the light chain and heavy chain are at least 90% identical or similar to SEQ ID NO:21 and 95, respectively.

18. The antibody or antigen-binding fragment thereof according to claim 13 or 14, wherein the antibody comprises a light chain comprising SEQ ID NO:21 and a heavy chain comprising SEQ ID NO:

95.

19. The antibody or antigen-binding fragment thereof according to any one of claims 13-18, wherein the antibody inhibits HLA-G-mediated immunosuppressive function and inhibits tumor growth by depleting tumor cells expressing HLA-G.

20. The antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody comprises a light chain comprising SEQ ID NO:21 and a heavy chain comprising SEQ ID NO:95, and wherein the antibody has improved ADCC and / or ADCP and / or CDC function and / or has an improved ability to deplete tumor cells expressing HLA-G.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, dsFv, scFv or dsscFv.

22. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-21.

23. The isolated polynucleotide of claim 22, wherein the polynucleotide encodes: a. a light chain variable region, where the polynucleotide: i. is at least 90% identical to SEQ ID NO:20, 16, or 24; or ii. comprises or consists of SEQ ID NO:20, 16 or 24; or b. a heavy chain variable region, where the polynucleotide: i. is at least 90% identical to SEQ ID NO:94, 28, 34, 58, 70, 76, 82, or 88; or ii. comprises or consists of SEQ ID NO:94, 28, 34, 58, 70, 76, 82 or 88.

24. The isolated polynucleotide of claim 22, wherein the polynucleotide encodes: a. light chain, where the polynucleotide: i. is at least 90% identical to SEQ ID NO:22, 18, or 26; or ii. comprises or consists of SEQ ID NO:22, 18 or 26; or b. heavy chain, where the polynucleotide: i. is at least 90% identical to SEQ ID NO:96, 30, 36, 60, 72, 78, 84, or 90; or ii. comprises or consists of SEQ ID NO:96, 30, 36, 60, 72, 78, 84 or 90.

25. A cloning vector comprising one or more polynucleotides according to any one of claims 22-24.

26. An expression vector comprising one or more polynucleotides according to any one of claims 22-24.

27. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1-21, wherein the host cell comprises one or more polynucleotides according to any one of claims 22-24 or one or more expression vectors according to claim 26.

28. The host cell of claim 27, wherein the host cell is genetically modified to reduce or eliminate the function of alpha 1,6-fucosyltransferase.

29. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1-21, comprising culturing a host cell according to claim 27 or 28 under conditions suitable for producing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

30. The method according to claim 29, wherein the method also comprises introducing the antibody or its antigen-binding fragment into the pharmaceutical composition.

31. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-21 for the production of a medicinal product for use in the treatment of a disease characterized by overexpression of HLA-G.

32. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-21 for the preparation of a medicinal product for use in the treatment of a solid tumor.

33. The use according to claim 32, wherein the solid tumor is clear cell renal cell carcinoma (RCC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, head and neck carcinoma, gastric cancer, or hepatocellular carcinoma.

34. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-21 for the preparation of a preparation for the diagnosis of clear cell renal cell carcinoma (RCC), colorectal carcinoma (CRC), pancreatic cancer, ovarian cancer, head and neck carcinoma, gastric cancer or hepatocellular carcinoma.

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