Proteins containing HLA-G antigen-binding domains and their use

Abstract

The present invention provides antigen-binding domains that bind to human leukocyte antigen G (HLA-G) protein, including antigen-binding domains that bind to HLA-G, polynucleotides encoding the same, vectors, host cells, and methods for producing and using the same.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 057,960, filed on 29 July 2020. Each of the disclosures of the aforementioned application is incorporated herein by reference in whole.

[0002] (Sequence Listing) This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on 12 July 2021, is named JBI6358WOPCT1_SL.txt and is 747KB in size.

[0003] (Field of invention) This disclosure provides proteins containing antigen-binding domains that bind to human leukocyte antigen G (HLA-G) proteins, encoding polynucleotides, vectors, host cells, and methods for producing and using them. [Background technology]

[0004] Human leukocyte antigen G (HLA-G) belongs to the non-classical MHC class Ib family of proteins. Seven alternative mRNAs encoding four membrane-bound (HLA-G1, G2, G3, G4) protein isoforms and three soluble (HLA-G5, G6, G7) protein isoforms have been described (Carosella et al., 2003). In contrast to the highly polymorphic classical HLA class I genes, HLA-G gene polymorphisms are very limited. Only 50 alleles have been listed for HLA-G, which encodes 16 full-length proteins. HLA-G1 and its soluble counterpart HLA-G5 are classical HLA class I-like, possessing identical extracellular structures: a heavy chain of three globular domains non-covalently bound to beta-2-microglobulin (β2m) and well-suited to binding to restriction peptides such as histone H2A peptides. 1-3 The HLA-G1 monomer differs from classical HLA class I molecules at the level of its peptide bond groove (a structural element important for the activation function of HLA molecules) and its α3 domain (a structural element important for the inhibitory function of HLA class I molecules). Proteolytic shedding of membrane isoforms further generates soluble molecules (Dong et al., 2003, Park et al., 2004). Monomers, homo and possible heteromultimers, and ubiquitinated proteins are the structures reported for HLA-G. The inhibitory function of HLA-G is thought to be mainly due to the dimer, not the monomer, which blocks human inhibitory receptors Ig-like transcripts 2 and 4 (ILT2 and ILT4), which are the best-characterized receptors. The HLA-G1 homodimer adopts an oblique orientation that exposes the ILT2 and ILT4 binding sites of the α3 domain, thereby binding with higher affinity and a slower dissociation rate than the HLA-G monomer.

[0005] Known HLA-G receptors include the major HLA-G receptor on peripheral immune cells (Colonna et al., 1997, 1998), the inhibitory receptors ILT2 (expressed on monocytes, DCs, B cells, and subsets of natural killer and T cells) and ILT4 (exclusively expressed by myeloid cells), the non-inhibitory receptors CD8 and CD160, and the KIR2DL4 receptor (whose status with respect to HLA-G remains ambiguous). ILT2 and ILT4 recognize and bind to the α3 domain and β2m of MHCI.

[0006] While most of the functions described for HLA-G are immune functions, non-immune functions such as inhibition of angiogenesis and bone formation have also been reported. The HLA-G-ILT2 interaction has repeatedly been shown to inhibit NK cell function and thus protect HLA-G expressing cells from NK-mediated cytolysis. Furthermore, the function of ILT-2 expressing T cells (activated and / or clonal T cells) and B cells is directly inhibited by HLA-G. HLA-G also induces alternative differentiation in myeloid APCs, induces differentiation of regulatory myeloid cells and regulatory T cells, and inhibits neutrophil phagocytosis.

[0007] The immunosuppressive ability of HLA-G, which inhibits signaling pathways required for NK-mediated cytolysis, T cell proliferation, and the induction of regulatory / suppressive cells, thereby evading the immune system, can be exploited by tumor cells to evade immune surveillance and lead to uncontrolled cell proliferation with increased invasiveness and metastatic potential. HLA-G not only allows evasion of host immune surveillance but also enhances metastasis during the progression of malignant tumors. Numerous studies have demonstrated tumor-mediated HLA-G expression (absent in normal surrounding regions) that can correlate with disease stage or worsening patient outcomes. HLA-G expression implies tumor evolution and progression, as well as advanced tumor stages, increased invasiveness and metastatic potential, and poor clinical prognosis. Its abnormal expression has been associated with reduced survival time.

[0008] While various cancers express HLA-G, its expression in normal tissues is mainly limited to the fetal-maternal interface on the extrachorionic trophoblast cell layer, the placenta, the amnion, and certain healthy adult tissues such as the thymus, cornea, bronchial epithelial cells, and pancreas, as well as specific cell types such as mesenchymal stem cells, activated monocytes, erythrocytes, and endothelial precursors (as outlined in Carosella et al., 2015).

[0009] Given its potent and widespread immunosuppressive function in normal tissues and its restricted expression pattern, HLA-G can be designated as an attractive therapeutic target in solid tumors and B-cell malignancies that overexpress HLA-G.

[0010] Of the few specific anti-HLA-G antibodies produced, only the following available antibodies (87G, MEM-G9, MEM-G11, G223) bind to native cell-expressed HLA-G. The degree of inhibition of HLA-G interactions with ligands ILT-2 and / or ILT-4 by these antibodies is not complete, even at high doses. Furthermore, while the 87G antibody in particular has been described as being able to enhance tumor killing in vitro, there are no reports of the effectiveness of blocking HLA-G by administering the antibody in vivo. [Overview of the project]

[0011] There is a need for next-generation HLA-G binding domains for therapeutic and diagnostic purposes.

[0012] The present invention provides a specific anti-HLA-G monoclonal antibody that potently blocks the binding of HLA-G to its homologous receptors ILT-2 and / or ILT-4. The generated anti-HLA-G antibody binds to either recombinant or endogenous HLA-G proteins in the absence of cross-reactivity with classical MHC class I molecules. The antibody of the present invention is to the inventors' knowledge the first HLA-G specific antibody to demonstrate specificity for HLA-G, absence of cross-reactivity with HLA-A, HLA-B, HLA-C, and HLA-E, and / or complete blockade of HLA-G interaction with ILT-2 / 4 ligands. In addition, the antibody of the present invention demonstrated tumor growth inhibition after in vivo administration.

[0013] The present invention also relates to the use of antibodies, or proteins containing variable domains derived from such antibodies, for eliminating tumor cells with elevated surface HLA-G expression by blocking the interaction between HLA-G and immunosuppressive ligands, or by engaging the immune system through either Fc or bispecific protein-mediated mobilization of immune cells. Therefore, antibodies are suitable for treating or alleviating a diagnosed condition in a patient when the condition utilizes induced HLA-G expression in the patient.

[0014] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to human leukocyte antigen G (HLA-G), wherein the antigen-binding domain that binds to HLA-G is a) Heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or b) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 52, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 53, or c) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 54, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 55, or d) HCDR1, HCDR2, and HCDR3 of VH in sequence number 56, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 57, or e) HCDR1, HCDR2, and HCDR3 of VH in sequence number 58, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 59, or f) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 60, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 61, or g) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 63, or h) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 65, or i) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 67, or j) Provided is an isolated protein comprising HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 69.

[0015] In a particular embodiment, the isolated protein is a) Sequence numbers 70, 71, 72, 88, 89, and 90, respectively b) Sequence numbers 73, 71, 74, 91, 89, and 92 respectively, c) Sequence numbers 75, 76, 77, 93, 89, and 94 respectively, d) Sequence numbers 78, 79, 80, 95, 89, and 96, respectively e) Sequence numbers 81, 82, 83, 97, 89, and 98 respectively, f) Sequence numbers 78, 71, 84, 99, 89, and 100, respectively g) Sequence numbers 78, 71, 84, 101, 89, and 100, respectively h) Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or i) Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0016] In certain embodiments, the antigen-binding domain that binds to HLA-G is scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0017] In other embodiments, the antigen-binding domain that binds to HLA-G is Fab.

[0018] In other embodiments, the antigen-binding domain that binds to HLA-G is VHH.

[0019] In other embodiments, the antigen-binding domain that binds to HLA-G is scFv.

[0020] In other embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus.

[0021] In a particular embodiment, L1 is a) Approximately 5 to 50 amino acids, b) Approximately 5 to 40 amino acids, c) Approximately 10 to 30 amino acids, or d) Contains approximately 10 to 20 amino acids.

[0022] In a particular embodiment, L1 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0023] In a particular embodiment, L1 includes the amino acid sequence of SEQ ID NO: 8.

[0024] In other embodiments, the antigen-binding domain that binds to HLA-G includes VH of SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68, and VL of SEQ ID NOs. 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0025] In other embodiments, the antigen-binding domain that binds to HLA-G is a) VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51 b) VH of sequence number 52 and VL of sequence number 53, c) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55 d) VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57 e) VH of sequence number 58 and VL of sequence number 59, f) VH of sequence number 60 and VL of sequence number 61, g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63 h) VH of sequence number 64 and VL of sequence number 65, i) VH of sequence number 66 and VL of sequence number 67, or j) Including VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69, In other embodiments, the antigen-binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[0026] In other embodiments, the protein of this disclosure is conjugated to the half-life extension portion.

[0027] In other embodiments, the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, an Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol.

[0028] In other embodiments, the isolated protein is a single-specific protein.

[0029] In other embodiments, the isolated protein is a multispecific protein.

[0030] In other embodiments, the multispecific protein is a bispecific protein.

[0031] In other embodiments, the multispecific protein is a triplespecific protein.

[0032] In other embodiments, the isolated protein of the Disclosure further comprises an immunoglobulin (Ig) constant region or a fragment thereof of the Ig constant region.

[0033] In other embodiments, the Ig steady-state region fragment includes an Fc region.

[0034] In other embodiments, the Ig constant region fragment includes a CH2 domain.

[0035] In other embodiments, the Ig constant region fragment includes a CH3 domain.

[0036] In other embodiments, the Ig constant region fragment includes a CH2 domain and a CH3 domain.

[0037] In other embodiments, the Ig steady-state region fragment includes at least a portion of the hinge, a CH2 domain, and a CH3 domain.

[0038] In other embodiments, the Ig steady-state region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0039] In other embodiments, the antigen-binding domain that binds to HLA-G is conjugated to the N-terminus of the Ig constant region or a fragment of the Ig constant region.

[0040] In other embodiments, the antigen-binding domain that binds to HLA-G is conjugated to the C-terminus of the Ig constant region or a fragment of the Ig constant region.

[0041] In other embodiments, the antigen-binding domain that binds to HLA-G is conjugated to the Ig constant region or a fragment of the Ig constant region via a second linker (L2).

[0042] In other embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0043] In other embodiments, the multispecific protein includes an antigen-binding domain that binds to an antigen on a lymphocyte.

[0044] In other embodiments, the lymphocytes are T cells.

[0045] In other embodiments, the T cells are CD8+ T cells.

[0046] In other embodiments, the lymphocytes are natural killer (NK) cells.

[0047] In other embodiments, the multispecific protein includes an antigen-binding domain that binds to CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

[0048] In other embodiments, the multispecific protein includes an antigen-binding domain that binds to CD3ε.

[0049] In other embodiments, the antigen-binding domain that binds to CD3ε is a) Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 361, HCDR2 of SEQ ID NO: 362, HCDR3 of SEQ ID NO: 363, Light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 367, LCDR2 of SEQ ID NO: 368, and LCDR3 of SEQ ID NO: 369 b) VH of sequence number 339 and VL of sequence number 340, c) HCDR1 of sequence number 361, HCDR2 of sequence number 362, HCDR3 of sequence number 363, LCDR1 of sequence number 367, LCDR2 of sequence number 368, and LCDR3 of sequence number 370 d) VH of sequence number 339 and VL of sequence number 341, e) VH of sequence number 339 and VL of sequence number 342, f) VH of sequence number 339 and VL of sequence number 343, g) VH of SEQ ID NO: 339 and VL of SEQ ID NO: 344, h) VH of sequence number 339 and VL of sequence number 345, i) HCDR1 of sequence number 364, HCDR2 of sequence number 365, HCDR3 of sequence number 366, LCDR1 of sequence number 371, LCDR2 of sequence number 372, and LCDR3 of sequence number 373, j) VH of SEQ ID NO: 346 and VL of SEQ ID NO: 347, or k) Includes VH of SEQ ID NO: 348 and VL of SEQ ID NO: 349.

[0050] In other embodiments, the Ig steady-state region or fragment of the Ig steady-state region is an IgG1, IgG2, IgG3, or IgG4 isotype.

[0051] In other embodiments, the Ig constant region or a fragment of the Ig constant region includes at least one mutation resulting in reduced binding of the protein to the Fcγ receptor (FcγR).

[0052] In other embodiments, at least one mutation resulting in reduced binding of the protein to FcγR is L235A / D265S, F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / The residues are selected from the group consisting of A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, and residue numbering follows the EU index.

[0053] In other embodiments, the Ig constant region or a fragment of the Ig constant region includes at least one mutation resulting in enhanced binding of the protein to FcγR.

[0054] In other embodiments, at least one mutation resulting in enhanced binding of the protein to FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, with residue numbering following the EU index.

[0055] In other embodiments, FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

[0056] In other embodiments, the Ig constant region of the Ig constant region fragment contains at least one mutation that modulates the protein's half-life.

[0057] In other embodiments, at least one mutation that modulates the protein's half-life is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, and the residue numbering follows the EU index.

[0058] In other embodiments, the protein contains at least one mutation in the CH3 domain of the Ig constant region.

[0059] In other embodiments, at least one mutation in the CH3 domain of the Ig constant region is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / The group is selected from Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, and the residue numbering follows the EU index.

[0060] In certain embodiments, the disclosure provides an isolated multispecific protein comprising a first antigen-binding domain that binds to HLA-G and a second antigen-binding domain that binds to a lymphocyte antigen.

[0061] In other embodiments, the lymphocyte antigen is a T cell antigen.

[0062] In other embodiments, the T cell antigen is the CD8+ T cell antigen.

[0063] In other embodiments, the lymphocyte antigen is the NK cell antigen.

[0064] In other embodiments, the lymphocyte antigens are CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

[0065] In other embodiments, the lymphocyte antigen is CD3ε.

[0066] In other embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to the lymphocyte antigen includes scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0067] In other embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens comprises Fab.

[0068] In other embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens include VHH.

[0069] In other embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens includes scFv.

[0070] In other embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus.

[0071] In other embodiments, L1 is a) Approximately 5 to 50 amino acids, b) Approximately 5 to 40 amino acids, c) Approximately 10 to 30 amino acids, or d) Contains approximately 10 to 20 amino acids.

[0072] In other embodiments, L1 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0073] In other embodiments, L1 includes the amino acid sequence of SEQ ID NO: 8.

[0074] In other embodiments, the first antigen-binding domain that binds to HLA-G includes HCDR1 of SEQ ID NO: 70, 73, 75, 78, 81, or 85, HCDR2 of SEQ ID NO: 71, 76, 79, 82, or 86, HCDR3 of SEQ ID NO: 72, 74, 77, 80, 83, 84, or 87, LCDR1 of SEQ ID NO: 88, 91, 93, 95, 97, 99, 101, or 102, LCDR2 of SEQ ID NO: 89 or 103, and LCDR3 of SEQ ID NO: 90, 92, 94, 96, 98, 100, or 104.

[0075] In other embodiments, the first antigen-binding domain that binds to HLA-G is a) Sequence numbers 70, 71, 72, 88, 89, and 90, respectively b) Sequence numbers 73, 71, 74, 91, 89, and 92 respectively, c) Sequence numbers 75, 76, 77, 93, 89, and 94 respectively, d) Sequence numbers 78, 79, 80, 95, 89, and 96, respectively e) Sequence numbers 81, 82, 83, 97, 89, and 98 respectively, f) Sequence numbers 78, 71, 84, 99, 89, and 100, respectively g) Sequence numbers 78, 71, 84, 101, 89, and 100, respectively h) Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or i) Includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0076] In other embodiments, the first antigen-binding domain that binds to HLA-G is a) VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51 b) VH of sequence number 52 and VL of sequence number 53, c) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55 d) VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57 e) VH of sequence number 58 and VL of sequence number 59, f) VH of sequence number 60 and VL of sequence number 61, g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63 h) VH of sequence number 64 and VL of sequence number 65, i) VH of sequence number 66 and VL of sequence number 67, or j) Includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0077] In other embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68, and VL of SEQ ID NOs. 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0078] In other embodiments, the first antigen-binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[0079] In other embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is a) HCDR1 of sequence number 361, HCDR2 of sequence number 362, HCDR3 of sequence number 363, LCDR1 of sequence number 367, LCDR2 of sequence number 368, and LCDR3 of sequence number 369 b) VH of sequence number 339 and VL of sequence number 340, c) HCDR1 of sequence number 361, HCDR2 of sequence number 362, HCDR3 of sequence number 363, LCDR1 of sequence number 367, LCDR2 of sequence number 368, and LCDR3 of sequence number 370 d) VH of sequence number 339 and VL of sequence number 341, e) VH of sequence number 339 and VL of sequence number 342, f) VH of sequence number 339 and VL of sequence number 343, g) VH of SEQ ID NO: 339 and VL of SEQ ID NO: 344, h) VH of sequence number 339 and VL of sequence number 345, i) HCDR1 of sequence number 364, HCDR2 of sequence number 365, HCDR3 of sequence number 366, LCDR1 of sequence number 371, LCDR2 of sequence number 372, and LCDR3 of sequence number 373, j) VH of SEQ ID NO: 346 and VL of SEQ ID NO: 347, or k) Includes VH of SEQ ID NO: 348 and VL of SEQ ID NO: 349.

[0080] In other embodiments, a first antigen-binding domain that binds to HLA-G is conjugated to a first immunoglobulin (Ig) constant region or a fragment of a first Ig constant region, and / or a second antigen-binding domain that binds to a lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of a second Ig constant region.

[0081] In other embodiments, the isolated multispecific protein further includes a second linker (L2) between a first antigen-binding domain that binds to HLA-G and a first Ig constant region or a fragment of the first Ig constant region, and between a second antigen-binding domain that binds to a lymphocyte antigen and a second Ig constant region or a fragment of the second Ig constant region.

[0082] In other embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0083] In other embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1, IgG2, IgG3, or IgG4 isotypes.

[0084] In other embodiments, the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, include at least one mutation resulting in reduced binding of the multispecific protein to FcγR.

[0085] In other embodiments, at least one mutation resulting in reduced binding of the multispecific protein to FcγR is L235A / D265S, F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 The group is selected from the deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, and residue numbering follows the EU index.

[0086] In other embodiments, the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, include at least one mutation that results in enhanced binding of the multispecific protein to the Fcγ receptor (FcγR).

[0087] In other embodiments, at least one mutation resulting in enhanced binding of the multispecific protein to FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, with residue numbering following the EU index.

[0088] In other embodiments, FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

[0089] In other embodiments, the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, include at least one mutation that modulates the half-life of the multispecific protein.

[0090] In other embodiments, at least one mutation that modulates the half-life of a multispecific protein is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, and residue numbering follows the EU index.

[0091] In other embodiments, at least one mutation in the CH3 domain of the first Ig constant region or the CH3 domain of a fragment of the first Ig constant region, and / or at least one mutation in the CH3 domain of the second Ig constant region or the CH3 domain of a fragment of the second Ig constant region.

[0092] In other embodiments, at least one mutation in the CH3 domain of the first Ig constant region or the CH3 domain of a fragment of the first Ig constant region and / or at least one mutation in the CH3 domain of the second Ig constant region or the CH3 domain of a fragment of the second Ig constant region is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407 The group is selected from V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, and residue numbering follows the EU index.

[0093] In other embodiments, the first Ig steady region or a fragment of the first Ig steady region and the second Ig steady region or a fragment of the second Ig steady region are modified as follows: a) L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig steady-state region, and L235A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig steady-state region, or b) Including L235A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig steady-state region and L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig steady-state region.

[0094] In certain embodiments, the present disclosure provides an immunoconjugate comprising an isolated protein of the present disclosure conjugated to a therapeutic agent or contrast agent.

[0095] In certain embodiments, the Disclosure provides a pharmaceutical composition comprising an isolated protein of the Disclosure and a pharmaceutically acceptable carrier.

[0096] In certain embodiments, the Disclosure provides polynucleotides encoding the isolated proteins of the Disclosure.

[0097] In certain embodiments, the Disclosure provides a vector comprising a polynucleotide encoding an isolated protein of the Disclosure.

[0098] In certain embodiments, the Disclosure provides a host cell comprising a vector encoding an isolated protein of the Disclosure.

[0099] In certain embodiments, the Disclosure provides a method for producing an isolated protein of the Disclosure, comprising culturing host cells of the Disclosure under conditions that express the protein, and collecting the protein produced by the host cells.

[0100] In certain embodiments, the present disclosure provides an immunoconjugate comprising an isolated multispecific protein of the present disclosure conjugated to a therapeutic agent or contrast agent.

[0101] In certain embodiments, the Disclosure provides a pharmaceutical composition comprising an isolated multispecific protein of the Disclosure and a pharmaceutically acceptable carrier.

[0102] In certain embodiments, the present disclosure provides polynucleotides encoding isolated, multispecific proteins of dosclosure.

[0103] In certain embodiments, the present disclosure provides a vector comprising a polynucleotide encoding an isolated, multispecific protein of dosclosure.

[0104] In certain embodiments, the present disclosure provides a host cell comprising a vector containing a polynucleotide encoding an isolated multispecific protein of dosclosure.

[0105] In certain embodiments, the Disclosure provides a method for producing an isolated multispecific protein of the Disclosure, comprising culturing host cells under conditions that express the multispecific protein, and collecting the multispecific protein produced by the host cells.

[0106] In certain embodiments, the Disclosure provides a method for treating an HLA-G expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of an isolated protein of the Disclosure, an isolated multispecific protein of the Disclosure, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure for a period of time sufficient to treat the HLA-G expressing cancer.

[0107] In certain embodiments, the Disclosure provides a method for reducing the amount of HLA-G expressing tumor cells in a subject, comprising administering to the subject an isolated protein of the Disclosure, an isolated multispecific protein of the Disclosure, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure for a period of time sufficient to reduce the amount of HLA-G expressing tumor cells.

[0108] In certain embodiments, the Disclosure provides a method for preventing the establishment of HLA-G expressing cancer in a subject, comprising administering to the subject an isolated protein of the Disclosure, an isolated multispecific protein of the Disclosure, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to prevent the establishment of HLA-G expressing cancer in the subject.

[0109] In certain embodiments, the Disclosure provides a method for treating a non-cancerous condition in a subject at risk of developing an HLA-G expressing cancer, the method comprising administering an isolated protein of the Disclosure, an isolated multispecific protein of the Disclosure, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to the subject to treat the non-cancerous condition.

[0110] In other embodiments, HLA-G expressing cancers include lung cancer, pancreatic cancer, kidney cancer, head and neck cancer, ovarian cancer, esophageal cancer, or breast cancer.

[0111] In other embodiments, the isolated protein or the isolated multispecific protein is administered in combination with a second therapeutic agent.

[0112] In other embodiments, the second therapeutic agent is surgery, chemotherapy, hormone receptor blockade therapy, or radiation, or any combination thereof.

[0113] In certain embodiments, the Disclosure provides a method for detecting the presence of cancer in a subject, comprising administering an immunoconjugate of the Disclosure to a subject suspected of having cancer, and visualizing the biological structure to which the immunoconjugate is bound, thereby detecting the presence of cancer.

[0114] In certain embodiments, the Disclosure provides a kit comprising an isolated protein of the Disclosure, an isolated multispecific protein of the Disclosure, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure.

[0115] In certain embodiments, the Disclosure provides an anti-idiotype antibody that binds to an isolated protein of the Disclosure.

[0116] In certain embodiments, the disclosure provides an isolated protein comprising an antigen-binding domain that binds to an epitope on HLA-G, wherein the epitope is a discontinuous epitope comprising the amino acid sequence of HHPVFDYE (SEQ ID NO: 485) and VPS.

[0117] In certain embodiments, the present disclosure provides an isolated protein comprising the amino acid sequence of SEQ ID NO: 478 or 479.

[0118] In a particular embodiment, the present disclosure provides an isolated protein comprising the amino acid sequence of SEQ ID NO: 478.

[0119] In a particular embodiment, the present disclosure provides an isolated protein comprising the amino acid sequence of SEQ ID NO: 479.

[0120] In a particular embodiment, the isolated protein contains the amino acid sequence of SEQ ID NO: 490.

[0121] In certain embodiments, the isolated protein contains the amino acid sequences of SEQ ID NOs. 489 and 447. In certain embodiments, the isolated protein contains the amino acid sequence of SEQ ID NOs. 439.

[0122] In certain embodiments, the present disclosure provides isolated proteins comprising the amino acid sequences of SEQ ID NOs. 465 and 468.

[0123] In certain embodiments, the present disclosure provides isolated proteins comprising the amino acid sequences of SEQ ID NOs. 466 and 469.

[0124] In certain embodiments, the present disclosure provides isolated proteins comprising the amino acid sequences of SEQ ID NOs. 467 and 470.

[0125] The foregoing will become clear from a more detailed description of the exemplary embodiments shown in the accompanying drawings below. [Brief explanation of the drawing]

[0126] [Figure 1] This shows the ability of the v region to bind to recombinant HLA-G after heat treatment when formatted as scFv. [Figure 2] Epitope mapping of selected antibodies on HLA-G (SEQ ID NO: 1) using hydrogen-deuterium exchange-based LC-MS is shown. The shown sequence is a fragment of SEQ ID NO: 1, with amino acid residue numbering starting from the first residue of the mature HLA-G (residues 183-274 are shown, SEQ ID NO: 497). [Figure 3A] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB665, which has been modified to target either IgG1 (MHGB665) or IgG4 (MHGB523). Figure 3A shows NKL cell-mediated cytotoxicity. [Figure 3B]This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB665, which has been modified to target either IgG1 (MHGB665) or IgG4 (MHGB523). Figure 3B shows NK-92 cell-mediated cytotoxicity. [Figure 4A] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB669 that has been modified to target either IgG1 (MHGB669) or IgG4 (MHGB526). Figure 4A shows NKL cell-mediated cytotoxicity. [Figure 4B] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB669 that has been modified to target either IgG1 (MHGB669) or IgG4 (MHGB526). Figure 4B shows NK-92 cell-mediated cytotoxicity. [Figure 5A] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB688 that has been modified to target either IgG1 (MHGB688) or IgG4 (MHGB596). Figure 5A shows NKL cell-mediated cytotoxicity. [Figure 5B] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB688 that has been manipulated against either IgG1 (MHGB688) or IgG4 (MHGB596). Figure 5B shows NK-92 cell-mediated cytotoxicity. [Figure 6A] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB694, modified to target either IgG1 (MHGB694) or IgG4 (MHGB616). Figure 6A shows NKL cell-mediated cytotoxicity. [Figure 6B] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB694, modified to target either IgG1 (MHGB694) or IgG4 (MHGB616). Figure 6B shows NK-92 cell-mediated cytotoxicity. [Figure 7A]This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB687 that has been manipulated against either IgG1 (MHGB687) or IgG4 (MHGB585). Figure 7A shows NKL cell-mediated cytotoxicity. [Figure 7B] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB687 that has been modified against either IgG1 (MHGB687) or IgG4 (MHGB585). Figure 7B shows NK-92 cell-mediated cytotoxicity. [Figure 8A] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB672 that has been modified to target either IgG1 (MHGB672) or IgG4 (MHGB508). Figure 8A shows NKL cell-mediated cytotoxicity. [Figure 8B] This shows the enhancement of NK cell-mediated cytotoxicity in K562-HLA-G cells by a variable region derived from MHGB672 that has been manipulated against either IgG1 (MHGB672) or IgG4 (MHGB508). Figure 8B shows NK-92 cell-mediated cytotoxicity. [Figure 9] The selected antibodies MHGB665 ("B665"), MHGB669 ("B669"), MHGB672 ("B672"), MHGB682 ("B682"), MHGB687 ("B687"), and MHGB688 ("B688") exhibit ADCC activity against JEG-3 cells. [Figure 10A] This shows the ADCC activity of the selected antibody. [Figure 10B] This shows the ADCC activity of the selected antibody. [Figure 10C] This shows the CDC activity of the selected antibody. [Figure 10D] This shows the CDC activity of the selected antibody. [Figure 11A] This shows the binding of hybridoma supernatant to primary human T cells. Clonal UCHT1 was used as a positive control (11B), and mouse IgG1 isotype (mIgG1) was used as a negative control. [Figure 11B] This shows the binding of hybridoma supernatant to primary human T cells. Clonal UCHT1 was used as a positive control (11B), and mouse IgG1 isotype (mIgG1) was used as a negative control. [Figure 12] This shows the binding of the anti-CD3 scFv variant expressed in E. coli to CD3. [Figure 13] The alignment of the VL regions of CD3B815 (sequence number 340), CD3W244 (sequence number 341), CD3W245 (sequence number 342), CD3W246 (sequence number 343), CD3W247 (sequence number 344), and CD3W248 (sequence number 345) is shown. [Figure 14] The hydrogen-deuterium exchange rates, determined using hydrogen-deuterium exchange mass spectrometry (HDX-MS), are shown for the complex of human CD3ε (CD3ε:CD3W245) bound to CD3W245 or the complex of human CD3ε (CD3ε:OKT3) bound to OKT3 (Sequence ID 484, a fragment of Sequence ID 375, is shown). A single underline indicates a segment with a 10%–30% reduction in deuteration level in the presence of the antibody compared to CD3ε alone, and a double underline indicates a segment with a >30% reduction in deuteration level. [Figure 15A] This shows the cytotoxicity and T cell activation percentage of HC3B125 against HLA-G expressing tumor cells (HUP-T3). [Figure 15B] This shows the cytotoxicity and T cell activation percentage of HC3B125 against HLA-G expressing tumor cells (HUP-T3). [Figure 15C] This shows the cytotoxicity and T cell activation percentage of HC3B125 against HLA-G expressing tumor cells RERF-LC-Ad-1. [Figure 15D] This shows the cytotoxicity and T cell activation percentage of HC3B125 against HLA-G expressing tumor cells RERF-LC-Ad-1. [Figure 16]HC3B258 and HC3B125 demonstrated cytotoxicity against RERF-LC-Ad-1 cells, with effector (T cells):target (RERF-LC-Ad1) ratios of 1:3, 1:1, or 3:1, as shown. [Figure 17A] This shows the group-average tumor volume (17A) and individual tumor volumes at day 27 of established pancreatic PDX in CD34+ cell-humanized NSG-SGM3 mice treated with either a control (HLA-G × null) or HCB125. [Figure 17B] This shows the group-average tumor volume (17A) and individual tumor volumes at day 27 of established pancreatic PDX in CD34+ cell-humanized NSG-SGM3 mice treated with either a control (HLA-G × null) or HCB125. [Figure 18] This shows the group-average tumor volume of established Hup-T3 xenografts in T-cell humanized NSG mice treated with either a control (CD3 × null) or HCB125. [Modes for carrying out the invention]

[0127] The methods of this disclosure can be more readily understood by referring to the following detailed description made in relation to the accompanying drawings, which form part of this disclosure. It should be understood that the methods of this disclosure are not limited to any particular methods described and / or shown herein, and furthermore, that the terms used herein are intended solely to illustrate particular embodiments by example and are not intended to limit them to the methods described in the claims.

[0128] All patents, published patent applications, and publications referenced herein are incorporated by reference as if they were described in their entirety herein.

[0129] Where a list is presented, it should be understood that, unless otherwise specified, each individual element of that list and all combinations of that list constitute a distinct embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0130] As used herein and in the attached Claims, the singular forms "a," "an," and "the" refer to multiple subjects unless specifically indicated otherwise. For example, the reference "a cell" includes combinations of two or more cells.

[0131] The transitional phrases “comprising,” “consisting essentially of,” and “consisting” are intended to imply meanings generally accepted in patent terminology, namely, (i) “comprising” is synonymous with “containing,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding other unlisted elements or process steps; (ii) “consisting of” excludes any elements, processes, or components not specified in the claims; and (iii) “consisting essentially of” limits the claims to specified materials or processes, and those that “do not substantially affect the basic and novel features” of the claimed invention. Embodiments described with the phrase “comprising” (or its equivalent) are also provided as embodiments described independently with “consisting” and “consisting essentially of.”

[0132] "Approximately" means that a particular value is within the acceptable margin of error for any value determined by those skilled in the art, and this depends to some extent on the method by which the value is measured or determined, i.e., on the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless otherwise expressly stated in the example or elsewhere in the specification, "approximately" means that a value is within the range of one standard deviation or up to 5%, whichever is greater, for the practice of the art.

[0133] "Activation," "stimulation," "activated," or "stimulated" refers to the induction of a change in the biological state of a cell that results in the expression of activation markers, cytokine production, proliferation, or mediation of cytotoxicity of target cells. Cells can be activated by a primary stimulatory signal. Co-stimulatory signals can amplify the magnitude of the primary signal and suppress cell death after initial stimulation, resulting in a more durable activated state and, consequently, higher cytotoxicity. A "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, induces the proliferation of T cells and / or NK cells, and / or the upregulation or downregulation of key molecules.

[0134] An "alternative scaffold" refers to a single-chain protein framework containing a structured core that associates with a highly conformationally tolerant variable domain. Because the variable domain allows for polymorphisms to be introduced without compromising scaffold integrity, it can be genetically engineered and selected to bind to specific antigens.

[0135] "Antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a mechanism that induces cell death in which antibody-coated target cells interact with effector cells with lytic activity, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via the Fc-gamma receptor (FcγR) expressed on the effector cells.

[0136] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated through internalization by phagocytic cells such as macrophages or dendritic cells.

[0137] "Antigen" refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, part thereof, or combination thereof) to which an antigen-binding domain or T cell receptor can bind, which can mediate an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens may be expressed by genes from biological samples such as tissue samples, tumor samples, cells, or fluids, organisms, protein / antigen subunits, killed or inactivated whole cells, or lysates, may be synthesized from such samples, or may be purified from such samples.

[0138] An "antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding fragments may be synthetic polypeptides, enzymatically available polypeptides, or genetically engineered polypeptides, and include portions of immunoglobulins that bind to antigens, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelid VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDR of an antibody, such as the FR3-CDR3-FR4 moiety, HCDR1, HCDR2, and / or HCDR3, as well as LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind to antigens, and multispecific proteins containing antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked to each other via synthetic linkers to form various types of single-chain antibody designs, where the VH / VL domains can pair intramolecularly or intermolecularly to form a monovalent antigen-binding domain, such as a single-chain Fv (scFv) or diabody, when the VH and VL domains are expressed as separate single-chain units. Antigen-binding fragments may also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which may be monospecific or multispecific, for genetic engineering of bispecific and multispecific proteins.

[0139] The term "antibody" has a broad meaning and includes monoclonal antibodies, including mouse, human, humanized, and chimeric monoclonal antibodies; antigen-binding fragments; multispecific antibodies such as bispecific, tripspecific, and quadrupspecific antibodies; dimers, tetramers, or multimers; single-chain antibodies; domain antibodies; and immunoglobulin molecules, including any other modified forms of immunoglobulin molecules containing antigen-binding sites of the required specificity. A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, and their multimers (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further classified into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0140] "Bispecificity" refers to a molecule (such as an antibody) that specifically binds to two different antigens within the same antigen, or to two different epitopes. Bispecific molecules may also cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, e.g., cynomolgus monkeys (Macaca cynomolgus) (cynomolgus, cyno) or chimpanzees (Pan troglodytes), and may bind to epitopes shared among two or more different antigens.

[0141] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0142] Complement-dependent cytotoxicity (CDC) refers to a mechanism of cell death induction in which the Fc effector domain of a target-bound protein binds to and activates complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the target cell surface, facilitating CDC through the binding of complement receptors (e.g., CR3) to leukocytes.

[0143] The "complementarity-determining region" (CDR) is the region of the antibody that binds to the antigen. There are three CDRs in the VH (Very High) antibody (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (Very Low) antibody (LCDR1, LCDR2, LCDR3). CDR can be defined using various descriptions such as Kabat (Wu et al. (1970) J Exp Med 132:211-50, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton J Bmol Biol 263:800-15, 1996). Correspondence between various descriptions and variable region numbering is described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77, Honegger and Pluckthun, J Mol Biol (2001) 309:657-70, the International ImMunoGeneTics (IMGT) database, web resources, http: / / www_imgt_org). CDRs can be described using available programs such as abYsis by UCL Business PLC. As used herein, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” include CDRs defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.

[0144] "Decrease," "decrease," "lower," "reduce," or "mitigate" generally refers to the ability of a test molecule to mediate a reduced response (i.e., a downstream effect) compared to a response mediated by a control or vehicle. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. A decrease may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or a decrease of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, for example, 1.5, 1.6, 1.7, 1.8, etc.).

[0145] "Differentiation" refers to a method of reducing the capacity or proliferation of cells, or moving them into a more developmentally restricted state.

[0146] "Code" or "coding" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that arise therefrom, serving as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, in a cell or other biological system, if the transcription and translation of mRNA corresponding to a gene produces a protein, then that gene, cDNA, or RNA codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence, and the non-coding strand used as a template for the transcription of a gene or cDNA, may be referred to as coding for a protein or other product of that gene or cDNA.

[0147] "Enhance," "promote," "increase," "expand," or "improve" generally refer to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) compared to the response mediated by the control or vehicle. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. Enhancement may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase in the measured response, such as an increase of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0148] "Expansion" refers to the result of cell division and cell death.

[0149] "Express" and "expression" refer to the well-known transcription and translation that occur intracellularly or in vitro. Therefore, the expression product, for example, a protein, may be expressed by a cell or in vitro, and may be intracellular, extracellular, or transmembrane protein.

[0150] An "expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0151] "dAb" or "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544 546 (1989)).

[0152] "Fab" or "Fab fragment" refers to an antibody fragment composed of VH, CH1, VL, and CL domains.

[0153] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide crosslinks within a hinge region.

[0154] "Fd" or "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains.

[0155] "Fv" or "Fv fragment" refers to an antibody fragment consisting of a VH domain and a VL domain derived from a single arm of the antibody.

[0156] A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as their polymers (e.g., IgM). Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain consisting of subdomains CH1, hinge, CH2, and CH3. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0157] "Genetic modification" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence and produces a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence and may include regulatory or control sequences that are manipulably ligated to a polynucleotide encoding a chimeric antigen receptor, such as an initiator, terminater, promoter, signaling, secretion, or other sequence used by the cell's genetic mechanism. The gene or sequence may include non-functional sequences that do not have a known function. A host cell that receives and expresses the introduced DNA or RNA is "genetically engineered." The DNA or RNA introduced into a host cell may originate from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0158] "Heterogeneous" refers to two or more polynucleotides or two or more polypeptides that are not found in the same relationship to each other in nature.

[0159] "Heterogeneic polynucleotides" refer to polynucleotides that do not exist in nature and encode two or more neoantigens as described herein.

[0160] "Heterogeneous polypeptide" refers to a polypeptide that does not exist in nature and contains two or more neoantigen polypeptides as described herein.

[0161] A "host cell" refers to any cell containing heterologous nucleic acids. An example of heterologous nucleic acid is a vector (e.g., an expression vector).

[0162] A “human antibody” refers to an antibody optimized to minimize the immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. A human antibody includes heavy-chain and light-chain variable regions that “derive” from a human-derived sequence if the variable region of the human antibody is obtained from a system using human germline immunoglobulin or a rearranged immunoglobulin gene. Such exemplary systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals possessing human immunoglobulin loci, such as mice or rats. A “human antibody” typically contains amino acid differences when compared to immunoglobulin expressed in humans, due to differences in the human antibody and the system used to obtain the human immunoglobulin locus, intentional introduction of somatic mutations or substitutions into the framework or CDR, or both. Typically, a “human antibody” is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain a consensus framework sequence derived from human framework sequence analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into a human immunoglobulin gene library presented on phages, for example, as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 2009 / 085462. Antibodies in which at least one CDR originates from a non-human species are not included in the definition of "human antibodies."

[0163] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Because humanized antibodies can contain substitutions in their framework, the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0164] "In combination with ~" means administering two or more therapeutic agents to the subject together as a mixture, simultaneously as individual agents, or sequentially as individual agents in any order.

[0165] "Isolated" refers to a homogeneous population of molecules (e.g., synthetic polynucleotides or polypeptides) that has been substantially separated and / or purified from other components of the system in which the molecules are produced, such as in recombinant cells, in addition to proteins that have undergone at least one purification or isolation step. "Isolated / Isolated" refers to molecules that are substantially free from other cellular material and / or chemicals, and includes molecules isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0166] "Human leukocyte antigen G" or "HLA-G" refers to a known protein also known as "HLA class I histocompatibility antigen, alpha chain G" or "MHC class I antigen G". All HLA-G isoforms and variants are encompassed under "HLA-G". The amino acid sequences of various isoforms can be searched using Uniprot ID numbers P17693-1 to P17693-7 and are shown in Table 1.

[0167] [Table 1]

[0168] "Modifying" refers to either an enhanced or reduced ability of a test molecule to mediate a greater or lesser response (i.e., a downstream effect) compared to the response mediated by the control or vehicle.

[0169] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules that is identical except for possible known modifications such as removal of C-terminal lysine from the antibody heavy chain, or post-translational modifications such as amino acid isomerization or deamidementation, methionine oxidation, or deamidementation of asparagine or glutamine; i.e., an individual antibody constituting a population. Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or polyvalent.

[0170] "Multispecificity" refers to molecules, such as antibodies, that specifically bind to two or more different antigens, or to two or more different epitopes within the same antigen. Multispecific molecules may also cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, e.g., cynomolgus monkeys (Macaca fascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes), and may bind to epitopes shared among two or more different antigens.

[0171] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. In this specification, NK cells refer to CD16 + CD56 + and / or CD57 + TCR -NK cells refer to differentiated lymphocytes that exhibit a specific phenotype. NK cells are characterized by their ability to bind to and kill cells that cannot express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0172] When used in relation to nucleic acids or amino acids, "operably linked" and similar terms refer to the operational linkage of nucleic acid sequences or amino acid sequences, respectively, that are arranged in a functional relationship with one another. For example, operable linked promoter, enhancer element, open reading frame, 5' and 3' UTR, and terminator sequences result in the precise production of nucleic acid molecules (e.g., RNA) and, in some cases, the production of polypeptides (i.e., expression of the open reading frame). An operable linked peptide refers to a peptide in which the functional domains of the peptide are arranged at appropriate distances from each other to confer the intended function of each domain.

[0173] A "pharmaceutical combination" refers to a combination of two or more active ingredients administered together or separately.

[0174] A "pharmaceutical composition" refers to a composition obtained by combining an active ingredient with a pharmaceutically acceptable carrier.

[0175] A "pharmaceutically acceptable carrier" or "excipient" refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the target. Exemplary pharmaceutically acceptable carriers are buffers, stabilizers, or preservatives.

[0176] A "polynucleotide" or "nucleic acid" refers to a synthetic molecule containing nucleotide chains covalently bonded by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. Polynucleotides can be DNA or RNA molecules.

[0177] "Preventing", "prevention", "prevent", or "prevention method" of a disease or disorder means preventing the occurrence of the disorder in a subject.

[0178] "Proliferation" refers to an increase in cell division, which can be either symmetric or asymmetric cell division.

[0179] "Promoter" refers to the minimum sequence necessary to initiate transcription. A promoter may also contain enhancer or repressor elements that enhance or suppress transcription, respectively.

[0180] As used interchangeably herein, "protein" or "polypeptide" refers to a molecule comprising one or more polypeptides each consisting of at least two amino acid residues linked by peptide bonds. A protein may be a monomer or a protein complex of two or more identical or different subunits. Small polypeptides consisting of less than 50 amino acids may be referred to as "peptides". A protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation or biotinylation. A protein can be recombinantly expressed.

[0181] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules prepared, expressed, created, or isolated by recombinant means. [[ID=第十七]]

[0182] "Regulatory element" refers to any cis- or trans-acting genetic element that controls an aspect of the expression of a nucleic acid sequence.

[0183] "Recurrent" refers to the recurrence of a disease or the signs and symptoms of a disease after a period of improvement following previous treatment with a therapeutic agent.

[0184] "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment before or at the start of treatment, or a refractory disease may become a resistant disease during treatment.

[0185] "Single-chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable light chain region (VL) and at least one antibody fragment comprising a variable heavy chain region (VH), wherein VL and VH are continuously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. Unless otherwise specified, as used herein, scFv may have the VL and VH variable regions in either order. For example, with respect to the N-terminus and C-terminus of the polypeptide, scFv may comprise VL-linker-VH, or may comprise VH-linker-VL.

[0186] "Specifically binds", "specific binding", "specifically bound", or "binds" refers to a proteinaceous molecule binding to an antigen or an epitope within an antigen with a higher affinity than to other antigens. Typically, a proteinaceous molecule binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (K -7 M) of about 1×10 -8 M or less, for example about 5×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less, and typically K D is at least 100-fold less than K D for binding to non-specific antigens (e.g., BSA, casein). In the context of the prostate neoantigens described herein, "specific binding" refers to a proteinaceous molecule binding to a prostate neoantigen without detectably binding to the wild-type protein of which the prostate neoantigen is a variant.

[0187] ​​"Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as non-human primates, mammals and non-mammals such as sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. The terms "subject" and "patient" may be used interchangeably herein.

[0188] The terms "T cell" and "T lymphocyte" are interchangeable and are used synonymously herein. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells may also be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may also be helper T cells (HTL, CD4) + T cells), CD4 + T cells, cytotoxic T cells (CTL, CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL, CD8 + T cells), CD4 + CD8 + This can be a T cell, or any other subset of T cells. It also includes "NKT cells," which refer to a special population of T cells that not only express the semi-variant αβ T cell receptor but also various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1 + and NK1.1 - , and CD4 + CD4 - CD8 + , and CD8 -The cells included are NKT cells, which are unique in that their TCR recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)," which refer to a special population of a small subset of T cells that have different TCRs on their surface. Unlike most T cells, whose TCR consists of two glycoprotein chains denoted as α and β-TCR chains, the TCR in γδ T cells consists of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immunomodulation, are an important source of IL-17, and are potent CD8 + It has been found that it induces a cytotoxic T cell response. It also includes "regulatory T cells" or "Tregs," which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4 cells. + T cells and IL-10 producing CD4 + This may also include T cells, specifically Foxp3-negative regulatory T cells.

[0189] In this specification, the terms “therapeutic effective dose” or “effective dose” as used interchangeably refer to the amount of medication and duration that is effective in achieving the desired therapeutic outcome. The therapeutic effective dose may vary depending on factors such as the individual’s condition, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improvement in the patient’s health, reduction in tumor burden, cessation or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body.

[0190] "Transduction" refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0191] "To treat," "to treat," or "to treat" a disease or disability such as cancer means achieving one or more of the following: reducing the severity and / or duration of the disability; inhibiting the worsening of characteristic symptoms of the disability being treated; limiting or preventing recurrence of the disability in a person who previously had the disability; or limiting or preventing recurrence of symptoms in a person who previously had symptoms of the disability.

[0192] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells that exhibit spontaneous or induced phenotypic changes in vivo, ex vivo, or tissue culture. These changes do not necessarily involve the uptake of new genetic material. Transformation can be induced by infection with transforming viruses and the incorporation of new genomic nucleic acids, or by the uptake of exogenous nucleic acids, and may occur spontaneously or after exposure to carcinogens, thereby resulting in mutations of endogenous genes. Transformation / cancer is exemplified by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, and tumor growth in suitable animal hosts such as nude mice, in vitro, in vivo, and ex vivo.

[0193] "Variant," "mutant," or "altered" refers to a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

[0194] Throughout this specification, unless otherwise explicitly stated herein, amino acid residue numbering in the antibody constant region follows the EU index described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0195] Mutations in the Ig constant region are referred to as follows: L351Y_F405A_Y407V refers to L351Y, F405A, and Y407V mutations in one immunoglobulin constant region. L351Y_F405A_Y407V / T394W refers to L351Y, F405A, and Y407V mutations in the first Ig constant region and a T394W mutation in the second Ig constant region present in one multimeric protein.

[0196] Antigen-binding domain that binds to HLA-G. This disclosure provides antigen-binding domains that bind to HLA-G, monospecific or multispecific proteins comprising HLA-G-binding antigen-binding domains, polynucleotides encoding them, vectors, host cells, and methods for producing and using them. The HLA-G-binding antigen-binding domains identified herein have demonstrated several unique properties, including 1) improved thermal stability, 2) improved scalability achieved by reducing the risk of deamidation, 3) reduced immunogenicity, 4) specificity to HLA-G with no cross-reactivity with HLA-A, HLA-B, HLA-C, and HLA-E, and 5) the ability to overcome immune checkpoint ligand expression on tumor cells and ensure tumor cell death via T cell-mediated cytotoxicity.

[0197] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to human leukocyte antigen G (HLA-G), wherein the antigen-binding domain that binds to HLA-G is Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 52, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 53, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 54, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 55, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 56, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 57, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 58, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 59, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 60, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 61, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 62, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 63, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 64, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 65, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 66, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 67, or This invention provides isolated proteins including HCDR1, HCDR2, and HCDR3 of the VH form of SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 of the VL form of SEQ ID NO: 69.

[0198] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G is Sequence numbers 70, 71, 72, 88, 89, and 90, respectively. Sequence numbers 73, 71, 74, 91, 89, and 92, respectively. Sequence numbers 75, 76, 77, 93, 89, and 94, respectively. Sequence numbers 78, 79, 80, 95, 89, and 96, respectively. Sequence numbers 81, 82, 83, 97, 89, and 98, respectively Sequence numbers 78, 71, 84, 99, 89, and 100, respectively. Sequence numbers 78, 71, 84, 101, 89, and 100, respectively. Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or Provided is an isolated protein comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 78, 71, 84, 95, 89, and 96, respectively.

[0199] The present disclosure provides an isolated protein comprising an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G comprises VH of SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of SEQ ID NO: 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0200] The present disclosure provides an isolated protein comprising an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G comprises VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51, comprises VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53, comprises VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55, comprises VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57, comprises VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59, comprises VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61, comprises VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63, comprises VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65, comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67, or comprises VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0201] The present disclosure provides an isolated protein comprising an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G comprises an amino acid sequence of SEQ ID NO: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[0202] <The disclosure also provides an isolated protein comprising an antigen-binding domain that binds to human leukocyte antigen G (HLA-G), wherein the antigen-binding domain that binds to HLA-G comprises a mutation engineered to provide germline optimization, the mutation being selected from the group consisting of E1Q, L5Q, E6Q, S71P, D46E, and H77N mutations in the VH domain, and K30E and G66V in the VL domain. The disclosure also provides an isolated protein comprising an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G comprises a mutation engineered to reduce the risk of post-translational modification, the mutation being N92H in the VL domain.

[0203] This disclosure also relates to an isolated protein comprising an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G is VH of sequence number 50 and VL of sequence number 51, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 53, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 55, VH of sequence number 50 and VL of sequence number 57, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 59 VH of SEQ ID NO: 50 and VL of SEQ ID NO: 61, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 63, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 65 VH of sequence number 50 and VL of sequence number 67, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 69 VH of sequence number 52 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of SEQ ID NO: 52 and VL of SEQ ID NO: 55 VH of sequence number 52 and VL of sequence number 57, VH of sequence number 52 and VL of sequence number 59, VH of sequence number 52 and VL of sequence number 61, VH of sequence number 52 and VL of sequence number 63, VH of SEQ ID NO: 52 and VL of SEQ ID NO: 65, VH of sequence number 52 and VL of sequence number 67, VH of sequence number 52 and VL of sequence number 69, VH of sequence number 54 and VL of sequence number 51, VH of sequence number 54 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 54 and VL of sequence number 57, VH of sequence number 54 and VL of sequence number 59, VH of sequence number 54 and VL of sequence number 61, VH of sequence number 54 and VL of sequence number 63, VH of SEQ ID NO: 54 and VL of SEQ ID NO: 65 VH of sequence number 54 and VL of sequence number 67, VH of sequence number 54 and VL of sequence number 69, VH of sequence number 56 and VL of sequence number 51, VH of sequence number 56 and VL of sequence number 53, VH of sequence number 56 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 56 and VL of sequence number 59, VH of sequence number 56 and VL of sequence number 61, VH of sequence number 56 and VL of sequence number 63, VH of sequence number 56 and VL of sequence number 65, VH of sequence number 56 and VL of sequence number 67, VH of sequence number 56 and VL of sequence number 69, VH of sequence number 58 and VL of sequence number 51, VH of sequence number 58 and VL of sequence number 53, VH of SEQ ID NO: 58 and VL of SEQ ID NO: 55 VH of sequence number 58 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 58 and VL of sequence number 61, VH of sequence number 58 and VL of sequence number 63, VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65, VH of sequence number 58 and VL of sequence number 67, VH of sequence number 58 and VL of sequence number 69, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 51, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 53, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 55, VH of sequence number 60 and VL of sequence number 57, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 60 and VL of sequence number 63, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 65 VH of sequence number 60 and VL of sequence number 67, VH of sequence number 60 and VL of sequence number 69, VH of sequence number 62 and VL of sequence number 51, VH of sequence number 62 and VL of sequence number 53, VH of sequence number 62 and VL of sequence number 55, VH of sequence number 62 and VL of sequence number 57, VH of sequence number 62 and VL of sequence number 59, VH of sequence number 62 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 62 and VL of sequence number 65, VH of sequence number 62 and VL of sequence number 67, VH of sequence number 62 and VL of sequence number 69, VH of sequence number 64 and VL of sequence number 51, VH of sequence number 64 and VL of sequence number 53, VH of sequence number 64 and VL of sequence number 55, VH of sequence number 64 and VL of sequence number 57, VH of sequence number 64 and VL of sequence number 59, VH of sequence number 64 and VL of sequence number 61, VH of sequence number 64 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 64 and VL of sequence number 67, VH of sequence number 64 and VL of sequence number 69, VH of sequence number 66 and VL of sequence number 51, VH of sequence number 66 and VL of sequence number 53, VH of sequence number 66 and VL of sequence number 55, VH of sequence number 66 and VL of sequence number 57, VH of sequence number 66 and VL of sequence number 59, VH of sequence number 66 and VL of sequence number 61, VH of sequence number 66 and VL of sequence number 63, VH of sequence number 66 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, VH of sequence number 66 and VL of sequence number 69, VH of sequence number 68 and VL of sequence number 51, VH of sequence number 68 and VL of sequence number 53, VH of sequence number 68 and VL of sequence number 55, VH of sequence number 68 and VL of sequence number 57, VH of sequence number 68 and VL of sequence number 59, VH of sequence number 68 and VL of sequence number 61, VH of sequence number 68 and VL of sequence number 63, VH of sequence number 68 and VL of sequence number 65, VH of SEQ ID NO: 68 and VL of SEQ ID NO: 67, or This provides isolated proteins including VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0204] The disclosure also provides an isolated protein comprising an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G comprises the amino acid sequence of SEQ ID NOs. 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[0205] In some embodiments, the antigen-binding domain that binds to HLA-G is scFv.

[0206] In some embodiments, the antigen-binding domain that binds to HLA-G is (scFv)2.

[0207] In some embodiments, the antigen-binding domain that binds to HLA-G is Fv.

[0208] In some embodiments, the antigen-binding domain that binds to HLA-G is Fab.

[0209] In some embodiments, the antigen-binding domain that binds to HLA-G is F(ab')2.

[0210] In some embodiments, the antigen-binding domain that binds to HLA-G is Fd.

[0211] In some embodiments, the HLA-G antigen-binding domain is a dAb.

[0212] In some embodiments, the HLA-G antigen-binding domain is VHH.

[0213] HLA-G binding scFv Either the VH domain or VL domain identified herein that binds to HLA-G can be manipulated into scFv formats of either VH-linker-VL or VL-linker-VH orientation. Furthermore, either the VH domain or VL domain identified herein can be used to generate sc(FV)2 structures such as VH-linker-VL-linker-VL-linker-VH, VH-linker-VL-linker-VH-linker-VL, VH-linker-VH-linker-VL-linker-VL, VL-linker-VH-linker-VL-linker-VH, or VL-linker-VL-linker-VH-linker-VH.

[0214] The VH and VL domains identified herein can be incorporated into the scFv format, and the binding and thermal stability of the resulting scFv to HLA-G can be evaluated using known methods. Binding can be evaluated using ProteOn XPR36, Biacore3000, or KinExA instruments, ELISA, or competitive binding assays known to those skilled in the art. Binding can be evaluated using purified scFv or E. coli supernatant or lysed cells containing expressed scFv. Measured affinity of test scFv to HLA-G may differ when measured under different conditions (e.g., molar osmotic concentration, pH). Therefore, affinity and other binding parameters (e.g., K) may differ. D , K on , K off The measurement of thermal stability is usually performed using standard conditions and standardized buffer. Thermal stability can be evaluated by heating the test scFv at a high temperature such as 50°C, 55°C, or 60°C for a period of time such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes, and measuring the binding of the test scFv to HLA-G. An scFv that retains equivalent binding to HLA-G compared to an unheated scFv sample is said to be thermally stable.

[0215] In recombinant expression systems, the linker is a peptide linker and may contain any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker needs to be of an appropriate length to link VH and VL in such a way that they form precise higher-order structures relative to each other, thereby maintaining desired activity such as binding to HLA-G.

[0216] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker is approximately 10 to 40 amino acids long. In some embodiments, the linker is approximately 10 to 35 amino acids long. In some embodiments, the linker is approximately 10 to 30 amino acids long. In some embodiments, the linker is approximately 10 to 25 amino acids long. In some embodiments, the linker is approximately 10 to 20 amino acids long. In some embodiments, the linker is approximately 15 to 20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acid lengths. In some embodiments, the linker is 16 amino acid lengths. In some embodiments, the linker is 17 amino acid lengths. In some embodiments, the linker is 18 amino acid lengths. In some embodiments, the linker is 19 amino acid lengths. In some embodiments, the linker is 20 amino acid lengths. In some embodiments, the linker is 21 amino acid lengths. In some embodiments, the linker is 22 amino acid lengths. In some embodiments, the linker is 23 amino acid lengths. In some embodiments, the linker is 24 amino acid lengths. In some embodiments, the linker is 25 amino acid lengths. In some embodiments, the linker is 26 amino acid lengths. In some embodiments, the linker is 27 amino acid lengths. In some embodiments, the linker is 28 amino acid lengths. In some embodiments, the linker is 29 amino acid lengths.In some embodiments, the linker is 30 amino acid lengths. In some embodiments, the linker is 31 amino acid lengths. In some embodiments, the linker is 32 amino acid lengths. In some embodiments, the linker is 33 amino acid lengths. In some embodiments, the linker is 34 amino acid lengths. In some embodiments, the linker is 35 amino acid lengths. In some embodiments, the linker is 36 amino acid lengths. In some embodiments, the linker is 37 amino acid lengths. In some embodiments, the linker is 38 amino acid lengths. In some embodiments, the linker is 39 amino acid lengths. In some embodiments, the linker is 40 amino acid lengths. Exemplary linkers that can be used are glycy-rich linkers, glycy and ser-containing linkers, glycy and ala-containing linkers, ala and ser-containing linkers, and other flexible linkers.

[0217] Other linker sequences may include immunoglobulin hinge regions, CL, or CH1 portions derived from immunoglobulin heavy or light chain isotypes. Alternatively, various non-protein polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may be found to be used as linkers. Exemplary linkers that may be used are shown in Table 2. Additional linkers are described, for example, in International Publication No. 2019 / 060695.

[0218] In some embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL) from the N-terminus to the C-terminus.

[0219] In some embodiments, scFv includes VL, L1, and VH (VL-L1-VH) from the N-terminus to the C-terminus.

[0220] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 8.

[0221] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 9.

[0222] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 10.

[0223] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 11.

[0224] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 12.

[0225] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 13.

[0226] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 14.

[0227] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 15.

[0228] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 16.

[0229] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 17.

[0230] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 18.

[0231] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 19.

[0232] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 20.

[0233] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 21.

[0234] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 22.

[0235] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 23.

[0236] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 24.

[0237] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 25.

[0238] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 26.

[0239] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 27.

[0240] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 28.

[0241] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 29.

[0242] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 30.

[0243] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 31.

[0244] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 32.

[0245] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 33.

[0246] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 34.

[0247] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 35.

[0248] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 36.

[0249] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 37.

[0250] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 38.

[0251] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 39.

[0252] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 40.

[0253] [Table 2]

[0254] In some embodiments, scFV is Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 52, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 53, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 54, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 55, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 56, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 57, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 58, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 59, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 60, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 61, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 62, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 63, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 64, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 65, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 66, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 67, or This includes HCDR1, HCDR2, and HCDR3 of VH in sequence number 68, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 69.

[0255] In some embodiments, scFv is Sequence numbers 70, 71, 72, 88, 89, and 90, respectively. Sequence numbers 73, 71, 74, 91, 89, and 92, respectively. Sequence numbers 75, 76, 77, 93, 89, and 94, respectively. Sequence numbers 78, 79, 80, 95, 89, and 96, respectively. Sequence numbers 81, 82, 83, 97, 89, and 98, respectively Sequence numbers 78, 71, 84, 99, 89, and 100, respectively. Sequence numbers 78, 71, 84, 101, 89, and 100, respectively. Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or This includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0256] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 70, 71, 72, 88, 89, and 90.

[0257] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 73, 71, 74, 91, 89, and 92, respectively.

[0258] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 75, 76, 77, 93, 89, and 94, respectively.

[0259] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 79, 80, 95, 89, and 96.

[0260] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 81, 82, 83, 97, 89, and 98.

[0261] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 99, 89, and 100.

[0262] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 101, 89, and 100.

[0263] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 85, 86, 87, 102, 103, and 104.

[0264] In some embodiments, the scFV includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 95, 89, and 96.

[0265] In some embodiments, scFV includes VH of sequence numbers 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of sequence numbers 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0266] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0267] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0268] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0269] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0270] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0271] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0272] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0273] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0274] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0275] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0276] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0277] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 53.

[0278] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 55.

[0279] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 57.

[0280] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 59.

[0281] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 61.

[0282] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 63.

[0283] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 65.

[0284] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 67.

[0285] In some embodiments, scFV includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 69.

[0286] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 51.

[0287] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0288] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 55.

[0289] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 57.

[0290] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 59.

[0291] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 61.

[0292] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 63.

[0293] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 65.

[0294] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 67.

[0295] In some embodiments, scFV includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 69.

[0296] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 51.

[0297] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 53.

[0298] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0299] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 57.

[0300] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 59.

[0301] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 61.

[0302] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 63.

[0303] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 65.

[0304] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 67.

[0305] In some embodiments, scFV includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 69.

[0306] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 51.

[0307] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 53.

[0308] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 55.

[0309] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0310] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 59.

[0311] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 61.

[0312] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 63.

[0313] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 65.

[0314] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 67.

[0315] In some embodiments, scFV includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 69.

[0316] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 51.

[0317] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 53.

[0318] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 55.

[0319] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 57.

[0320] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0321] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 61.

[0322] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 63.

[0323] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65.

[0324] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 67.

[0325] In some embodiments, scFV includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 69.

[0326] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 51.

[0327] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 53.

[0328] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 55.

[0329] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 57.

[0330] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 59.

[0331] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0332] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 63.

[0333] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 65.

[0334] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 67.

[0335] In some embodiments, scFV includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 69.

[0336] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 51.

[0337] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 53.

[0338] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 55.

[0339] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 57.

[0340] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 59.

[0341] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 61.

[0342] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0343] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 65.

[0344] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67.

[0345] In some embodiments, scFV includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69.

[0346] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 51.

[0347] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 53.

[0348] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 55.

[0349] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 57.

[0350] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 59.

[0351] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 61.

[0352] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 63.

[0353] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0354] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 67.

[0355] In some embodiments, scFV includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 69.

[0356] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 51.

[0357] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 53.

[0358] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 55.

[0359] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 57.

[0360] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 59.

[0361] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 61.

[0362] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 63.

[0363] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 65.

[0364] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0365] In some embodiments, scFV includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 69.

[0366] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 51.

[0367] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 53.

[0368] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 55.

[0369] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 57.

[0370] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 59.

[0371] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 61.

[0372] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 63.

[0373] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 65.

[0374] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 67.

[0375] In some embodiments, scFV includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0376] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 248.

[0377] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 249.

[0378] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 250.

[0379] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 251.

[0380] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 252.

[0381] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 253.

[0382] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 254.

[0383] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 255.

[0384] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 256.

[0385] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 257.

[0386] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 258.

[0387] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 259.

[0388] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 260.

[0389] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 261.

[0390] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 262.

[0391] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 263.

[0392] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 264.

[0393] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 265.

[0394] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 266.

[0395] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 267.

[0396] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 268.

[0397] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 269.

[0398] Other antigen-binding domains that bind to HLA-G Any of the VH and VL domains identified herein that bind to HLA-G can be manipulated into Fab, F(ab')2, Fd, or Fv formats, and their binding to HLA-G and thermal stability can be evaluated using the assays described herein.

[0399] In some embodiments, Fab is Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 52, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 53, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 54, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 55, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 56, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 57, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 58, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 59, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 60, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 61, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 62, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 63, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 64, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 65, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 66, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 67, or This includes HCDR1, HCDR2, and HCDR3 of VH in sequence number 68, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 69.

[0400] In some embodiments, Fab is Sequence numbers 70, 71, 72, 88, 89, and 90, respectively. Sequence numbers 73, 71, 74, 91, 89, and 92, respectively. Sequence numbers 75, 76, 77, 93, 89, and 94, respectively. Sequence numbers 78, 79, 80, 95, 89, and 96, respectively. Sequence numbers 81, 82, 83, 97, 89, and 98, respectively Sequence numbers 78, 71, 84, 99, 89, and 100, respectively. Sequence numbers 78, 71, 84, 101, 89, and 100, respectively. Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or This includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0401] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 70, 71, 72, 88, 89, and 90.

[0402] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 73, 71, 74, 91, 89, and 92.

[0403] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 75, 76, 77, 93, 89, and 94.

[0404] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 79, 80, 95, 89, and 96.

[0405] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 81, 82, 83, 97, 89, and 98.

[0406] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 99, 89, and 100.

[0407] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 101, 89, and 100.

[0408] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 85, 86, 87, 102, 103, and 104.

[0409] In some embodiments, the Fab includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 95, 89, and 96.

[0410] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0411] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0412] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0413] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0414] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0415] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0416] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0417] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0418] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0419] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0420] In some embodiments, Fab includes VH of SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of SEQ ID NOs. 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0421] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0422] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 53.

[0423] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 55.

[0424] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 57.

[0425] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 59.

[0426] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 61.

[0427] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 63.

[0428] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 65.

[0429] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 67.

[0430] In some embodiments, Fab includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 69.

[0431] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 51.

[0432] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0433] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 55.

[0434] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 57.

[0435] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 59.

[0436] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 61.

[0437] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 63.

[0438] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 65.

[0439] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 67.

[0440] In some embodiments, Fab includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 69.

[0441] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 51.

[0442] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 53.

[0443] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0444] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 57.

[0445] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 59.

[0446] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 61.

[0447] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 63.

[0448] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 65.

[0449] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 67.

[0450] In some embodiments, Fab includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 69.

[0451] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 51.

[0452] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 53.

[0453] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 55.

[0454] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0455] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 59.

[0456] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 61.

[0457] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 63.

[0458] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 65.

[0459] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 67.

[0460] In some embodiments, Fab includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 69.

[0461] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 51.

[0462] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 53.

[0463] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 55.

[0464] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 57.

[0465] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0466] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 61.

[0467] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 63.

[0468] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65.

[0469] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 67.

[0470] In some embodiments, Fab includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 69.

[0471] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 51.

[0472] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 53.

[0473] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 55.

[0474] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 57.

[0475] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 59.

[0476] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0477] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 63.

[0478] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 65.

[0479] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 67.

[0480] In some embodiments, Fab includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 69.

[0481] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 51.

[0482] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 53.

[0483] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 55.

[0484] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 57.

[0485] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 59.

[0486] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 61.

[0487] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0488] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 65.

[0489] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67.

[0490] In some embodiments, Fab includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69.

[0491] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 51.

[0492] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 53.

[0493] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 55.

[0494] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 57.

[0495] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 59.

[0496] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 61.

[0497] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 63.

[0498] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0499] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 67.

[0500] In some embodiments, Fab includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 69.

[0501] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 51.

[0502] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 53.

[0503] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 55.

[0504] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 57.

[0505] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 59.

[0506] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 61.

[0507] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 63.

[0508] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 65.

[0509] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0510] In some embodiments, Fab includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 69.

[0511] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 51.

[0512] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 53.

[0513] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 55.

[0514] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 57.

[0515] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 59.

[0516] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 61.

[0517] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 63.

[0518] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 65.

[0519] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 67.

[0520] In some embodiments, Fab includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0521] In some embodiments, F(ab')2 is Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 52, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 53, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 54, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 55, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 56, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 57, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 58, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 59, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 60, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 61, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 62, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 63, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 64, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 65, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 66, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 67, or This includes HCDR1, HCDR2, and HCDR3 of VH in sequence number 68, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 69.

[0522] In some embodiments, F(ab')2 is Sequence numbers 70, 71, 72, 88, 89, and 90, respectively. Sequence numbers 73, 71, 74, 91, 89, and 92, respectively. Sequence numbers 75, 76, 77, 93, 89, and 94, respectively. Sequence numbers 78, 79, 80, 95, 89, and 96, respectively. Sequence numbers 81, 82, 83, 97, 89, and 98, respectively Sequence numbers 78, 71, 84, 99, 89, and 100, respectively. Sequence numbers 78, 71, 84, 101, 89, and 100, respectively. Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or This includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0523] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 70, 71, 72, 88, 89, and 90, respectively.

[0524] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 73, 71, 74, 91, 89, and 92, respectively.

[0525] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 75, 76, 77, 93, 89, and 94, respectively.

[0526] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 79, 80, 95, 89, and 96, respectively.

[0527] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 81, 82, 83, 97, 89, and 98, respectively.

[0528] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 99, 89, and 100, respectively.

[0529] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 101, 89, and 100, respectively.

[0530] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 85, 86, 87, 102, 103, and 104, respectively.

[0531] In some embodiments, F(ab')2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0532] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0533] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0534] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0535] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0536] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0537] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0538] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0539] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0540] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0541] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0542] In some embodiments, F(ab')2 includes VH of sequence numbers 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of sequence numbers 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0543] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0544] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 53.

[0545] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 55.

[0546] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 57.

[0547] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 59.

[0548] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 61.

[0549] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 63.

[0550] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 65.

[0551] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 67.

[0552] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 69.

[0553] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 51.

[0554] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0555] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 55.

[0556] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 57.

[0557] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 59.

[0558] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 61.

[0559] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 63.

[0560] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 65.

[0561] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 67.

[0562] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 69.

[0563] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 51.

[0564] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 53.

[0565] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0566] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 57.

[0567] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 59.

[0568] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 61.

[0569] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 63.

[0570] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 65.

[0571] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 67.

[0572] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 69.

[0573] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 51.

[0574] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 53.

[0575] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 55.

[0576] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0577] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 59.

[0578] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 61.

[0579] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 63.

[0580] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 65.

[0581] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 67.

[0582] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 69.

[0583] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 51.

[0584] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 53.

[0585] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 55.

[0586] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 57.

[0587] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0588] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 61.

[0589] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 63.

[0590] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65.

[0591] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 67.

[0592] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 69.

[0593] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 51.

[0594] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 53.

[0595] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 55.

[0596] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 57.

[0597] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 59.

[0598] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0599] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 63.

[0600] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 65.

[0601] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 67.

[0602] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 69.

[0603] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 51.

[0604] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 53.

[0605] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 55.

[0606] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 57.

[0607] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 59.

[0608] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 61.

[0609] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0610] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 65.

[0611] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67.

[0612] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69.

[0613] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 51.

[0614] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 53.

[0615] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 55.

[0616] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 57.

[0617] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 59.

[0618] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 61.

[0619] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 63.

[0620] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0621] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 67.

[0622] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 69.

[0623] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 51.

[0624] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 53.

[0625] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 55.

[0626] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 57.

[0627] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 59.

[0628] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 61.

[0629] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 63.

[0630] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 65.

[0631] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0632] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 69.

[0633] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 51.

[0634] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 53.

[0635] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 55.

[0636] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 57.

[0637] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 59.

[0638] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 61.

[0639] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 63.

[0640] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 65.

[0641] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 67.

[0642] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0643] In some embodiments, Fv is Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 52, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 53, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 54, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 55, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 56, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 57, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 58, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 59, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 60, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 61, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 62, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 63, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 64, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 65, or HCDR1, HCDR2, and HCDR3 of VH in sequence number 66, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 67, or This includes HCDR1, HCDR2, and HCDR3 of VH in sequence number 68, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 69.

[0644] In some embodiments, Fv is Sequence numbers 70, 71, 72, 88, 89, and 90, respectively. Sequence numbers 73, 71, 74, 91, 89, and 92, respectively. Sequence numbers 75, 76, 77, 93, 89, and 94, respectively. Sequence numbers 78, 79, 80, 95, 89, and 96, respectively. Sequence numbers 81, 82, 83, 97, 89, and 98, respectively Sequence numbers 78, 71, 84, 99, 89, and 100, respectively. Sequence numbers 78, 71, 84, 101, 89, and 100, respectively. Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or This includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0645] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 70, 71, 72, 88, 89, and 90, respectively.

[0646] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 73, 71, 74, 91, 89, and 92, respectively.

[0647] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 75, 76, 77, 93, 89, and 94, respectively.

[0648] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 79, 80, 95, 89, and 96, respectively.

[0649] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 81, 82, 83, 97, 89, and 98, respectively.

[0650] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 99, 89, and 100.

[0651] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 78, 71, 84, 101, 89, and 100.

[0652] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 85, 86, 87, 102, 103, and 104, respectively.

[0653] In some embodiments, Fv includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0654] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0655] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0656] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0657] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0658] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0659] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0660] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0661] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0662] In some embodiments, Fv includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0663] In some embodiments, Fv includes VH of sequence number 68 and VL of sequence number 69.

[0664] In some embodiments, Fv includes VH of sequence numbers 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of sequence numbers 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0665] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0666] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 53.

[0667] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 55.

[0668] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 57.

[0669] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 59.

[0670] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 61.

[0671] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 63.

[0672] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 65.

[0673] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 67.

[0674] In some embodiments, Fv includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 69.

[0675] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 51.

[0676] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0677] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 55.

[0678] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 57.

[0679] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 59.

[0680] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 61.

[0681] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 63.

[0682] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 65.

[0683] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 67.

[0684] In some embodiments, Fv includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 69.

[0685] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 51.

[0686] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 53.

[0687] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0688] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 57.

[0689] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 59.

[0690] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 61.

[0691] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 63.

[0692] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 65.

[0693] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 67.

[0694] In some embodiments, Fv includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 69.

[0695] In some embodiments, Fv includes VH of sequence number 56 and VL of sequence number 51.

[0696] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 53.

[0697] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 55.

[0698] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0699] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 59.

[0700] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 61.

[0701] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 63.

[0702] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 65.

[0703] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 67.

[0704] In some embodiments, Fv includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 69.

[0705] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 51.

[0706] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 53.

[0707] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 55.

[0708] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 57.

[0709] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0710] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 61.

[0711] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 63.

[0712] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65.

[0713] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 67.

[0714] In some embodiments, Fv includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 69.

[0715] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 51.

[0716] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 53.

[0717] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 55.

[0718] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 57.

[0719] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 59.

[0720] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0721] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 63.

[0722] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 65.

[0723] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 67.

[0724] In some embodiments, Fv includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 69.

[0725] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 51.

[0726] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 53.

[0727] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 55.

[0728] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 57.

[0729] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 59.

[0730] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 61.

[0731] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0732] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 65.

[0733] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67.

[0734] In some embodiments, Fv includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 69.

[0735] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 51.

[0736] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 53.

[0737] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 55.

[0738] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 57.

[0739] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 59.

[0740] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 61.

[0741] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 63.

[0742] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0743] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 67.

[0744] In some embodiments, Fv includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 69.

[0745] In some embodiments, Fv includes VH of sequence number 66 and VL of sequence number 51.

[0746] In some embodiments, Fv includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 53.

[0747] In some embodiments, Fv includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 55.

[0748] In some embodiments, Fv includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 57.

[0749] In some embodiments, Fv includes VH of sequence number 66 and VL of sequence number 59.

[0750] In some embodiments, Fv includes VH of sequence number 66 and VL of sequence number 61.

[0751] In some embodiments, Fv includes VH of sequence number 66 and VL of sequence number 63.

[0752] In some embodiments, Fv includes VH of sequence number 66 and VL of sequence number 65.

[0753] In some embodiments, Fv includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0754] In some embodiments, Fv includes VH of sequence number 66 and VL of sequence number 69.

[0755] In some embodiments, Fv includes VH of sequence number 68 and VL of sequence number 51.

[0756] In some embodiments, Fv includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 53.

[0757] In some embodiments, Fv includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 55.

[0758] In some embodiments, Fv includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 57.

[0759] In some embodiments, Fv includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 59.

[0760] In some embodiments, Fv includes VH of sequence number 68 and VL of sequence number 61.

[0761] In some embodiments, Fv includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 63.

[0762] In some embodiments, Fv includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 65.

[0763] In some embodiments, Fv includes VH of sequence number 68 and VL of sequence number 67.

[0764] In some embodiments, Fv includes VH of sequence number 68 and VL of sequence number 69.

[0765] In some embodiments, Fd includes VH of sequence number 50.

[0766] In some embodiments, Fd includes VH of sequence number 52.

[0767] In some embodiments, Fd includes VH of sequence number 54.

[0768] In some embodiments, Fd includes VH of sequence number 56.

[0769] In some embodiments, Fd includes VH of sequence number 58.

[0770] In some embodiments, Fd includes VH of sequence number 60.

[0771] In some embodiments, Fd includes VH of sequence number 62.

[0772] In some embodiments, Fd includes VH of sequence number 64.

[0773] In some embodiments, Fd includes VH of sequence number 66.

[0774] In some embodiments, Fd includes VH of sequence number 68.

[0775] Homologous antigen-binding domains and antigen-binding domains with conservative substitutions Variants of the antigen-binding domain that binds to HLA-G are within the scope of this disclosure. For example, a variant may contain 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, or 29 amino acid substitutions in the antigen-binding domain that binds to HLA-G, insofar as it retains or has improved functional properties compared to the parent antigen-binding domain. In some embodiments, sequence identity may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to the antigen-binding domain that binds to HLA-G of this disclosure. In some embodiments, the diversity resides within the framework domain. In some embodiments, variants are generated by conservative substitution.

[0776] For example, the antigen-binding domain that binds to HLA-G may include substitutions at the residue positions of E1Q, L5Q, E6Q, S71P, E1Q, L5Q, E6Q, and S71P in VH (residue numbering according to MHGB688-VH in SEQ ID NO: 52) and K30E and G66V in VL (residue numbering according to MHGB688-VL in SEQ ID NO: 53). Conservative substitutions can be made at any designated position, and the resulting variants of the antigen-binding domain that bind to HLA-G are tested for desired properties using the assays described herein.

[0777] Also, VH of sequence number 50 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, or An antigen-binding domain that binds to HLA-G is also provided, which includes VH and VL that are at least 80% identical to VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0778] In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%. In some embodiments, the identity is 94%. In some embodiments, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

[0779] The identity percentage between two arrays is a function of the number of identical positions shared by the arrays, taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap (i.e., identity % = number of identical positions / total number of positions × 100).

[0780] The percentage of identity between two amino acid sequences can be determined using the algorithm by E. Meyers and W. Miller (Comput Appl Biosci 4:11-17 (1988)), which is incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage of identity between two amino acid sequences can also be determined using the algorithm by Needleman and Wunsch (J Mol Biol 48:444-453 (1970)), which is incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, gap weightings 16, 14, 12, 10, 8, 6, or 4, and length weightings 1, 2, 3, 4, 5, or 6.

[0781] In some embodiments, variants of the antigen-binding domain that bind to HLA-G include one or two conservative substitutions in any of the CDR regions while retaining the desired functional properties of the parent antigen-binding fragment that binds to HLA-G.

[0782] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody, including amino acid modifications. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is a substitution in which an amino acid is replaced by an amino acid residue with a similar side chain. The families of amino acid residues with similar side chains are clearly defined and include amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), non-charged side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), β-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions in the antibodies of the present invention can be carried out by known methods, such as PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, a library of variants may be generated, for example, using random codons (NNK) or non-random codons (e.g., DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)). The resulting variants can be tested for their characteristics using the assays described herein.

[0783] Method for generating antigen-binding fragments that bind to HLA-G. The HLA-G-binding antigen-binding domains provided in this disclosure can be generated using a variety of techniques. For example, the Kohler and Milstein hybridoma method can be used to identify VH / VL pairs that bind to HLA-G. In the hybridoma method, mice, or other host animals such as hamsters, rats, or chickens, are immunized with human and / or cynomolgus monkey HLA-G, and then spleen cells derived from the immunized animals are fused with myeloma cells using standard methods to form hybridoma cells. Colonies arising from a single immortalized hybridoma cell can be screened for the generation of antibodies containing antigen-binding domains that bind to HLA-G with desired properties such as binding specificity, cross-reactivity or lack thereof, affinity for the antigen, and any desired functionality.

[0784] Antigen-binding domains that bind to HLA-G produced by immunizing non-human animals may be humanized. Exemplary humanization techniques, including the selection of a human acceptor framework, include CDR transplantation (U.S. Patent No. 5,225,539), SDR transplantation (U.S. Patent No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28:489-499), specificity-determining residue resurfacing (U.S. Patent Application Publication No. 2010 / 0261620), human framework adaptation (U.S. Patent No. 8,748,356), or hyperhumanization (U.S. Patent No. 7,709,226). These methods involve transplanting a CDR or a subset of CDR residues from a parent antibody into a human framework that can be selected based on overall homology to the parent framework, based on similarity in CDR length, identity of canonical structure, or a combination thereof.

[0785] The humanized antigen-binding domain may be further optimized to improve its selectivity or affinity for a desired antigen by incorporating altered framework-supporting residues (reverse mutations) to maintain binding affinity, or by introducing diversity into either the CDR, for example, to improve the affinity of the antigen-binding domain, using techniques such as those described in International Publication Nos. 1090 / 007861 and 1992 / 22653.

[0786] Using transgenic animals such as mice, rats, or chickens that possess a human immunoglobulin (Ig) locus in their own genome, antigen-binding fragments that bind to HLA-G can be generated, as described, for example, in U.S. Patent No. 6,150,584, International Publication No. 1999 / 45962, 2002 / 066630, 2002 / 43478, 2002 / 043478, and 1990 / 04036. The endogenous immunoglobulin locus of such animals may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the animal genome using homologous or non-homologous recombination, using a transchromosome, or using a minigene. Companies such as Regeneron (http: / / _www_regeneron_com), Harbour Antibodies (http: / / _www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc_net), KyMab (http: / / _www_kymab_com), Trianni (http: / / _www.trianni_com), and Ablexis (http: / / _www_ablexis_com) may be using the above technologies to provide human antibodies that target selected antigens.

[0787] Antigen-binding domains that bind to HLA-G can be selected from phage display libraries in which phages are engineered to express human immunoglobulin or a portion thereof, such as Fab, single-chain antibodies (scFv), or unpaired or paired antibody variable regions. Antigen-binding domains that bind to HLA-G can be isolated from phage display libraries that express antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coated proteins, for example, as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 09 / 085462. The libraries may be screened for phage binding to human and / or cynomolgus monkey HLA-G, positive clones obtained may be further characterized, Fab may be isolated from clone lysates and converted to scFv or other components of the antigen-binding fragment.

[0788] The preparation of immunogenic antigens and the expression and generation of the antigen-binding domains of this disclosure can be carried out using any preferred technique, such as recombinant protein synthesis. The immunogenic antigen may be administered to an animal in the form of a purified protein or a protein mixture containing whole cells or cell or tissue extracts, or the antigen may be de novoly formed in the animal's body from nucleic acids encoding the antigen or a portion thereof.

[0789] Conjugation to the half-life extension portion The antigen-binding domain that binds to HLA-G in this disclosure may be conjugated to a half-life extension region. Exemplary half-life extension regions include albumin, albumin variants, albumin-binding proteins and / or domains, transferrin and its fragments and analogs, immunoglobulin (Ig) or its fragments, such as the Fc region. The amino acid sequences of the aforementioned half-life extension regions are known. Ig or its fragments include all isotypes, namely IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0790] Additional half-life extension moieties that can be conjugated to the antigen-binding domain that binds to the HLA-G of this disclosure include polyethylene glycol (PEG) molecules, e.g., PEG5000 or PEG20000, fatty acids and fatty acid esters of different chain lengths, e.g., laurate, myristic acid, stearate, arachidic acid, behenate, oleate, arachidonic acid, octanodioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, and carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides), for desired properties. These moieties may be directly fused to the antigen-binding domain that binds to the HLA-G of this disclosure and can be generated by standard cloning and expression techniques. Alternatively, the moieties may be conjugated to the recombinant antigen-binding domain that binds to the HLA-G of this disclosure using well-known chemical coupling methods.

[0791] For example, the pegyl moiety can be conjugated to the antigen-binding domain of the HLA-G of the present disclosure by incorporating a cysteine ​​residue into the C-terminus of the antigen-binding domain of the present disclosure, or by manipulating it to a residue position facing away from the HLA-G binding site, and then using a well-known method to conjugate a pegyl group to the cysteine.

[0792] In some embodiments, the antigen-binding fragment that binds to HLA-G is conjugated to the half-life extension portion.

[0793] In some embodiments, the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, the Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol. In some embodiments, the half-life extension portion is the Ig constant region.

[0794] In some embodiments, the half-life extension portion is Ig.

[0795] In some embodiments, the half-life extension portion is a fragment of Ig.

[0796] In some embodiments, the half-life extension portion is the Ig steady-state region.

[0797] In some embodiments, the half-life extension portion is a fragment of the Ig steady-state region.

[0798] In some embodiments, the half-life extension portion is the Fc region.

[0799] In some embodiments, the half-life extension portion is albumin.

[0800] In some embodiments, the half-life extension portion is the albumin-binding domain.

[0801] In some embodiments, the half-life extension portion is transferrin.

[0802] In some embodiments, the half-life extension portion is polyethylene glycol.

[0803] The antigen-binding domains that bind to HLA-G, which are conjugated to the half-life extension portion, can be evaluated for their pharmacokinetic properties using known in vivo models.

[0804] Conjugation to the constant region of immunoglobulin (Ig) or fragments of the constant region of Ig The HLA-G-binding antigen-binding domains of this disclosure can be conjugated to an Ig constant region or fragment of an Ig constant region to confer antibody-like properties, including Fc effector function (C1q binding), complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or downregulation of cell surface receptors (e.g., B cell receptors (BCRs)). The Ig constant region or fragment of an Ig constant region can also function as a half-life extension portion, as discussed herein. The HLA-G-binding antigen-binding domains of this disclosure can be manipulated using standard methods to form conventional full-length antibodies. Full-length antibodies containing the HLA-G-binding antigen-binding domain may be further manipulated as described herein.

[0805] The constant region of the immunoglobulin heavy chain consists of subdomains CH1, hinge, CH2, and CH3. In residue numbering according to the EU index, the CH1 domain in the heavy chain extends from residues A118 to V215, the CH2 domain from A231 to K340, and the CH3 domain from G341 to K447. In some cases, G341 is referred to as the CH2 domain residue. The hinge is generally defined as containing E216 and terminating at P230 in human IgG1. The Ig Fc region includes at least the CH2 and CH3 domains of the Ig constant region and therefore includes at least the region from approximately A231 to K447 of the Ig heavy chain constant region.

[0806] The present invention also provides an antigen-binding domain that binds to an immunoglobulin (Ig) constant region or a fragment of the Ig constant region conjugated to an HLA-G.

[0807] In some embodiments, the Ig steady-state region is the heavy chain steady-state region.

[0808] In some embodiments, the Ig steady-state region is the light chain steady-state region.

[0809] In some embodiments, the Ig steady-state region fragment includes an Fc region.

[0810] In some embodiments, the Ig constant region fragment includes a CH2 domain.

[0811] In some embodiments, the Ig constant region fragment includes a CH3 domain.

[0812] In some embodiments, the Ig constant region fragment includes a CH2 domain and a CH3 domain.

[0813] In some embodiments, the Ig constant region fragment includes at least a portion of the hinge, the CH2 domain, and the CH3 domain. The portion of the hinge refers to one or more amino acid residues of the Ig hinge.

[0814] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0815] In some embodiments, the antigen-binding domain that binds to HLA-G is conjugated to the N-terminus of the Ig constant region or a fragment of the Ig constant region.

[0816] In some embodiments, the antigen-binding domain that binds to HLA-G is conjugated to the C-terminus of the Ig constant region or a fragment of the Ig constant region.

[0817] In some embodiments, the antigen-binding domain that binds to HLA-G is conjugated to the Ig constant region or a fragment of the Ig constant region via a second linker (L2).

[0818] In some embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0819] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 8.

[0820] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 9.

[0821] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 10.

[0822] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 11.

[0823] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 12.

[0824] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 13.

[0825] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 14.

[0826] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 15.

[0827] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 16.

[0828] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 17.

[0829] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 18.

[0830] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 20.

[0831] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 21.

[0832] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 22.

[0833] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 23.

[0834] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 24.

[0835] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 25.

[0836] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 26.

[0837] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 27.

[0838] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 28.

[0839] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 29.

[0840] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 30.

[0841] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 31.

[0842] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 32.

[0843] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 33.

[0844] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 34.

[0845] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 35.

[0846] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 36.

[0847] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 37.

[0848] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 38.

[0849] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 39.

[0850] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 40.

[0851] Antigen-binding domains conjugated to the Ig constant region or fragments of the Ig constant region of the present disclosure that bind to HLA-G can be evaluated for their functionality using several known assays. Binding to HLA-G can be evaluated using the methods described herein. Altered properties conferred by the Ig constant region or fragments of the Ig constant region, such as the Fc region, can be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII, or FcRn receptors, or using cell-based assays that measure ADCC, CDC, or ADCP, for example.

[0852] ADCC can be evaluated using an in vitro assay that uses HLA-G expressing cells as target cells and NK cells as effector cells. Cell lysis can be detected by the release of a label (e.g., radioactive substrate, fluorescent dye, or native intracellular protein) from the lysed cells. In an exemplary assay, target cells are used in a ratio of one target cell to four effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and a test antibody. Cell lysis is measured by incubating the sample for 2 hours and measuring the BATDA released into the supernatant. Data are normalized to the maximum cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich), and the minimum control is determined by the spontaneous release of BATDA from target cells in the absence of any antibody.

[0853] ADCP can be evaluated by using monocyte-derived macrophages as effector cells and any HLA-G expressing cells engineered to express GFP or other labeled molecules as target cells. In an exemplary assay, the effector:target cell ratio may be, for example, 4:1. Effector cells may be incubated with target cells for 4 hours with or without the antibodies of the present invention. After incubation, the cells can be detached using actase. Macrophages can be identified using fluorescently labeled anti-CD11b and anti-CD14 antibodies, and the rate of phagocytosis can be determined using standard methods for CD11 + CD14 + This can be determined based on the GFP fluorescence percentage in macrophages.

[0854] For example, the CDC of cells is 1 × 10⁶ Daudi cells in RPMI-B (RPMI supplemented with 1% BSA). 5The reaction can be measured by seeding cells per well (50 μL / well), adding 50 μL of test protein to the well at a final concentration of 0–100 μg / mL, incubating the reaction at room temperature for 15 minutes, adding 11 μL of pooled human serum to the well, and incubating the reaction at 37°C for 45 minutes. The percentage of lysed cells (%) can be detected as the percentage of propidium iodide-stained cells in the FACS assay using a standard method.

[0855] Proteins comprising an antigen-binding domain that binds to HLA-G as disclosed herein The HLA-G-binding antigen-binding domains of this disclosure can be engineered into monospecific or multispecific proteins of various designs using standard methods.

[0856] This disclosure also provides a single-specific protein comprising an isolated antigen-binding domain that binds to the HLA-G of this disclosure.

[0857] In some embodiments, the single-specific protein is an antibody.

[0858] This disclosure also provides a multispecific protein comprising an antigen-binding domain that binds to the HLA-G of this disclosure.

[0859] In some embodiments, the multispecific protein is bispecific.

[0860] In some embodiments, the multispecific protein is triple specific.

[0861] In some embodiments, the multispecific protein is quadruplespecific.

[0862] In some embodiments, the multispecific protein is monovalent in terms of binding to HLA-G.

[0863] In some embodiments, the multispecific protein is divalent with respect to binding to HLA-G.

[0864] This disclosure also provides an isolated multispecific protein comprising a first antigen-binding domain that binds to HLA-G and a second antigen-binding domain that binds to a lymphocyte antigen.

[0865] In some embodiments, the lymphocyte antigen is a T cell antigen.

[0866] In some embodiments, the T cell antigen is CD8 + It is a T cell antigen.

[0867] In some embodiments, the lymphocyte antigen is the NK cell antigen.

[0868] In some embodiments, the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

[0869] In some embodiments, the lymphocyte antigen is CD3ε.

[0870] In some embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to the lymphocyte antigen includes scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0871] In some embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens comprises Fab.

[0872] In some embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens includes F(ab')2.

[0873] In some embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens includes VHH.

[0874] In some embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens includes Fv.

[0875] In some embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens includes Fd.

[0876] In some embodiments, the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to lymphocyte antigens includes scFv.

[0877] In some embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus.

[0878] In some embodiments, L1 contains approximately 5 to 50 amino acids.

[0879] In some embodiments, L1 contains approximately 5 to 40 amino acids.

[0880] In some embodiments, L1 contains approximately 10 to 30 amino acids.

[0881] In some embodiments, L1 contains approximately 10 to 20 amino acids.

[0882] In some embodiments, L1 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0883] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 8.

[0884] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 9.

[0885] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 10.

[0886] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 11.

[0887] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 12.

[0888] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 13.

[0889] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 14.

[0890] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 15.

[0891] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 16.

[0892] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 17.

[0893] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 18.

[0894] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 19.

[0895] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 20.

[0896] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 21.

[0897] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 22.

[0898] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 23.

[0899] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 24.

[0900] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 25.

[0901] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 26.

[0902] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 27.

[0903] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 28.

[0904] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 29.

[0905] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 30.

[0906] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 31.

[0907] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 32.

[0908] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 33.

[0909] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 34.

[0910] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 35.

[0911] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 36.

[0912] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 37.

[0913] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 38.

[0914] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 39.

[0915] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 40.

[0916] In some embodiments, the first antigen-binding domain that binds to HLA-G includes HCDR1 of SEQ ID NO: 70, 73, 75, 78, 81, or 85, HCDR2 of SEQ ID NO: 71, 76, 79, 82, or 86, HCDR3 of SEQ ID NO: 72, 74, 77, 80, 83, 84, or 87, LCDR1 of SEQ ID NO: 88, 91, 93, 95, 97, 99, 101, or 102, LCDR2 of SEQ ID NO: 89 or 103, and LCDR3 of SEQ ID NO: 90, 92, 94, 96, 98, 100, or 104.

[0917] In some embodiments, the first antigen-binding domain that binds to HLA-G is Sequence numbers 70, 71, 72, 88, 89, and 90, respectively. Sequence numbers 73, 71, 74, 91, 89, and 92, respectively. Sequence numbers 75, 76, 77, 93, 89, and 94, respectively. Sequence numbers 78, 79, 80, 95, 89, and 96, respectively. Sequence numbers 81, 82, 83, 97, 89, and 98, respectively Sequence numbers 78, 71, 84, 99, 89, and 100, respectively. Sequence numbers 78, 71, 84, 101, 89, and 100, respectively. Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or This includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

[0918] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51.

[0919] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53.

[0920] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55.

[0921] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57.

[0922] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59.

[0923] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

[0924] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

[0925] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

[0926] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

[0927] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0928] In some embodiments, the first antigen-binding domain that binds to HLA-G includes VH of SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68, and VL of SEQ ID NOs. 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0929] In some embodiments, the first antigen-binding domain that binds to HLA-G is VH of sequence number 50 and VL of sequence number 51, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 53, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 55, VH of sequence number 50 and VL of sequence number 57, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 59 VH of SEQ ID NO: 50 and VL of SEQ ID NO: 61, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 63, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 65 VH of sequence number 50 and VL of sequence number 67, VH of SEQ ID NO: 50 and VL of SEQ ID NO: 69 VH of sequence number 52 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of SEQ ID NO: 52 and VL of SEQ ID NO: 55 VH of sequence number 52 and VL of sequence number 57, VH of sequence number 52 and VL of sequence number 59, VH of sequence number 52 and VL of sequence number 61, VH of sequence number 52 and VL of sequence number 63, VH of SEQ ID NO: 52 and VL of SEQ ID NO: 65, VH of sequence number 52 and VL of sequence number 67, VH of sequence number 52 and VL of sequence number 69, VH of sequence number 54 and VL of sequence number 51, VH of sequence number 54 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 54 and VL of sequence number 57, VH of sequence number 54 and VL of sequence number 59, VH of sequence number 54 and VL of sequence number 61, VH of sequence number 54 and VL of sequence number 63, VH of SEQ ID NO: 54 and VL of SEQ ID NO: 65 VH of sequence number 54 and VL of sequence number 67, VH of sequence number 54 and VL of sequence number 69, VH of sequence number 56 and VL of sequence number 51, VH of sequence number 56 and VL of sequence number 53, VH of sequence number 56 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 56 and VL of sequence number 59, VH of sequence number 56 and VL of sequence number 61, VH of sequence number 56 and VL of sequence number 63, VH of sequence number 56 and VL of sequence number 65, VH of sequence number 56 and VL of sequence number 67, VH of sequence number 56 and VL of sequence number 69, VH of sequence number 58 and VL of sequence number 51, VH of sequence number 58 and VL of sequence number 53, VH of SEQ ID NO: 58 and VL of SEQ ID NO: 55 VH of sequence number 58 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 58 and VL of sequence number 61, VH of sequence number 58 and VL of sequence number 63, VH of SEQ ID NO: 58 and VL of SEQ ID NO: 65, VH of sequence number 58 and VL of sequence number 67, VH of sequence number 58 and VL of sequence number 69, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 51, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 53, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 55, VH of sequence number 60 and VL of sequence number 57, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 60 and VL of sequence number 63, VH of SEQ ID NO: 60 and VL of SEQ ID NO: 65 VH of sequence number 60 and VL of sequence number 67, VH of sequence number 60 and VL of sequence number 69, VH of sequence number 62 and VL of sequence number 51, VH of sequence number 62 and VL of sequence number 53, VH of sequence number 62 and VL of sequence number 55, VH of sequence number 62 and VL of sequence number 57, VH of sequence number 62 and VL of sequence number 59, VH of sequence number 62 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 62 and VL of sequence number 65, VH of sequence number 62 and VL of sequence number 67, VH of sequence number 62 and VL of sequence number 69, VH of sequence number 64 and VL of sequence number 51, VH of sequence number 64 and VL of sequence number 53, VH of sequence number 64 and VL of sequence number 55, VH of sequence number 64 and VL of sequence number 57, VH of sequence number 64 and VL of sequence number 59, VH of sequence number 64 and VL of sequence number 61, VH of sequence number 64 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 64 and VL of sequence number 67, VH of sequence number 64 and VL of sequence number 69, VH of sequence number 66 and VL of sequence number 51, VH of sequence number 66 and VL of sequence number 53, VH of sequence number 66 and VL of sequence number 55, VH of sequence number 66 and VL of sequence number 57, VH of sequence number 66 and VL of sequence number 59, VH of sequence number 66 and VL of sequence number 61, VH of sequence number 66 and VL of sequence number 63, VH of sequence number 66 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, VH of sequence number 66 and VL of sequence number 69, VH of sequence number 68 and VL of sequence number 51, VH of sequence number 68 and VL of sequence number 53, VH of sequence number 68 and VL of sequence number 55, VH of sequence number 68 and VL of sequence number 57, VH of sequence number 68 and VL of sequence number 59, VH of sequence number 68 and VL of sequence number 61, VH of sequence number 68 and VL of sequence number 63, VH of sequence number 68 and VL of sequence number 65, VH of SEQ ID NO: 68 and VL of SEQ ID NO: 67, or Includes VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[0930] In some embodiments, the first antigen-binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[0931] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 248.

[0932] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 249.

[0933] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 250.

[0934] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 251.

[0935] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 252.

[0936] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 253.

[0937] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 254.

[0938] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 255.

[0939] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 256.

[0940] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 257.

[0941] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 258.

[0942] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 259.

[0943] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 260.

[0944] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 261.

[0945] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 262.

[0946] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 263.

[0947] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 264.

[0948] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 265.

[0949] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 266.

[0950] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 267.

[0951] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 268.

[0952] In some embodiments, the first binding domain that binds to HLA-G includes the amino acid sequence of SEQ ID NO: 269.

[0953] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is HCDR1 (sequence number 361), HCDR2 (sequence number 362), HCDR3 (sequence number 363), LCDR1 (sequence number 367), LCDR2 (sequence number 368), and LCDR3 (sequence number 370), VH of sequence number 339, and VL of sequence numbers 340, 341, 342, 343, 344, or 345, HCDR1 of sequence number 364, HCDR2 of sequence number 365, HCDR3 of sequence number 366, LCDR1 of sequence number 371, LCDR2 of sequence number 372, and LCDR3 of sequence number 373, or Includes VH of sequence number 346 or 348 and VL of sequence number 347 or 349.

[0954] In some embodiments, a first antigen-binding domain that binds to HLA-G is conjugated to a first immunoglobulin (Ig) constant region or a fragment of a first Ig constant region, and / or a second antigen-binding domain that binds to a lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of a second Ig constant region.

[0955] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include an Fc region.

[0956] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH2 domain.

[0957] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH3 domain.

[0958] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH2 domain and a CH3 domain.

[0959] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include at least a portion of the hinge, a CH2 domain, and a CH3 domain.

[0960] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0961] In some embodiments, the multispecific protein further includes a second linker (L2) between a first antigen-binding domain that binds to HLA-G and a first Ig constant region or a fragment of the first Ig constant region, and between a second antigen-binding domain that binds to lymphocyte antigens and a second Ig constant region or a fragment of the second Ig constant region.

[0962] In some embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0963] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1, IgG2, IgG3, or IgG4 isotypes.

[0964] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1 isotypes.

[0965] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG2 isotypes.

[0966] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG3 isotypes.

[0967] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG4 isotypes.

[0968] The first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region may be further genetically modified as described herein.

[0969] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, contain at least one mutation resulting in reduced binding of the multispecific protein to FcγR.

[0970] In some embodiments, at least one mutation resulting in reduced binding of the multispecific protein to FcγR is found in L235A / D265S, F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G23 The group is selected from the following: 6 deletions / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, and residue numbering follows the EU index. In some embodiments, the L234A / L235A / D265S mutation is the one that results in reduced binding of multispecific proteins to FcγR.

[0971] In some embodiments, the first Ig constant region or fragment of the first Ig constant region, and the second Ig constant region or fragment of the second Ig constant region, contain at least one mutation that results in enhanced binding of the multispecific protein to the Fcγ receptor (FcγR).

[0972] In some embodiments, at least one mutation resulting in enhanced binding of the multispecific protein to FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, with residue numbering following the EU index.

[0973] In some embodiments, FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

[0974] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, include at least one mutation that modulates the half-life of the multispecific protein.

[0975] In some embodiments, at least one mutation that modulates the half-life of a multispecific protein is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, and residue numbering follows the EU index.

[0976] In some embodiments, the multispecific protein comprises at least one mutation in the CH3 domain of a first Ig constant region or a fragment of the first Ig constant region and / or at least one mutation in the CH3 domain of a second Ig constant region or a fragment of the second Ig constant region.

[0977] In some embodiments, at least one mutation in the CH3 domain of the first Ig constant region or the CH3 domain of a fragment of the first Ig constant region and / or at least one mutation in the CH3 domain of the second Ig constant region or the CH3 domain of a fragment of the second Ig constant region is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y40 The residues are selected from the group consisting of 7V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, and the residue numbering follows the EU index.

[0978] In some embodiments, the first Ig steady region or a fragment of the first Ig steady region and the second Ig steady region or a fragment of the second Ig steady region are modified as follows: L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig steady-state region, and L235A_D265S_T350V_T366L_K392L_T394W in the second Ig steady-state region, or This includes L235A_D265S_T350V_T366L_K392L_T394W in the first Ig steady-state region and L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig steady-state region.

[0979] Generation of multispecific proteins containing antigen-binding fragments that bind to HLA-G. The antigen-binding fragments that bind to HLA-G in this disclosure may be modified into multispecific antibodies, which are also included within the scope of the present invention.

[0980] The antigen-binding fragment that binds to HLA-G may be engineered into a full-length, multispecific antibody generated using Fab-arm exchange, by introducing substitutions into two monospecific bivalent antibodies within the Ig constant region CH3 domain to facilitate Fab-arm exchange in vitro. In this method, two monospecific bivalent antibodies are genetically engineered to have specific substitutions in the CH3 domain to promote heterodimer stability. These antibodies are incubated together under sufficiently reducing conditions for cysteine ​​in the hinge region to isomerize the disulfide bond, thereby generating a bispecific antibody via Fab-arm exchange. The incubation conditions can, optimally, be returned to non-reducing conditions. Typical reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation can be performed at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at a pH of 5 to 8, for example, pH 7.0 or pH 7.4, for at least 90 minutes.

[0981] Possible CH3 mutations include techniques such as knob-in-hole mutations (Genentech), electrostatic match mutations (Chugai, Amgen, NovoNordisk, Oncomed), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).

[0982] Knob-in-hole mutations, disclosed for example in International Publication No. 1996 / 027011, include interfacial mutations of the CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in a preferential interaction between the first and second CH3 regions. Exemplary CH3 region mutations that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0983] Heavy chain heterodimer formation can be facilitated by using electrostatic interactions by substituting a positively charged residue on the first CH3 region and a negatively charged residue on the second CH3 region, as described in U.S. Patent Application Publications 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532.

[0984] Other asymmetric mutations that can be used to promote heavy chain heterodimerization include L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T3 described in U.S. Patent Application Publication No. 2012 / 0149876 or 2013 / 0195849 (Zymeworks). These are 66L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0985] SEED body mutations, as described in U.S. Patent Application Publication No. 2007 / 0287170, involve the substitution of selected IgG residues with IgA residues to promote heavy chain heterodimerization.

[0986] Other exemplary variants that may be used include R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366W, and Y349C_T366W / S354C_T366S, as described in International Publication No. 2007 / 147901, International Publication No. 2011 / 143545, International Publication No. 2013 / 157954, International Publication No. 2013 / 096291, and U.S. Patent Application Publication No. 2018 / 0118849. 6S_L368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E_D282K_K322D / D239K_E240K_K292D, K392D_K409D / D356K_D399K.

[0987] Duobody® variants (Genmab) are disclosed, for example, in U.S. Patent No. 9,150,663 and U.S. Patent Application Publication No. 2014 / 0303356, and include variants such as F405L / K409R, wild-type / F405L_R409K, T350I_K370T_F405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH, and Y407LWQ / K409AGRH.

[0988] Further dual or multispecific structures that can incorporate an antigen-binding domain that binds to HLA-G include: Dual Variable Domain Immunoglobulin (DVD) (International Publication No. 2009 / 134776; DVD is a full-length antibody comprising a heavy chain having a VH1-linker-VH2-CH structure and a light chain having a VL1-linker-VL2-CL structure, with the linker being optional); structures containing various dimerization domains for conjugating two antibody arms with different specificities, e.g., leucine zipper or collagen dimerization domain (International Publication No. 2012 / 022811, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441); two or more domain antibodies (dAbs) conjugated together; diabodies; heavy-chain-only antibodies such as camelid antibodies and genetically modified camelid antibodies; Dual Targeting (DT)-Ig (GSK / Domantis); Two-in-one Antibody (Genentech), Cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche), ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion,Examples include Zymogenetics / BMS, Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), Dual (ScFv)2-Fab (National Research Center for Antibody Medicine--China), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech). Examples of ScFv antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (Affimed), dual-affinity retargeting technology (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), dual-target nanobodies (Ablynx), and dual-target heavy-chain-only domain antibodies.

[0989] The HLA-G-binding antigen-binding domain of this disclosure may also be engineered into a multispecific protein comprising three polypeptide chains. In such a design, at least one antigen-binding domain is in the form of an scFv. Examples of designs include designs 1-4 below (where "1" represents the first antigen-binding domain, "2" represents the second antigen-binding domain, and "3" represents the third antigen-binding domain). Design 1: Chain A) scFv1-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 2: Chain A) scFv1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 3: Chain A) scFv1-CH1-hinge-CH2-CH3, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 Design 4: Chain A) CH2-CH3-scFv1, Chain B) VL2-CL, Chain C) VH2-CH1-hinge-CH2-CH3 CH3 operation is described in U.S. Patent Publication No. 2012 / 0149876 or U.S. Patent Publication No. 2013 / 0195849 (Zymeworks) as L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F40 Variations such as 5A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W may be incorporated into designs 1-4.

[0990] Isotype, allotype, and Fc gene manipulation The Ig constant region or fragments of the Ig constant region, such as the Fc region, present in the protein of this disclosure may be any allotype or isotype.

[0991] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG1 isotype.

[0992] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG2 isotype.

[0993] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG3 isotype.

[0994] In some embodiments, the Ig steady-state region or a fragment of the Ig steady-state region is an IgG4 isotype.

[0995] The Ig constant region or fragments of the Ig constant region can be any allotype. The allotype is not expected to affect the properties of the Ig constant region, such as binding or Fc-mediated effector function. The immunogenicity of therapeutic proteins containing fragments of the Ig constant region is associated with an increased risk of infusion reactions and a shortened therapeutic response (Baert et al., (2003) N Engl J Med 348:602-08). The extent to which therapeutic proteins containing fragments of the Ig constant region induce an immune response in the host may be determined to some extent by the allotype of the Ig constant region (Stickler et al., (2011) Genes and Immunity 12:213-21). The allotype of the Ig constant region is related to amino acid sequence mutations at specific positions in the antibody's constant region sequence. Table 3 shows the selected IgG1, IgG2, and IgG4 allotypes.

[0996] [Table 3]

[0997] C-terminal lysine (CTL) can be removed from the Ig constant region by endogenous circulating carboxypeptidase in the bloodstream (Cai et al., (2011) Biotechnol Bioeng 108:404-412). During manufacturing, extracellular Zn is used as described in U.S. Patent Application Publication No. 2014 / 0273092. 2+ EDTA, or EDTA-Fe 3+ By controlling the concentration of [the substance], CTL removal can be controlled to below the maximum level. The CTL content of a protein can be measured using known methods.

[0998] In some embodiments, the antigen-binding fragment conjugated to the Ig constant region and bound to HLA-G has a C-terminal lysine content of about 10% to about 90%. In some embodiments, the C-terminal lysine content is about 20% to about 80%. In some embodiments, the C-terminal lysine content is about 40% to about 70%. In some embodiments, the C-terminal lysine content is about 55% to about 70%. In some embodiments, the C-terminal lysine content is about 60%.

[0999] By introducing Fc region mutations into the antigen-binding domain that binds to the Ig constant region or a fragment of the Ig constant region conjugated to an HLA-G, the effector functions of ADCC, ADCP, and / or ADCP, as well as / or pharmacokinetic properties, can be regulated. This can be achieved by introducing mutations into Fc that regulate the binding of mutated Fc to activated FcγR (FcγRI, FcγRIIa, FcγRIII), inhibitory FcγRIIb, and / or FcRn.

[1000] In some embodiments, the antigen-binding domain that binds to an Ig constant region or a fragment of an Ig constant region conjugates to an HLA-G includes at least one mutation in the Ig constant region or fragment of an Ig constant region.

[1001] In some embodiments, at least one mutation is located in the Fc region.

[1002] In some embodiments, the antigen-binding domain that binds to an Ig constant region or an HLA-G conjugated to a fragment of an Ig constant region contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations in the Fc region.

[1003] In some embodiments, the antigen-binding domain that binds to an Ig constant region or an HLA-G conjugated to a fragment of the Ig constant region includes at least one mutation in the Fc region that modulates the binding of the antibody to FcRn.

[1004] Fc positions that can be mutated to regulate the half-life (e.g., binding to FcRn) include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that can be performed individually or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R. Exemplary mutations, either alone or in combination, that can be performed to increase the half-life of an antibody are the mutations M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, and T307A / E380A / N434A. Exemplary mutations, either alone or in combination, that can be performed to decrease the half-life are the mutations H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R.

[1005] In some embodiments, the antigen-binding domain that binds to the Ig constant region or an HLA-G conjugated to a fragment of the Ig constant region contains the M252Y / S254T / T256E mutation.

[1006] In some embodiments, an antigen-binding domain that binds to an Ig constant region or an HLA-G conjugated to a fragment of an Ig constant region contains at least one mutation in the Fc region that reduces the binding of the protein to the activated Fcγ receptor (FcγR) and / or reduces Fc effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[1007] Fc positions that can be mutated to reduce the binding of the protein to the activated FcγR, and subsequently to reduce its effector function, include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331, and 365. Exemplary mutations that can be added individually or in combination include the K214T, E233P, L234V, L234A, G236 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S, and P331S mutations in IgG1, IgG2, IgG3, or IgG4. Exemplary mutation combinations that result in reduced ADCC protein include L234A / L235A in IgG1, L234A / L235A / D265S in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, and K214T / E233P / L234V / L235A / G in IgG1. These are mutations in IgG2: 236 deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S. Alternatively, a hybrid IgG2 / 4 Fc domain, such as Fc having residues 117-260 derived from IgG2 and residues 261-447 derived from IgG4, may be used.

[1008] An example of a mutation that results in reduced CDC protein is the K322A mutation.

[1009] The well-known S228P mutation can be added to an IgG4 antibody to enhance the stability of IgG4.

[1010] In some embodiments, the antigen-binding domain that binds to an Ig constant region or an HLA-G conjugated to a fragment of an Ig constant region contains at least one mutation selected from the group consisting of deletions of K214T, E233P, L234V, L234A, G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, K322, A330S, and P331S.

[1011] In some embodiments, the antigen-binding domain that binds to the Ig constant region or an HLA-G conjugated to a fragment of the Ig constant region contains the L234A / L235A / D265S mutation.

[1012] In some embodiments, the antigen-binding domain that binds to the Ig constant region or an HLA-G conjugated to a fragment of the Ig constant region contains the L234A / L235A mutation.

[1013] In some embodiments, an antigen-binding domain that binds to an Ig constant region or an HLA-G conjugated to a fragment of an Ig constant region contains at least one mutation in the Fc region that enhances the binding of the protein to the Fcγ receptor (FcγR) and / or enhances Fc effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[1014] Fc positions that can be mutated to increase the binding of the protein to the activated FcγR and / or enhance Fc effector function include positions 236, 239, 243, 256, 290, 292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396, or 430 (residue numbering according to the EU index). Exemplary mutations that can be performed individually or in combination include G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T, and P396L. Exemplary mutation combinations that result in increased ADCC or ADCP proteins include S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E.

[1015] Fc positions that can be mutated to enhance CDC include positions 267, 268, 324, 326, 333, 345, and 430. Exemplary mutations that can occur individually or in combination are S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F, and E430T. Exemplary combinations of mutations that result in proteins with increased CDC are K326A / E333A, K326W / E333A, H268F / S324T, S267E / H268F, S267E / S324T, and S267E / H268F / S324T.

[1016] The specific mutations described herein are those observed when compared to the wild-type amino acid sequences of IgG1, IgG2, and IgG4, respectively, in sequence numbers 130, 131, and 132.

[1017] Sequence ID 486, wild-type IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAK GQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1018] Sequence ID 487, wild-type IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPSREEMTKN QVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

[1019] Sequence ID 488, wild-type IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK

[1020] The binding of antibodies to FcγR or FcRn can be evaluated using flow cytometry in cells engineered to express each receptor. In an exemplary binding assay, 2 × 10⁶ cells were used in a 96-well plate. 5Cells are seeded at a rate of 1 / well and blocked in BSA Stain Buffer (BD Biosciences, San Jose, USA) at 4°C for 30 minutes. Cells are incubated with the test antibody on ice at 4°C for 1.5 hours. After washing twice with BSA stain buffer, cells are incubated with R-PE labeled anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories) at 4°C for 45 minutes. Cells are washed twice with stain buffer and then resuspended in 150 μL of Stain Buffer containing a 1:200 dilution of DRAQ7 live / dead cell staining reagent (Cell Signaling Technology, Danvers, USA). PE and DRAQ7 signals from stained cells are detected using a Miltenyi MACSQuant flow cytometer (Miltenyi Biotec, Auburn, USA) with B2 and B4 channels, respectively. Live cells were gated out by DRAQ7 exclusion, and the geometric mean fluorescence signal was determined for at least 10,000 live cell events collected. FlowJo software (Tree Star) was used for the analysis. The data was plotted as the logarithm of antibody concentration against the mean fluorescence signal. Nonlinear regression analysis was performed.

[1021] Glycan gene manipulation The ability of an antigen-binding domain conjugated to an Ig constant region or fragment of an Ig constant region to mediate ADCC (Adverse Drug Continuity Coagulation) can be enhanced by manipulating the oligosaccharide component of the Ig constant region or fragment of an Ig constant region. Human IgG1 or IgG3 is N-glycosylated at Asn297, where the majority of the glycan is in the form of the known bifurcated G0, G0F, G1, G1F, G2, or G2F. Ig constant region-containing proteins that can be produced by unmanipulated CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from a bifurcated complex oligosaccharide bound to an antigen-binding domain conjugated to an Ig constant region or fragment of an Ig constant region enhances the protein's ADCC via improved FcγRIIIa binding without altering antigen binding or CDC activity.Such proteins can be achieved using different methods reported to lead to the successful expression of relatively high defucosylated immunoglobulins with branched complex-type Fc oligosaccharides, including: control of culture osmotic pressure (Konno et al., Cytotechnology 64(:249-65, 2012), application of variant CHO strain Lec13 as host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), application of variant CHO strain EB66 as host cell line (Olivier et al., MAbs;2(4):405-415, 2010;PMID:20562582), and application of rat hybridoma cell line YB2 / 0 as host cell line (Shinkawa et al., J Biol This could involve: Chem278:3466-3473,2003; introduction of specific small interfering RNA to the 1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908,2004); or co-expression of β-1,4-N-acetylglucosaminyltransferase III and kifunensin, a potent inhibitor of Golgi α-mannosidase II or α-mannosidase I (Ferrara et al., J Biol Chem 281:5032-5036,2006; Ferrara et al., Biotechnol Bioeng 93:851-861,2006; Xhou et al., Biotechnol Bioeng 99:652-65,2008).

[1022] In some embodiments, the antigen-binding domain that binds to an Ig constant region or a fragment of an Ig constant region of the Disclosure has a branched glycan structure having a fucose content of about 1% to about 15%, for example, about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the antigen-binding domain that binds to an Ig constant region or a fragment of an Ig constant region of the Disclosure has a glycan structure having a fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.

[1023] "Fucose content" refers to the amount of fucose monosaccharides in the sugar chain of Asn297. The relative amount of fucose is the ratio of the fucose-containing structure to the total sugar structure. These sugar structures can be determined by several methods, e.g., 1) the use of MALDI-TOF on N-glycosidase F-treated samples (e.g., complex, hybrid, and oligo- and high-mannose structures) as described in International Publication No. 2008 / 077546 2, 2) enzymatic release of Asn297 glycans, subsequent derivatization, and detection / quantification by HPLC (UPLC) and / or HPLC-MS (UPLC-MS) with fluorescence detection, and 3) cleavage between the first GlcNAc monosaccharide and the second GlcNAc monosaccharide, leaving the fucose attached to the first GlcNAc, Endo Intact protein analysis of natural or reduced mAbs with or without treatment of Asn297 glycan with or without S or other enzymes; 4) digestion of mAbs into constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), followed by separation, detection and quantification by HPLC-MS (UPLC-MS); 5) separation of mAb oligosaccharides from mAb proteins by specific enzymatic deglycosylation of Asn 297 with PNGase F. The oligosaccharides released in this manner can be separated and identified by various supplemental techniques that enable detailed characterization of the glycan structure by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, which involves labeling with fluorophores and comparing the measured mass with the theoretical mass; determination of the degree of sialylation by ion-exchange HPLC (GlycoSep C); separation and quantification of oligosaccharide types according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N); and separation and quantification of oligosaccharides by high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LIF).

[1024] As used herein, "low fucose" or "low fucose content" refers to an antigen-binding domain that binds to an Ig constant region or an HLA-G conjugated to a fragment of an Ig constant region having a fucose content of approximately 1% to 15%.

[1025] As used herein, "normal fucose" or "normal fucose content" refers to a state in which the antigen-binding domain conjugated to the Ig constant region or a fragment of the Ig constant region has a fucose content of more than approximately 50%, typically more than 80%, or more than 85%.

[1026] Anti-idiotype antibody The anti-idiotype antibody is an antibody that specifically binds to the antigen-binding domain that binds to HLA-G as disclosed herein.

[1027] The present invention also provides an anti-idiotype antibody that specifically binds to the antigen-binding domain of HLA-G according to the present disclosure.

[1028] The present invention also, VH of sequence number 50 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, or This invention provides anti-idiotype antibodies that specifically bind to the antigen-binding domain that binds to HLA-G, including VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[1029] An anti-idiotypic (Id) antibody is an antibody that recognizes an antigenic determinant (e.g., a paratope or CDR). Id antibodies may or may not block the antigen. Antigen-blocking Ids may be used to detect free antigen-binding domains in a sample (e.g., antigen-binding domains that bind to HLA-G as described herein). Non-blocking Ids can be used to detect all antibodies in a sample (free, partially bound to the antigen, or fully bound to the antigen). Id antibodies can be prepared by immunizing animals with an antibody that has been prepared as an anti-Id.

[1030] Furthermore, so-called anti-anti-Id antibodies can be generated by using an anti-Id antibody as an immunogen to induce an immune response in yet another animal. The anti-anti-Id may have the same epitope as the original antigen-binding domain that induced the anti-Id. Therefore, by using an antibody against the idiotype determinant of the antigen-binding domain, it is possible to identify other clones expressing the same specific antigen-binding domain. Anti-Id antibodies can be modified (thereby producing anti-Id antibody variants) and / or induced by any preferred technique, such as those described elsewhere in this specification.

[1031] Immunoconjugates The antigen-binding domains that bind to HLA-G, proteins containing antigen-binding domains that bind to HLA-G, or multispecific proteins containing antigen-binding domains that bind to HLA-G (collectively referred to herein as HLA-G-binding proteins) can be conjugated to heterologous molecules.

[1032] In some embodiments, the heterologous molecule is a detectable label or a cytotoxic agent.

[1033] The present invention also provides an antigen-binding domain that binds to an HLA-G conjugated with a detectable label.

[1034] The present invention also provides a protein comprising an antigen-binding domain that binds to an HLA-G conjugated with a detectable label.

[1035] The present invention also provides a multispecific protein comprising an antigen-binding domain that binds to HLA-G conjugated with a detectable label.

[1036] The present invention also provides an antigen-binding domain that binds to an HLA-G conjugated to a cytotoxic agent.

[1037] The present invention also provides a protein comprising an antigen-binding domain that binds to an HLA-G conjugated to a cytotoxic agent.

[1038] The present invention also provides a multispecific protein comprising an antigen-binding domain that binds to an HLA-G conjugated to a cytotoxic agent.

[1039] The HLA-G binding proteins of this disclosure can be used to direct therapeutic agents to HLA-G expressing cells, such as prostate cancer or breast cancer cells. Alternatively, HLA-G expressing cells can be targeted using the HLA-G binding proteins of this disclosure, which, after internalization, are conjugated to therapeutic agents intended to modify cellular function.

[1040] In some embodiments, the detectable label is also a cytotoxic agent.

[1041] The HLA-G binding proteins of this disclosure, conjugated to a detectable label, can be used to evaluate HLA-G expression in a variety of samples.

[1042] The detectable label comprises a composition that, when conjugated to the HLA-G binding protein of this disclosure, makes the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

[1043] Examples of detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron density reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescent quenchers, colored molecules, radioisotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or their fragments, polyhistidine, Ni 2+ Examples include Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatases, peroxidases, luciferases, electron donors / receptors, acridinium esters, and colorimetric substrates.

[1044] A detectable label may spontaneously emit a signal, for example, when the detectable label is a radioactive isotope. In other cases, a detectable label emits a signal as a result of being stimulated by an external field.

[1045] Exemplary radioactive isotopes may be γ-emitting, Auger-emitting, β-emitting, α-emitting, or positron-emitting radioactive isotopes. Exemplary radioactive isotopes include: 3 H, 11 C, 13 C, 15 N, 18 F, 19 F, 55 Co, 57 Co, 60 Co, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 68 Ga, 72 As, 75 Br, 86 Y, 89 Zr, 90 Sr, 94m Tc, 99m Tc, 115 In, 123 1. 124 1. 125 I, 131 1. 211 At, 212 Bi, 213 Bi,223 Ra, 226 Ra, 225 Ac, and 227 Ac is one example.

[1046] Exemplary metal atoms include metals with atomic numbers greater than 20, such as calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, bromine, krypton, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, iodine, xenon, cesium, barium, lanthanum, hafnium, tantalum, tungsten, and rhenium atoms. These atoms are osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, francium, radium, actinium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium.

[1047] In some embodiments, the metal atoms may be alkaline earth metals having an atomic number greater than 20.

[1048] In some embodiments, the metal atom may be a lanthanide.

[1049] In some embodiments, the metal atom may be an actinide.

[1050] In some embodiments, the metal atom can be a transition metal.

[1051] In some embodiments, the metal atom can be a base metal.

[1052] In some embodiments, the metal atoms can be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

[1053] In some embodiments, the metal atom can be a metal having an atomic number from 53 (i.e., iodine) to 83 (i.e., bismuth).

[1054] In some embodiments, the metal atom can be an atom suitable for magnetic resonance imaging.

[1055] The metal atom is Ba 2+ , Bi 3+ , Cs + , Ca 2+ , Cr 2+ , Cr 3+ , Cr 6+ , Co 2+ , Co 3+ , Cu + , Cu 2+ , Cu 3+ , Ga 3+ , Gd 3+ , Au + , Au 3+ , Fe 2+ , Fe 3+ , F 3+ , Pb 2+ , Mn 2+ , Mn 3+ , Mn 4+ , Mn 7+ , Hg 2+ , Ni 2+ , Ni 3+ , Ag + , Sr 2+ , Sn 2+ , Sn 4+ , and Zn 2+These can be metal ions in the form of +1, +2, or +3 oxidation states. The metal atoms may include metal oxides, such as iron oxide, manganese oxide, or gadolinium oxide.

[1056] Suitable dyes include, for example, any commercially available dye such as 5(6)-carboxyfluorescein, IRDye 680RD maleimide, or IRDye 800CW, or ruthenium polypyridyl dye.

[1057] Suitable fluorophores include fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594, Alexa647), near-infrared (NIR) (700-900 nm) fluorescent dyes, as well as carbocyanin and aminostyryl dyes.

[1058] An antigen-binding domain that binds to HLA-G, conjugated with a detectable label, can be used as a contrast agent.

[1059] Proteins containing an antigen-binding domain that binds to HLA-G conjugated with a detectable label can be used as contrast agents.

[1060] Multispecific proteins containing an antigen-binding domain that binds to HLA-G conjugated with a detectable label can be used as contrast agents.

[1061] In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a toxin or fragment thereof having enzymatic activity derived from bacteria, fungi, plants, or animals), or a radioisotope (i.e., a radioconjugate).

[1062] In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, lysine, geldanamycin, mytansinoids, or calicheamicin. The cytotoxic agent may induce its cytotoxic or cell proliferation inhibitory effect through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[1063] In some embodiments, the cytotoxic agents are enzymatic toxins such as diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), bitter melon (momordica charantia) inhibitor, curcin, crotin, soapwort (sapaonaria officinalis) inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecenes.

[1064] In some embodiments, the cytotoxic agent is 212 Bi, 131 I, 131 In, 90 Y, and 186 These are radioactive nuclides such as Re.

[1065] In some embodiments, the cytotoxic agent is drostatin or a peptide analog and derivative of drostatin, auristatin, or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Patents 5,635,483 and 5,780,588. Drostatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob Agents and Chemother. 45(12):3580~3584) and possess anticancer and antifungal activity. The drostatin and auristatin drug sites can be bound to the antibodies of the present invention via the N (amino) or C (carboxyl) terminus of the peptide drug site (International Publication No. 02 / 088172), or via any genetically engineered cysteine ​​within the antibody.

[1066] The HLA-G binding proteins of this disclosure can be conjugated to detectable labels using known methods.

[1067] In some embodiments, the detectable label forms a complex with a chelating agent.

[1068] In some embodiments, a detectable label is conjugated to the HLA-G binding protein of this disclosure via a linker.

[1069] A detectable label or cytotoxic moiety can be directly or indirectly linked to the HLA-G binding protein of this disclosure using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenol linkers (derivatives of N-succimidyl benzoate, dodecaborate), chelate moieties of both macrocyclic and acyclic chelating agents, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), and diethylenetriaminepentaacetic acid. avid, derivatives of DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters ( Examples include disuccinimidyl sverat, aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-activated fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), as well as other chelate moieties. Suitable peptide linkers are well known.

[1070] In some embodiments, the HLA-G binding proteins of this disclosure are removed from the blood via renal clearance.

[1071] kit The present invention also provides a kit comprising an antigen-binding domain that binds to HLA-G.

[1072] The present invention also provides a kit comprising a protein containing an antigen-binding domain that binds to HLA-G.

[1073] The present invention also provides a kit comprising a multispecific protein containing an antigen-binding domain that binds to HLA-G.

[1074] The kit can be used for therapeutic purposes and as a diagnostic kit.

[1075] The kit can be used to detect the presence of HLA-G in a sample.

[1076] In some embodiments, the kit comprises the HLA-G binding protein of the Disclosure and a reagent for detecting the HLA-G binding protein. The kit may also comprise one or more other elements, including instructions for use; other reagents, e.g., labels, therapeutic agents, or agents useful for chelation or other coupling methods, antibodies against labels or therapeutic agents, or radioprotective compositions; devices or other materials for preparing antibodies for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[1077] In some embodiments, the kit includes an antigen-binding domain that binds to an HLA-G in a container, and instructions for using the kit.

[1078] In some embodiments, the kit includes a protein containing an antigen-binding domain that binds to HLA-G in a container, and instructions for using the kit.

[1079] In some embodiments, the kit includes a multispecific protein containing an antigen-binding domain that binds to HLA-G in a container, and instructions for using the kit.

[1080] In some embodiments, the antigen-binding domain that binds to HLA-G in the kit is labeled.

[1081] In some embodiments, the protein containing the antigen-binding domain that binds to HLA-G in the kit is labeled.

[1082] In some embodiments, the multispecific protein containing the antigen-binding domain that binds to HLA-G in the kit is labeled.

[1083] In some embodiments, the kit is VH of sequence number 50 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, or It contains antigen-binding domains that bind to HLA-G, including VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[1084] In some embodiments, the kit includes an HLA-G-binding antigen-binding domain, including SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[1085] Method for detecting HLA-G The present invention also provides a method for detecting HLA-G in a sample, comprising: obtaining a sample; contacting the sample with an antigen-binding domain that binds to HLA-G of the present disclosure; and detecting the bound HLA-G in the sample.

[1086] In some embodiments, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, synovial fluid, cells not associated with circulating cells or tissue (i.e., free cells), tissue (e.g., surgically excised tissue, biopsy material including fine-needle aspiration), tissue slides, etc.

[1087] The antigen-binding domains that bind to HLA-G in this disclosure can be detected using known methods. Exemplary methods include directly labeling the antibody using fluorescent or chemiluminescent labeling, or radiolabeling, or conjugating the antibody of the present invention to an easily detectable moiety such as biotin, an enzyme, or an epitope tag. Exemplary labels and moieties include ruthenium, 111 In-DOTA, 111 These include in-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluoron dyes, oxazine dyes, phenanthidine dyes, rhodamine dyes, and Alexafluor® dyes.

[1088] The antigen-binding domains of the HLA-G present disclosure can be used in a variety of assays for detecting HLA-G in a sample. Exemplary assays include Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemical analysis, fluorescence-activated cell sorting (FACS), or ELISA assay.

[1089] Polynucleotides, vectors, and host cells The Disclosure also provides isolated polynucleotides encoding any of the HLA-G binding proteins of the Disclosure. The HLA-G binding proteins include an antigen-binding domain that binds to HLA-G, a protein comprising an antigen-binding domain that binds to HLA-G, and a multispecific protein comprising an antigen-binding domain that binds to HLA-G.

[1090] The present invention also provides isolated polynucleotides encoding either an HLA-G binding protein or a fragment thereof.

[1091] The present invention also provides an isolated polynucleotide encoding VH of SEQ ID NO: 50.

[1092] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 51.

[1093] The present invention also provides an isolated polynucleotide encoding VH of SEQ ID NO: 52.

[1094] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 53.

[1095] The present invention also provides an isolated polynucleotide encoding VH of SEQ ID NO: 54.

[1096] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 55.

[1097] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 56.

[1098] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 57.

[1099] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 58.

[1100] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 59.

[1101] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 60.

[1102] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 61.

[1103] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 62.

[1104] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 63.

[1105] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 64.

[1106] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 65.

[1107] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 66.

[1108] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 67.

[1109] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 68.

[1110] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 69.

[1111] The present invention also provides isolated polynucleotides encoding VH of SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68.

[1112] The present invention also provides isolated polynucleotides encoding VLs of SEQ ID NOs. 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[1113] The present invention also provides isolated polynucleotides encoding VH of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[1114] The present invention also, VH of sequence number 50 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, or This provides isolated polynucleotides encoding VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[1115] The present invention also provides isolated polynucleotides encoding polypeptides of SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[1116] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 248.

[1117] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 249.

[1118] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 250.

[1119] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 251.

[1120] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 252.

[1121] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 253.

[1122] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 254.

[1123] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 255.

[1124] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 256.

[1125] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 257.

[1126] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 258.

[1127] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 259.

[1128] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 260.

[1129] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 261.

[1130] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 262.

[1131] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 263.

[1132] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 264.

[1133] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 265.

[1134] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 266.

[1135] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 267.

[1136] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 268.

[1137] The present invention also provides an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 269.

[1138] Some embodiments of the present disclosure also provide isolated or purified nucleic acids comprising a polynucleotide encoding the HLA-G binding protein of the present disclosure or a polynucleotide complementary to the polynucleotide that hybridizes to the HLA-G binding protein of the present disclosure under stringent conditions.

[1139] Polynucleotides that hybridize under stringent conditions may hybridize under high-stringency conditions. “High-stringency conditions” means that the polynucleotide hybridizes specifically to a target sequence (a nucleotide sequence of any of the nucleic acids described herein) in a detectable amount stronger than non-specific hybridization. High-stringency conditions include conditions that distinguish polynucleotides having precisely complementary sequences or containing only a few scattered mismatches from random sequences that end up having several sub-regions (e.g., 3–12 bases) that match the nucleotide sequence. Such complementary sub-regions melt more readily than full-length complements of 14–17 or more bases, and high-stringency hybridization makes them readily identifiable. Relatively high-stringency conditions include low-salt and / or high-temperature conditions, such as those provided by about 0.02–0.1 M NaCl or equivalent at a temperature of about 50–70°C. Such highly stringent conditions tolerate virtually no mismatch between the nucleotide sequence and the template or target chain, if any. It is generally understood that these conditions can be made even stringier by the addition of gradually increasing amounts of formamide.

[1140] The polynucleotide sequences of this disclosure can be operably ligated to one or more regulatory elements (e.g., promoters or enhancers) that express the nucleotide sequence in an intended host cell. The polynucleotides may be cDNAs. The promoters may be strong, weak, tissue-specific, inducible, or developmental stage-specific. Exemplary promoters that may be used include hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, and human muscle creatine. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the embodiments described. Examples of such viral promoters include the earliest promoter of cytomegalovirus (CMV), the early and late promoters of SV40, the promoter of mouse mammary tumor virus (MMTV), Moloney's leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and long terminal repeats (LTRs) of other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Inducible promoters may also be used, such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, and promoters containing one or more interferon-stimulated response elements (ISREs), such as the protein kinase R2',5'-oligoadenylate synthase, the Mx gene, and ADAR1.

[1141] The present invention also provides vectors comprising the polynucleotides of the present invention. This disclosure also provides expression vectors comprising the polynucleotides of the present invention. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vectors suitable for introducing the synthetic polynucleotides of the present invention into a given biological or genetic background by any means. The polynucleotides encoding the HLA-G binding protein of this disclosure may be operably ligated to regulatory sequences in the expression vector to ensure the expression of the HLA-G binding protein. Such regulatory elements may include transcription promoters, sequences encoding suitable mRNA ribosome binding sites, and sequences that control the termination of transcription and translation. The expression vector may also include one or more non-transcription elements, such as origins of replication, suitable promoters and enhancers ligated to the gene to be expressed, other 5' or 3' adjacent non-transcription sequences, 5' or 3' untranslated sequences (e.g., essential ribosome binding sites), polyadenylation sites, splice donor and acceptor sites, or transcription termination sequences. Origins of replication that confer the ability to replicate in a host may also be incorporated.

[1142] Expression vectors may contain naturally occurring or non-naturally occurring nucleotide linkages, or both. Non-naturally occurring or modified nucleotide linkages do not inhibit the transcription or replication of the vector.

[1143] After the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the HLA-G binding protein of this disclosure encoded by the incorporated polynucleotide. The transcriptional and translational regulatory sequences in the expression vector used to transform vertebrate cells can be provided by a viral source. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[1144] The vectors of this disclosure may also contain one or more internal ribosome entry sites (IRESs). Including IRES sequences in the fusion vector may be beneficial for enhancing the expression of certain proteins. In some embodiments, the vector system may include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. The components of the vector may be linked in close proximity, or arranged to provide optimal spacing for gene product expression (i.e., by introducing “spacer” nucleotides between ORFs), or otherwise positioned. Regulatory elements, such as IRES motifs, may also be arranged to provide optimal spacing for expression.

[1145] The vectors of this disclosure may be circular or linear. They may be prepared to contain a functional replication system in a prokaryotic or eukaryotic host cell. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[1146] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be constructed for constitutive or inducible expression.

[1147] Furthermore, recombinant expression vectors may be constructed to contain suicide genes. As used herein, the term “suicide gene” refers to a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to a drug (e.g., a pharmacokinetic) to cells expressing the gene, causing the cells to die when they come into contact with or are exposed to the drug. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[1148] The vector may also contain selection markers well known in the art. These selection markers include positive and negative selection markers. Marker genes include those for biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.) and complementation for providing protonutrient supply in nutritionally dependent hosts. Exemplary marker genes include antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, histidinol resistance gene, histidinol × resistance gene), glutamine synthase genes, HSV-TK and HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase genes for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence or cloning site encoding the selection marker may be upstream or downstream of the nucleic acid sequence or cloning site encoding the polypeptide of interest.

[1149] Examples of vectors that may be used include: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), pEE6.4 (Lonza), and pEE12.4 (Lonza). Additional vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can also be used. Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The expression vector may be a viral vector, such as a retroviral vector, such as a gamma retroviral vector.

[1150] In some embodiments, the vector includes a polynucleotide encoding VH of SEQ ID NO: 50.

[1151] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 51.

[1152] In some embodiments, the vector includes a polynucleotide encoding VH of SEQ ID NO: 52.

[1153] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 53.

[1154] In some embodiments, the vector includes a polynucleotide encoding VH of SEQ ID NO: 54.

[1155] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 55.

[1156] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 56.

[1157] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 57.

[1158] In some embodiments, the vector includes a polynucleotide encoding the VL of sequence number 58.

[1159] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 59.

[1160] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 60.

[1161] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 61.

[1162] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 62.

[1163] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 63.

[1164] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 64.

[1165] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 65.

[1166] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 66.

[1167] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 67.

[1168] In some embodiments, the vector includes a polynucleotide encoding the VL of sequence number 68.

[1169] In some embodiments, the vector includes a polynucleotide encoding the VL of SEQ ID NO: 69.

[1170] In some embodiments, the vector includes a polynucleotide encoding VH of sequence numbers 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68.

[1171] In some embodiments, the vector includes a polynucleotide encoding the VL of sequence numbers 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[1172] In some embodiments, the vector comprises polynucleotides encoding VH of SEQ ID NOs. 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of SEQ ID NOs. 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[1173] In some embodiments, the vector is VH of sequence number 50 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, or It contains polynucleotides encoding VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[1174] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of sequence numbers 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[1175] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 248.

[1176] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 249.

[1177] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 250.

[1178] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 251.

[1179] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 252.

[1180] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 253.

[1181] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 254.

[1182] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 255.

[1183] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 256.

[1184] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 257.

[1185] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 258.

[1186] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 259.

[1187] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 260.

[1188] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 261.

[1189] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 262.

[1190] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 263.

[1191] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 264.

[1192] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 265.

[1193] In some embodiments, the vector includes a polynucleotide encoding the polypeptide of SEQ ID NO: 266.

[1194] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 267.

[1195] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 268.

[1196] In some embodiments, the vector contains a polynucleotide encoding the polypeptide of SEQ ID NO: 269.

[1197] The present invention also provides host cells containing one or more vectors of the present invention. “Host cell” refers to a cell into which a vector has been introduced. The term “host cell” is understood to refer not only to a specific target cell, but also to the progeny of such cells, and stable cell lines generated from a specific target cell. Such progeny may not be identical to the parent cell, as certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they are still included within the scope of the term “host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells, or archaeal cells. Examples of prokaryotic host cells include bacilli such as Escherichia coli and Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. Other microorganisms, such as yeast, are also useful for expression. Examples of suitable yeast host cells include Saccharomyces (e.g., S. cerevisiae) and Pichia. Exemplary eukaryotic cells may be derived from mammals, insects, birds, or other animals. Examples of mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines, including SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics (Walkersville, MD)), CHO-K1 (ATCC CRL-61), or DG44.

[1198] The disclosure also provides a method for producing the HLA-G binding protein of the disclosure, comprising culturing the host cells of the disclosure under conditions that express the K2 binding protein, and recovering the HLA-G binding protein produced by the host cells. Methods for preparing and purifying the protein are known. After synthesis (chemically or recombinantly), the HLA-G binding protein can be purified by standard procedures including ammonium sulfate precipitation, affinity columns, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (Generally, see Scopes, Protein Purification (Springer-Verlag, NY, (1982))). The target protein may be substantially pure, e.g., with a purity of at least about 80%–85%, at least about 85%–90%, at least about 90%–95%, or at least about 98%–99%, or higher, and may be free from contaminants such as cell debris, macromolecules other than the target protein.

[1199] The polynucleotide encoding the HLA-G binding protein of this disclosure can be incorporated into a vector using standard molecular biological methods. Transformation, culture, antibody expression, and purification of host cells are performed using well-known methods.

[1200] The polynucleotides of this disclosure can be generated using modified nucleotides. Exemplary modified nucleotides include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N 6- Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquosin, 5″-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, quosin, beta-D-galactosylquosin, inosine, N 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[1201] Pharmaceutical composition / administration This disclosure also provides a pharmaceutical composition comprising the HLA-G binding protein of this disclosure and a pharmaceutically acceptable carrier.

[1202] The Disclosure also provides a pharmaceutical composition comprising an antigen-binding domain that binds to the HLA-G of the Disclosure and a pharmaceutically acceptable carrier.

[1203] The Disclosure also provides a pharmaceutical composition comprising a protein containing an antigen-binding domain that binds to the HLA-G of the Disclosure, and a pharmaceutically acceptable carrier.

[1204] The Disclosure also provides a pharmaceutical composition comprising a multispecific protein containing an antigen-binding domain that binds to the HLA-G of the Disclosure, and a pharmaceutically acceptable carrier.

[1205] The disclosure also provides a pharmaceutical composition comprising a multispecific protein having an antigen-binding domain that binds to HLA-G and an antigen-binding domain that binds to a tumor antigen other than HLA-G, and a pharmaceutically acceptable carrier.

[1206] The HLA-G binding proteins of this disclosure can be prepared as pharmaceutical compositions containing an effective amount of antibody as the active ingredient in a pharmaceutically acceptable carrier. These solutions are sterilized and generally free of particulate matter. They can be sterilized by conventionally known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants.

[1207] When used herein in reference to pharmaceutical compositions, the term “pharmaceutically acceptable” means that it is approved for use in animals and / or humans by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or any other generally accepted pharmacopoeia.

[1208] Treatment and Usage The Disclosure also provides a bispecific or multispecific protein for therapeutic use, comprising a first antigen-binding domain that specifically binds to HLA-G and a second antigen-binding domain that specifically binds to a second antigen.

[1209] The disclosure also provides a bispecific or multispecific protein for use in the treatment of cell proliferation disorders, comprising a first antigen-binding domain that specifically binds to HLA-G and a second antigen-binding domain that specifically binds to a second antigen.

[1210] The Disclosure also provides a bispecific or multispecific protein for use in the treatment of cancer, comprising a first antigen-binding domain that specifically binds to HLA-G and a second antigen-binding domain that specifically binds to a second antigen.

[1211] The Disclosure also provides a bispecific or multispecific protein for use in the manufacture of a pharmaceutical product for therapeutic use, comprising a first antigen-binding domain that specifically binds to an HLA-G and a second antigen-binding domain that specifically binds to a second antigen.

[1212] The Disclosure also provides a bispecific or multispecific protein for use in the manufacture of a pharmaceutical product for use in the treatment of cell proliferation disorders, comprising a first antigen-binding domain that specifically binds to an HLA-G and a second antigen-binding domain that specifically binds to a second antigen.

[1213] The Disclosure also provides a bispecific or multispecific protein for use in the manufacture of a drug for treating cancer, comprising a first antigen-binding domain that specifically binds to HLA-G and a second antigen-binding domain that specifically binds to a second antigen.

[1214] This disclosure also includes a method for treating cancer in a subject, comprising administering to the subject a multispecific protein comprising an antigen-binding domain that binds to HLA-G in a therapeutically effective amount, wherein the antigen-binding domain that binds to HLA-G is VH of sequence number 50 and VL of sequence number 51, VH of sequence number 52 and VL of sequence number 53, VH of sequence number 54 and VL of sequence number 55, VH of sequence number 56 and VL of sequence number 57, VH of sequence number 58 and VL of sequence number 59, VH of sequence number 60 and VL of sequence number 61, VH of sequence number 62 and VL of sequence number 63, VH of sequence number 64 and VL of sequence number 65, VH of sequence number 66 and VL of sequence number 67, or The present invention provides a method comprising VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

[1215] The disclosure also provides a method for treating cancer in a subject, comprising administering to the subject a multispecific protein comprising a therapeutically effective amount of an antigen-binding domain that binds to HLA-G, wherein the antigen-binding domain that binds to HLA-G comprises the amino acid sequence of SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269.

[1216] Further embodiments of the present disclosure are methods for treating a cell proliferation disorder in a subject requiring treatment, comprising administering a therapeutically effective dose of a bispecific or multispecific protein of the present disclosure comprising a first antigen-binding domain that specifically binds to HLA-G and a second antigen-binding domain that specifically binds to a second antigen. In other embodiments, a bispecific or multispecific protein of the present disclosure comprising a first antigen-binding domain that specifically binds to HLA-G and a second antigen-binding domain that specifically binds to a second antigen is administered to the subject.

[1217] In any of the uses or methods described above, the cell proliferative disorder is cancer. In other embodiments, cancer is selected from the group consisting of lung cancer, pancreatic cancer, kidney cancer, head and neck cancer, ovarian cancer, esophageal cancer, breast cancer, uterine cancer, melanoma, neuroblastoma, glioblastoma, colorectal cancer, gastric cancer, parathyroid cancer, bladder cancer, liver cancer, hepatocellular carcinoma, pleural mesothelioma, prostate cancer, bile duct cancer, thyroid cancer, fetal cancer, seminoma, uveal melanoma, pheochromocytoma, teratoma, thymoma, adrenocortical carcinoma, astrocytoma, synovial sarcoma, myelodysplastic syndrome, acute myeloid leukemia (AML), Hodgkin lymphoma, multiple myeloma (MM), non-Hodgkin lymphoma, and B-cell chronic lymphocytic leukemia.

[1218] In other embodiments, the cancer is lung cancer. In other embodiments, the lung cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), or lung adenocarcinoma. In other embodiments, the cancer is pancreatic cancer. In other embodiments, the cancer is adenocarcinoma, for example, metastatic adenocarcinoma (e.g., lung adenocarcinoma, gastric adenocarcinoma, or pancreatic adenocarcinoma).

[1219] In other embodiments, the kidney cancer is clear cell renal cell carcinoma (CCRCC).

[1220] In other embodiments, the kidney cancer is of the papillary type.

[1221] In other embodiments, ovarian cancer is a high-grade serous cancer of the ovary, peritoneum, or fallopian tube.

[1222] In other embodiments, ovarian cancer may have elevated blood markers (e.g., CA125) or cancer-related fluids that can be monitored.

[1223] In other embodiments, the breast cancer is triple-negative breast cancer (TNBC).

[1224] In another embodiment, the Disclosure features a kit comprising (a) a composition comprising any one of the aforementioned bi- or multi-specific proteins, comprising a first antigen-binding domain that specifically binds to HLA-G and a second antigen-binding domain that specifically binds to a second antigen, and (b) a package insert including instructions for administering the composition to a subject to treat or delay the progression of a cell proliferation disorder.

[1225] In any of the uses or methods described above, the subject may be a human being.

[1226] Embodiments: The present invention provides the following non-limiting embodiments. 1) An isolated protein containing an antigen-binding domain that binds to human leukocyte antigen G (HLA-G), wherein the antigen-binding domain that binds to HLA-G is a) Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or b) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 52, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 53, or c) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 54, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 55, or d) HCDR1, HCDR2, and HCDR3 of VH in sequence number 56, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 57, or e) HCDR1, HCDR2, and HCDR3 of VH in sequence number 58, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 59, or f) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 60, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 61, or g) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 63, or h) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 65, or i) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 67, or j) Isolated proteins comprising HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 69. 2) a) Sequence numbers 70, 71, 72, 88, 89, and 90, respectively b) Sequence numbers 73, 71, 74, 91, 89, and 92 respectively, c) Sequence numbers 75, 76, 77, 93, 89, and 94 respectively, d) Sequence numbers 78, 79, 80, 95, 89, and 96, respectively e) Sequence numbers 81, 82, 83, 97, 89, and 98 respectively, f) Sequence numbers 78, 71, 84, 99, 89, and 100, respectively g) Sequence numbers 78, 71, 84, 101, 89, and 100, respectively h) Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or i) The isolated protein according to Embodiment 1, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively. 3) The isolated protein according to Embodiment 1 or 2, wherein the antigen-binding domain that binds to HLA-G is scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH. 4) The isolated protein according to Embodiment 3, wherein the antigen-binding domain that binds to HLA-G is Fab. 5) The isolated protein according to Embodiment 3, wherein the antigen-binding domain that binds to HLA-G is scFv. 6) The isolated protein according to Embodiment 5, wherein scFv comprises VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus. 7) L1 is a) Approximately 5 to 50 amino acids, b) Approximately 5 to 40 amino acids, c) Approximately 10 to 30 amino acids, or d) The isolated protein according to Embodiment 6, comprising approximately 10 to 20 amino acids. 8) The isolated protein according to Embodiment 6, wherein L1 contains the amino acid sequence of SEQ ID NOs. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. 9) The isolated protein according to Embodiment 8, wherein L1 contains the amino acid sequence of SEQ ID NO: 8. 10) An isolated protein according to any one of Embodiments 1 to 9, wherein the antigen-binding domain that binds to HLA-G comprises VH of SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of SEQ ID NO: 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69. 11) The antigen-binding domain that binds to HLA-G, a) VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51 b) VH of sequence number 52 and VL of sequence number 53, c) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55 d) VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57 e) VH of sequence number 58 and VL of sequence number 59, f) VH of sequence number 60 and VL of sequence number 61, g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63 h) VH of sequence number 64 and VL of sequence number 65, i) VH of sequence number 66 and VL of sequence number 67, or j) The isolated protein according to Embodiment 10, comprising VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69 12) An isolated protein according to any one of Embodiments 1 to 11, wherein the antigen-binding domain that binds to HLA-G contains the amino acid sequence of SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269. 13) An isolated protein according to any one of Embodiments 1 to 12, wherein the protein is conjugated to the half-life extension portion. 14) The isolated protein according to Embodiment 13, wherein the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, an Ig constant region, a fragment of an Ig constant region, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol. 15) The isolated protein according to any one of Embodiments 1 to 14, wherein the isolated protein is a single-specific protein. 16) The isolated protein according to any one of Embodiments 1 to 14, wherein the isolated protein is a multispecific protein. 17) The isolated protein according to Embodiment 16, wherein the multispecific protein is a bispecific protein. 18) The isolated protein according to Embodiment 16, wherein the multispecific protein is a tripspecific protein. 19) The isolated protein according to any one of Embodiments 1 to 18, further comprising an immunoglobulin (Ig) constant region or a fragment thereof of the Ig constant region. 20) The isolated protein according to Embodiment 19, wherein the fragment of the Ig constant region includes an Fc region. 21) The isolated protein according to Embodiment 19, wherein a fragment of the Ig constant region contains a CH2 domain. 22) The isolated protein according to Embodiment 19, wherein the fragment of the Ig constant region contains a CH3 domain. 23) The isolated protein according to Embodiment 19, wherein the fragment of the Ig constant region includes a CH2 domain and a CH3 domain. 24) The isolated protein according to Embodiment 19, wherein the fragment of the Ig constant region comprises at least a portion of the hinge, a CH2 domain, and a CH3 domain. 25) The isolated protein according to Embodiment 19, wherein the Ig constant region fragment comprises a hinge, a CH2 domain, and a CH3 domain. 26) The isolated protein according to any one of embodiments 19 to 25, wherein the antigen-binding domain that binds to HLA-G is conjugated to the N-terminus of the Ig constant region or a fragment of the Ig constant region. 27) The isolated protein according to any one of embodiments 19 to 25, wherein the antigen-binding domain that binds to HLA-G is conjugated to the C-terminus of the Ig constant region or a fragment of the Ig constant region. 28) The isolated protein according to any one of embodiments 19 to 27, wherein the antigen-binding domain that binds to HLA-G is conjugated to an Ig constant region or a fragment of an Ig constant region via a second linker (L2). 29) The isolated protein according to Embodiment 41, wherein L2 contains the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. 30) An isolated protein according to any one of embodiments 16 to 29, wherein the multispecific protein includes an antigen-binding domain that binds to an antigen on a lymphocyte. 31) The isolated protein according to Embodiment 30, wherein the lymphocyte is a T lymphocyte. 32) T cells, CD8 + The isolated protein described in Embodiment 30 is a T cell. 33) The isolated protein according to Embodiment 30, wherein the lymphocytes are natural killer (NK) cells. 34) An isolated protein according to any one of Embodiments 16 to 33, wherein the multispecific protein includes an antigen-binding domain that binds to CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C. 35) The isolated protein according to Embodiment 34, wherein the multispecific protein includes an antigen-binding domain that binds to CD3ε. 36) The antigen-binding domain that binds to CD3ε, a) Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 361, HCDR2 of SEQ ID NO: 362, HCDR3 of SEQ ID NO: 363, Light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 367, LCDR2 of SEQ ID NO: 368, and LCDR3 of SEQ ID NO: 369 b) VH of sequence number 339 and VL of sequence number 340, c) HCDR1 of sequence number 361, HCDR2 of sequence number 362, HCDR3 of sequence number 363, LCDR1 of sequence number 367, LCDR2 of sequence number 368, and LCDR3 of sequence number 370 d) VH of sequence number 339 and VL of sequence number 341, e) VH of sequence number 339 and VL of sequence number 342, f) VH of sequence number 339 and VL of sequence number 343, g) VH of SEQ ID NO: 339 and VL of SEQ ID NO: 344, h) VH of sequence number 339 and VL of sequence number 345, i) HCDR1 of sequence number 364, HCDR2 of sequence number 365, HCDR3 of sequence number 366, LCDR1 of sequence number 371, LCDR2 of sequence number 372, and LCDR3 of sequence number 373, j) VH of SEQ ID NO: 346 and VL of SEQ ID NO: 347, or k) The isolated protein according to Embodiment 48, comprising VH of SEQ ID NO: 348 and VL of SEQ ID NO: 349. 37) The isolated protein according to any one of embodiments 19 to 36, wherein the Ig constant region or a fragment of the Ig constant region is an IgG1, IgG2, IgG3, or IgG4 isotype. 38) The isolated protein according to any one of embodiments 19 to 37, wherein the Ig constant region or a fragment of the Ig constant region contains at least one mutation resulting in reduced binding of the protein to the Fcγ receptor (FcγR). 39) At least one mutation resulting in reduced binding of the protein to FcγR is present in L235A / D265S, F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / An isolated protein according to Embodiment 38, selected from the group consisting of D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, wherein the residue numbering follows the EU index. 40) The isolated protein according to any one of Embodiments 19 to 37, wherein the Ig constant region or a fragment of the Ig constant region contains at least one mutation resulting in enhanced binding of the protein to FcγR. 41) The isolated protein according to Embodiment 40, wherein at least one mutation resulting in enhanced binding of the protein to FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, and residue numbering follows the EU index. 42) The isolated protein according to any one of Embodiments 38 to 41, wherein FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof. 43) An isolated protein according to any one of embodiments 19 to 42, wherein the Ig constant region or a fragment of the Ig constant region contains at least one mutation that modulates the protein's half-life. 44) The isolated protein according to Embodiment 43, wherein at least one mutation that modulates the protein's half-life is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, and the residue numbering follows the EU index. 45) An isolated protein according to any one of embodiments 19 to 44, wherein the protein contains at least one mutation in the CH3 domain of the Ig constant region. 46) At least one mutation in the CH3 domain of the Ig constant region is present in T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / An isolated protein according to Embodiment 45, selected from the group consisting of K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, wherein the residue numbering follows the EU index. 47) An isolated, multispecific protein comprising a first antigen-binding domain that binds to HLA-G and a second antigen-binding domain that binds to lymphocyte antigens. 48) The isolated multispecific protein according to Embodiment 47, wherein the lymphocyte antigen is a T cell antigen. 49) T cell antigen is CD8 + The isolated multispecific protein described in Embodiment 47 is a T cell antigen. 50) The isolated multispecific protein according to Embodiment 47, wherein the lymphocyte antigen is an NK cell antigen. 51) An isolated multispecific protein according to any one of Embodiments 47 to 50, wherein the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C. 52) The isolated multispecific protein according to Embodiment 51, wherein the lymphocyte antigen is CD3ε. 53) An isolated multispecific protein according to any one of Embodiments 47 to 52, wherein the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to a lymphocyte antigen comprises scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH. 54) The isolated multispecific protein according to Embodiment 53, wherein the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to a lymphocyte antigen comprises Fab. 55) The isolated multispecific protein according to Embodiment 53, wherein the first antigen-binding domain that binds to HLA-G and / or the second antigen-binding domain that binds to a lymphocyte antigen comprises scFv. 56) The isolated multispecific protein according to Embodiment 55, wherein scFv comprises VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus. 57) L1 is, a) Approximately 5 to 50 amino acids, b) Approximately 5 to 40 amino acids, c) Approximately 10 to 30 amino acids, or d) An isolated multispecific protein according to Embodiment 56, comprising approximately 10 to 20 amino acids. 58) The isolated multispecific protein according to Embodiment 57, wherein L1 contains the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. 59) The isolated multispecific protein according to Embodiment 58, wherein L1 contains the amino acid sequence of SEQ ID NO: 8. 60) An isolated multispecific protein according to any one of Embodiments 47 to 59, wherein the first antigen-binding domain that binds to HLA-G comprises HCDR1 of SEQ ID NO: 70, 73, 75, 78, 81, or 85, HCDR2 of SEQ ID NO: 71, 76, 79, 82, or 86, HCDR3 of SEQ ID NO: 72, 74, 77, 80, 83, 84, or 87, LCDR1 of SEQ ID NO: 88, 91, 93, 95, 97, 99, 101, or 102, LCDR2 of SEQ ID NO: 89 or 103, and LCDR3 of SEQ ID NO: 90, 92, 94, 96, 98, 100, or 104. 61) The first antigen-binding domain that binds to HLA-G, a) Sequence numbers 70, 71, 72, 88, 89, and 90, respectively b) Sequence numbers 73, 71, 74, 91, 89, and 92 respectively, c) Sequence numbers 75, 76, 77, 93, 89, and 94 respectively, d) Sequence numbers 78, 79, 80, 95, 89, and 96, respectively e) Sequence numbers 81, 82, 83, 97, 89, and 98 respectively, f) Sequence numbers 78, 71, 84, 99, 89, and 100, respectively g) Sequence numbers 78, 71, 84, 101, 89, and 100, respectively h) Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, or i) An isolated multispecific protein according to any one of embodiments 47 to 60, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively. 62) The antigen-binding domain that binds to HLA-G, a) VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51 b) VH of sequence number 52 and VL of sequence number 53, c) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55 d) VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57 e) VH of sequence number 58 and VL of sequence number 59, f) VH of sequence number 60 and VL of sequence number 61, g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63 h) VH of sequence number 64 and VL of sequence number 65, i) VH of sequence number 66 and VL of sequence number 67, or j) An isolated multispecific protein according to any one of embodiments 47 to 61, comprising VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69. 63) An isolated multispecific protein according to any one of embodiments 47 to 61, wherein the first antigen-binding domain that binds to HLA-G comprises VH of SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of SEQ ID NO: 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69. 64) An isolated multispecific protein according to any one of embodiments 47 to 63, wherein the first antigen-binding domain that binds to HLA-G comprises the amino acid sequence of SEQ ID NOs: 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, or 269. 65) The second antigen-binding domain that binds to lymphocyte antigens, a) HCDR1 of sequence number 361, HCDR2 of sequence number 362, HCDR3 of sequence number 363, LCDR1 of sequence number 367, LCDR2 of sequence number 368, and LCDR3 of sequence number 369 b) VH of sequence number 339 and VL of sequence number 340, c) HCDR1 of sequence number 361, HCDR2 of sequence number 362, HCDR3 of sequence number 363, LCDR1 of sequence number 367, LCDR2 of sequence number 368, and LCDR3 of sequence number 370 d) VH of sequence number 339 and VL of sequence number 341, e) VH of sequence number 339 and VL of sequence number 342, f) VH of sequence number 339 and VL of sequence number 343, g) VH of SEQ ID NO: 339 and VL of SEQ ID NO: 344, h) VH of sequence number 339 and VL of sequence number 345, i) HCDR1 of sequence number 364, HCDR2 of sequence number 365, HCDR3 of sequence number 366, LCDR1 of sequence number 371, LCDR2 of sequence number 372, and LCDR3 of sequence number 373, j) VH of SEQ ID NO: 346 and VL of SEQ ID NO: 347, or k) An isolated multispecific protein according to any one of embodiments 47 to 56, comprising VH of SEQ ID NO: 348 and VL of SEQ ID NO: 349. 66) An isolated multispecific protein according to any one of embodiments 47 to 65, wherein a first antigen-binding domain that binds to HLA-G is conjugated to a first immunoglobulin (Ig) constant region or a fragment of a first Ig constant region, and / or a second antigen-binding domain that binds to a lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of a second Ig constant region. 67) The isolated multispecific protein according to Embodiment 66, further comprising a second linker (L2) between a first antigen-binding domain that binds to HLA-G and a first Ig constant region or a fragment of the first Ig constant region, and between a second antigen-binding domain that binds to a lymphocyte antigen and a second Ig constant region or a fragment of the second Ig constant region. 68) The isolated multispecific p...

Claims

1. An isolated protein comprising an antigen-binding domain that binds to human leukocyte antigen G (HLA-G), wherein the antigen-binding domain that binds to HLA-G is a) Heavy chain complementarity determination regions (HCDRs) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 50, and light chain complementarity determination regions (LCDRs) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 51, or b) HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 52, and LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 53, or c) HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 54, and LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 55, or d) HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 56, and LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 57, or e) HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 58, and LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 59, or f) HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 60, and LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 61, or g) HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 63, or h) HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 64, and LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 65, or i) HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 66, and LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 67, or j) Isolated protein comprising HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO:

69.

2. a) Sequence numbers 70, 71, 72, 88, 89, and 90, respectively b) Sequence numbers 73, 71, 74, 91, 89, and 92, respectively c) Sequence numbers 75, 76, 77, 93, 89, and 94, respectively d) Sequence numbers 78, 79, 80, 95, 89, and 96, respectively e) Sequence numbers 81, 82, 83, 97, 89, and 98, respectively f) Sequence numbers 78, 71, 84, 99, 89, and 100, respectively g) Sequence IDs 78, 71, 84, 101, 89, and 100, respectively h) Sequence numbers 85, 86, 87, 102, 103, and 104, respectively, i) The isolated protein according to claim 1, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 78, 71, 84, 95, 89, and 96, respectively.

3. The isolated protein according to claim 1, wherein the antigen-binding domain that binds to HLA-G is scFv or Fab.

4. The isolated protein according to claim 3, wherein the scFv comprises VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus.

5. The isolated protein according to claim 4, wherein L1 comprises the amino acid sequence of SEQ ID NO:

8.

6. The isolated protein according to claim 1, wherein the antigen-binding domain that binds to HLA-G comprises VH of SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, or 68 and VL of SEQ ID NO: 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

7. The antigen-binding domain that binds to HLA-G is a) VH of sequence number 50 and VL of sequence number 51, b) VH of sequence number 52 and VL of sequence number 53, c) VH of sequence number 54 and VL of sequence number 55, d) VH of sequence number 56 and VL of sequence number 57, e) VH of sequence number 58 and VL of sequence number 59, f) VH of sequence number 60 and VL of sequence number 61, g) VH of sequence number 62 and VL of sequence number 63, h) VH of sequence number 64 and VL of sequence number 65, i) VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67, or j) The isolated protein according to claim 6, comprising VH of SEQ ID NO: 68 and VL of SEQ ID NO:

69.

8. The isolated protein according to claim 6, wherein the antigen-binding domain that binds to HLA-G comprises the amino acid sequence of SEQ ID NOs: 265, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 266, 267, 268, or 269.

9. The isolated protein according to claim 1, wherein the protein is a bispecific protein.

10. The isolated protein according to claim 9, further comprising an Fc region.

11. The isolated protein according to claim 9, wherein the bispecific protein includes an antigen-binding domain that binds to CD3ε.

12. The antigen-binding domain that binds to the aforementioned CD3ε, a) HCDR1 of SEQ ID NO: 364, HCDR2 of SEQ ID NO: 365, HCDR3 of SEQ ID NO: 366, LCDR1 of SEQ ID NO: 371, LCDR2 of SEQ ID NO: 372, and LCDR3 of SEQ ID NO: 373 b) VH of sequence number 346 and VL of sequence number 347, c) VH of sequence number 348 and VL of sequence number 349, d) Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 361, HCDR2 of SEQ ID NO: 362, HCDR3 of SEQ ID NO: 363, Light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 367, LCDR2 of SEQ ID NO: 368, and LCDR3 of SEQ ID NO: 369 e) VH of sequence number 339 and VL of sequence number 340, f) HCDR1 of sequence number 361, HCDR2 of sequence number 362, HCDR3 of sequence number 363, LCDR1 of sequence number 367, LCDR2 of sequence number 368, and LCDR3 of sequence number 370, g) VH of sequence number 339 and VL of sequence number 341, h) VH of sequence number 339 and VL of sequence number 342, i) VH of sequence number 339 and VL of sequence number 343, j) VH of sequence number 339 and VL of sequence number 344, or k) The isolated protein according to claim 11, comprising VH of SEQ ID NO: 339 and VL of SEQ ID NO:

345.

13. The isolated protein according to claim 10, wherein the Fc region comprises at least one mutation resulting in reduced binding of the protein to the Fcγ receptor (FcγR).

14. The isolated protein according to claim 13, wherein the at least one mutation resulting in reduced binding of the protein to the FcγR is L234A / L235A / D265S, and the residue numbering follows the EU index.

15. The isolated protein according to claim 10, wherein the protein comprises at least one mutation in the Fc domain, the mutation promoting heavy chain heterodimerization.

16. The isolated protein according to claim 15, wherein the at least one mutation of the Fc domain is selected from the group consisting of T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W, and the residue numbering follows the EU index.

17. A pharmaceutical composition comprising the isolated protein described in claim 1 and a pharmaceutically acceptable carrier.

18. A polynucleotide encoding the isolated protein described in claim 1.

19. A vector comprising the polynucleotide described in claim 18.

20. A host cell comprising the vector according to claim 19.

21. A method for producing the isolated protein described in claim 1, comprising: culturing the host cells described in claim 20 under conditions for expressing the protein; and collecting the protein produced by the host cells.

22. A composition comprising the isolated protein according to claim 1 for treating HLA-G expressing cancer in a subject.

23. A composition comprising the isolated protein according to claim 1 for reducing the amount of HLA-G expressing tumor cells in a target.

24. The composition according to claim 22, wherein the HLA-G expressing cancer is lung cancer, pancreatic cancer, kidney cancer, head and neck cancer, ovarian cancer, esophageal cancer, colorectal cancer, uterine cancer, or breast cancer.

25. The isolated protein according to claim 1, comprising the amino acid sequence of sequence number 478.

26. The isolated protein according to claim 25, comprising the amino acid sequence of SEQ ID NO:

490.

27. The isolated protein according to claim 25, further comprising the amino acid sequences of SEQ ID NOs. 489 and 447.

28. The isolated protein according to claim 27, further comprising the amino acid sequence of SEQ ID NO: 439.

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