Bispecific antigen-binding protein

Abstract

The present invention relates to a bispecific antigen protein that induces endocytosis and lysosomal degradation of PD-L1 upon contact with cells expressing PD-L1 and RNF128. The present invention further relates to the use of such a bispecific antigen-binding protein in the treatment of cancer.

Description

Technology Field

[0001] The present invention relates to the field of medicine, particularly to oncology. Specifically, the present invention relates to a bispecific antigen-binding protein that induces the endocytosis and lysosomal degradation of PD-L1 upon contact with cells expressing PD-L1 and RNF128. The present invention further relates to the use of such a bispecific antigen-binding protein in the treatment of cancer. Background Technology

[0002] Cells communicate with their environment through the activity of plasma membrane-embedded receptors that capture external chemical signals and initiate intracellular signaling cascades to induce cellular responses. Receptor availability on the cell surface is a critical determinant of signal specificity and sensitivity, and the dysregulation of these events is frequently associated with the development or progression of therapy resistance, as well as diseases such as cancer, autoimmune diseases, neurological disorders, and inflammatory disorders.

[0003] For example, mutational activation or overexpression of receptors is a major and widely recognized cancer-promoting mechanism in many tissues (e.g., EGFR, ERBB2, PDGFR, TGFβR, IGFR1, GHR, FZD, LRP6). Due to the dependence of cancer cells on abnormal receptor activity, various neutralizing antibodies and small molecule inhibitors have been developed. However, successful neutralization of receptor activity requires the production of potent binders capable of reaching plasma concentrations sufficient to demonstrate high efficacy without inducing toxicity, which can prove difficult in the case of non-covalent interactors. Furthermore, compensatory receptor stabilization or upregulation is a major resistance pathway.

[0004] Post-translational modification of the cytosolic domain of membrane-bound receptors using ubiquitin leads to their rapid removal from the cell surface via induced endocytosis. The internalized receptors may subsequently undergo lysosomal degradation. In healthy stem cells, high levels of Wnt signaling induce the expression of two homologous membrane-bound ubiquitin ligases, RNF43 and ZNRF3, which are known to mediate the removal and ubiquitination of Frizzled (FZD), a receptor for Wnt, from the cell surface (Koo et al, Nature 2012, 488(7413):665-9). Thus, this negative feedback loop plays a role in regulating the sensitivity of stem cells to Wnt by controlling the effective number of Frizzled (FZD) receptors on the cell surface. The activity of RNF43 / ZNRF3 toward FZD is neutralized in stem cell niches by secreted protein R-spondin (RSPO), which forms a complex with RNF43 / ZNRF3 as well as LGR4 / 5 receptors (Hao et al, Nature 2012, 485(7397):195-200). Subsequently, this trimer RSPO-LGR4 / 5-RNF43 / ZNRF3 complex is cleared from the cell surface, leading to the stabilization of FZD receptor expression and increased levels of Wnt signaling. Wnt signaling is frequently misregulated in cancers. These cancers exhibit increased expression of Wnt target genes, including RNF43 and ZNRF3.

[0005] E3 ubiquitin ligase recruits ubiquitin-loaded E2 ubiquitin-conjugating enzymes to protein substrates and assists or directly catalyzes the transfer of ubiquitin to protein substrates.

[0006] Ubiquitination of receptors mediated by transmembrane ubiquitin E3 ligases is known to result in endocytosis and subsequent degradation of the ubiquitinated substrate. It is known in the art that such degradation preferably occurs in lysosomes. Lysosomal degradation requires the ligation of monoubiquitin, multiubiquitin, Lys11-, Lys29-, Lys48-, or Lys63-linked polyubiquitin chains into membrane-coupled receptors. This is in contrast to the activity of cytosolic ubiquitin ligases, which utilize the proteasome degradation pathway, namely, through the coupling of Lys11-, Lys29-, or Lys48-linked polyubiquitin chains into cytosolic target proteins.

[0007] Therefore, transmembrane E3 ubiquitin ligases can interact with different members of the E2 enzyme family to selectively target membrane-bound substrates. The ubiquitinated substrate will be internalized and then preferably degraded through lysosome degradation.

[0008] There is still a strong need in the industry to effectively target membrane-coupled receptors, particularly those involved in the development or progression of diseases, and to inhibit their activity. In particular, there is a strong demand in the industry to effectively target membrane-coupled receptors involved in the development or progression of, for example, cancer, autoimmune diseases, neurological disorders, rare diseases, and inflammatory disorders, as well as therapy resistance, and to inhibit their activity.

[0009] In a first embodiment, the present invention relates to a bispecific antigen-binding protein comprising the following:

[0010] i) a first immunoglobulin monovariable domain (ISVD) that specifically binds to the extracellular portion of RNF128 transmembrane E3 ubiquitin ligase (RNF128); and

[0011] ii) A second ISVD that specifically binds to the extracellular portion of a tumor-associated antigen (TAA).

[0012] In one embodiment, in a bispecific antigen-binding protein,

[0013] i) The first ISVD is

[0014] a) a complementary-determining region (CDR)1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3;

[0015] b) CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4, CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6; and c) CDR1 comprising the amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 9.

[0016] It includes a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from a group consisting of;

[0017] ii) The second ISVD is PD-L1, PD1, PSCA, L1-CAM, EpCAM, HER2 (ErbB2 / Neu), Receptor Tyrosine Kinase-like Rare Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171, CTLA-4 (CD152), PSMA (Prostate-specific Membrane Antigen), BCMA, TROP2, STEAP1, CD52, CD56, CD80, CD70, E-Selectin, EphB2, EPHA4, Melanotransferrin, Mud 6, TMEFF2, Killer Ig-like Receptor, Killer Ig-like Receptor 3DL2 (KIR3DL2), B7.1, B7.2, B7-H3, B7-H4, B7-H6, IL-6 receptor, IL-1 accessory protein, MAGE, MART-1 / Melan-A, gp100, MICA, MICB, Colorectal-Rectal Associated Antigen (CRC)-C017-1A / GA733, Protein Tyrosine Kinase 7 (PTK7), Receptor Protein Tyrosine Kinase 3 (TYRO-3), NaPi2b, TYRP1, Nectin-4, UL16-Binding Protein (ULBP), RAET1 Protein, Carcinoembryonic Antigen (CEA), CEACAM5, etv6, Prostate-Specific Antigen (PSA), T-cell receptor / CD3-Zeta chain, MAGE-A3, GAGE-Tumor Antigen, Anti-Müllerian Hormone Type II Receptor, Delta-like Ligand 3 (DLL3), Delta-like Ligand 4(DLL4), DR5, NTRKR1(EC 2.7.10.1), SLAMF7, TRAILR1, TRAILR2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC1, MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR / ERBB1), HER-3 / ERBB3, HER-4 / ERBB4, heteromeric receptor composed of at least one HER subunit, gastrin-releasing peptide receptor antigen, cMET, CA125, integrin receptor, a5β3 integrin, a5β1 integrin, α-llbβ3-integrin, PDGF alpha receptor, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, IL-1 accessory protein, CSF1R, α-fetal protein, mesothelin (MSLN), Claudin-18 isotype 2 (Claudin 18.2, CLDN18), folate receptor alpha (FRα, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, Flexin-A1, TNFRSF10B, AXL, EDNRB, OLR1, ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1, cdc27, CDCP1, fodrin, Connexin 37, Ig-ideotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, imp-1, P1A, SCP-1 CT-7, c-erbB-2, FcRL5 / FcRH5, Flt3, muc16, muc17, mmp9, It specifically binds to a TAA selected from the group consisting of FAP, Lewis-Y, EGFRvIII, GPC3, GPRC5D, gpA33, 5T4, SSTR2, CD73, CD25, CD45, and CD133.

[0018] In a further embodiment, the present invention provides a bispecific antigen-binding protein comprising the following:

[0019] i) A first immunoglobulin monovariable domain (ISVD) that specifically binds to the extracellular portion of RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the first ISVD is

[0020] a) a complementary-determining region (CDR)1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3;

[0021] b) CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4; CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5; and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6; and

[0022] c) CDR1 comprising the amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 9.

[0023] A first immunoglobulin monovariable domain (ISVD) comprising a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of;

[0024] and

[0025] ii) a second ISVD that specifically binds to the extracellular portion of PD-L1, wherein the second ISVD is

[0026] d) a complementary-determining region (CDR) 1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR 2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and a CDR 3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 12;

[0027] e) CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13, CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 15;

[0028] and

[0029] f) CDR1 comprising the amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 18 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 18

[0030] A second ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from a group consisting of

[0031] In one embodiment, in a bispecific antigen-binding protein,

[0032] i) The first ISVD is

[0033] a) Amino acid sequence listed in SEQ ID NO: 19;

[0034] b) the amino acid sequence listed in SEQ ID NO: 20; and

[0035] c) Amino acid sequence listed in SEQ ID NO: 21

[0036] It comprises an amino acid sequence selected from the group consisting of, and an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

[0037] ii) The 2nd ISVD is

[0038] d) Amino acid sequence listed in SEQ ID NO: 22;

[0039] e) The amino acid sequence listed in SEQ ID NO: 23; and

[0040] f) Amino acid sequence listed in SEQ ID NO: 24

[0041] It includes an amino acid sequence selected from the group consisting of, and an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0042] In one embodiment, at least one of the first and second ISVDs is humanized.

[0043] In one embodiment, i) the first humanized ISVD is

[0044] a) Amino acid sequence listed in SEQ ID NO: 114;

[0045] b) Amino acid sequence listed in SEQ ID NO: 115;

[0046] c) the amino acid sequence listed in SEQ ID NO: 116; and

[0047] d) Amino acid sequence listed in SEQ ID NO: 117

[0048] An amino acid sequence selected from the group consisting of, and an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

[0049] and ii) the second humanized ISVD is

[0050] e) The amino acid sequence listed in SEQ ID NO: 108; and

[0051] f) Amino acid sequence listed in SEQ ID NO: 110

[0052] It is at least one of an amino acid sequence selected from the group consisting of, and an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0053] In one embodiment, i) the first humanized ISVD comprises an amino acid sequence having at least one of SEQ ID NO: 114, 115, 116 and 117, and ii) the second humanized ISVD comprises an amino acid sequence having at least one of SEQ ID NO: 108 and 110.

[0054] In one embodiment, the bispecific antigen-binding protein is

[0055] i) a first polypeptide comprising a first human Fc fragment and a first ISVD; and

[0056] ii) A second polypeptide comprising a second human Fc fragment and a second ISVD

[0057] It is a bispecific antibody containing,

[0058] Preferably, the antibody does not contain an antibody light chain.

[0059] In one embodiment, each Fc fragment comprises a CH2 domain and a CH3 domain. In one embodiment, the first Fc fragment and the second Fc fragment comprise different amino acid sequences comprising a complementary knob-into-hole modification that promotes the association of the first and second polypeptide chains of the Fc region to form a heteromeric Fc region.

[0060] In one embodiment, the bispecific antigen-binding protein has at least one biological activity selected from the following:

[0061] a) Bispecific antigen-binding proteins induce endocytosis and lysosomal degradation of PD-L1 when in contact with cells expressing PD-L1 and RNF128; and

[0062] b) When a bispecific antigen-binding protein is added to a co-culture of tumor cells and human PBMCs, it induces at least one of the secretion of cytokines and the death of tumor cells expressing PD-L1 and RNF128, wherein the secreted cytokines include at least one of IL-2 and interferon γ.

[0063] In a further embodiment, the present invention provides a pharmaceutical composition comprising a bispecific antigen-binding protein as described herein and a pharmaceutically acceptable carrier.

[0064] In another additional aspect, the present invention provides a bispecific antigen-binding protein as described herein for use as a medicine, or a pharmaceutical composition as described herein. In one embodiment, a bispecific antigen-binding protein as described herein, or a pharmaceutical composition as described herein, for use in the treatment of cancer, preferably cancer comprising tumor cells expressing PD-L1.

[0065] In another additional aspect, the present invention provides a nucleic acid molecule comprising one or more nucleotide sequences encoding at least one polypeptide chain of a bispecific antigen-binding protein as described herein, preferably one or more nucleotide sequences are operably linked to a regulatory sequence for the expression of one or more polypeptide chains in a host cell.

[0066] In another additional aspect, the present invention provides a host cell comprising a nucleic acid molecule as described herein.

[0067] In another additional aspect, the present invention provides a method for producing a bispecific antigen-binding protein as described herein, the method comprising the step of culturing a host cell as described herein so that one or more nucleotide sequences are expressed and a bispecific antigen-binding protein is produced. In one embodiment, the present invention further comprises the step of recovering the bispecific antigen-binding protein, and optionally, the step of formulating the bispecific antigen-binding protein using a pharmaceutically acceptable carrier.

[0068] In another additional aspect, the present invention provides an RNF128 binding protein comprising an immunoglobulin monovariable domain (ISVD) that specifically binds to the extracellular portion of an RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the ISVD comprises a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of:

[0069] a) CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 1, CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 3;

[0070] b) CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4; CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5; and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6; and

[0071] c) CDR1 comprising an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 7, CDR2 comprising an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 9.

[0072] In one embodiment, the immunoglobulin single variable domain comprises a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of:

[0073] a) CDR1 comprising the amino acid sequence described in SEQ ID NO: 1, CDR2 comprising the amino acid sequence described in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 3;

[0074] b) CDR1 comprising the amino acid sequence described in SEQ ID NO: 4, CDR2 comprising the amino acid sequence described in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 6; and

[0075] c) CDR1 comprising the amino acid sequence described in SEQ ID NO: 7, CDR2 comprising the amino acid sequence described in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 9.

[0076] In one embodiment, the ISVD comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from the group consisting of:

[0077] a) Amino acid sequence listed in SEQ ID NO: 19;

[0078] b) the amino acid sequence listed in SEQ ID NO: 20; and

[0079] c) Amino acid sequence listed in SEQ ID NO: 21.

[0080] In one embodiment, the ISVD comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence described in SEQ ID NO: 19.

[0081] In one embodiment, the ISVD is humanized. In one embodiment, the humanized ISVD comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from the group consisting of:

[0082] a) Amino acid sequence listed in SEQ ID NO: 114;

[0083] b) Amino acid sequence listed in SEQ ID NO: 115;

[0084] c) the amino acid sequence listed in SEQ ID NO: 116; and

[0085] d) Amino acid sequence listed in SEQ ID NO: 117.

[0086] Description of the present invention

[0087] definition

[0088] Various terms relating to the methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. Unless otherwise indicated, these terms are given their ordinary meanings in the field to which the present invention belongs. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein. Any method and material similar or equivalent to that described herein may be used in the practice of testing the present invention, but preferred materials and methods are described herein.

[0089] "One" ("a", "an") and "the" ("the"): These singular terms include multiple referents unless the content clearly indicates otherwise. Therefore, the indefinite article "one" ("a" or "an") generally means "at least one." Thus, for example, a reference to "one cell" includes a combination of two or more cells, etc.

[0090] “Approximately” and “roughly”: These terms, when referring to measurable values ​​such as quantities, temporal durations, etc., mean that they include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and much more preferably ±0.1% from the specified value, because such variation is appropriate for carrying out the disclosed method. Additionally, quantities, ratios, and other numerical values ​​are sometimes presented herein in the form of ranges. It will be understood that these range forms are used for convenience and brevity and should be flexibly understood to include not only the numerical values ​​explicitly specified as limits of the range, but also all individual numerical values ​​or sub-ranges included within the range, as each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include not only the explicitly stated limits of about 1 and about 200, but also individual ratios such as about 2, about 3 and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, etc.

[0091] "And / or": The term "and / or" refers to a situation in which one or more of the mentioned cases may occur alone or in combination with at least one of the mentioned cases and all of the mentioned cases.

[0092] "Comprising": This term is understood to be inclusive, open, and non-exclusive. Specifically, this term and its variations mean that the specified features, stages, or components are included. These terms should not be interpreted as excluding the presence of other features, stages, or components.

[0093] "Exemplary": This term means "serving as an example, case, or illustration" and should not be interpreted as excluding other forms disclosed herein.

[0094] As used herein, “cancer” and “cancerous” refer to or describe physiological diseases in mammals characterized by typically uncontrolled cell growth. Cancer is also referred to as a malignant neoplasm.

[0095] As used herein, "in combination with" is intended to refer to any form of administration in which the first drug is provided together with additional (second, third) drugs. The drugs may be administered simultaneously, individually, sequentially, or in any order. The drugs administered in combination have biological activity in the target to which the drugs are delivered.

[0096] As used herein, "simultaneous" administration refers to the simultaneous administration of more than one drug, but not necessarily through the same route of administration or in the form of a single combination formulation. For example, one drug may be administered orally, while another drug may be administered intravenously during the patient's hospital visit. "Separate" includes administering drugs in separate forms and / or at separate times, but again, not necessarily through the same route of administration. "Sequential" indicates that the administration of the second drug follows immediately or at a time after the administration of the first drug.

[0097] As used herein, “compositions,” “products,” or “combinations” useful for the methods of the present disclosure include those suitable for various routes of administration, including but not limited to intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and / or parenteral or mucosal application. Compositions, formulations, and products according to the present disclosure typically comprise a drug (alone or in combination) and one or more suitable pharmaceutically acceptable excipients.

[0098] As used herein, "effective dose" refers to the amount of agent required to improve the symptoms of a disease compared to an untreated patient. The effective dose of the agent(s) used to carry out the invention for the therapeutic treatment of cancer depends on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as the "effective" dose. Accordingly, in the context of this disclosure, regarding the administration of a drug that is "effective against" a disease or condition, it indicates that administration in a clinically appropriate manner results in beneficial effects for at least a statistically significant fraction of patients, such as improvement of symptoms, cure, reduction of at least one sign or symptom of the disease, extension of life, improvement of quality of life, or other effects generally recognized positively by a physician familiar with treating a particular type of disease or condition.

[0099] "Sequence identity" is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences determined by comparing sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, such as as determined by the match between these sequence strings in some cases. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substituents with the sequence of a second polypeptide. "Identity" and "similarity" can be easily calculated by known methods. The terms “sequence identity” or “sequence similarity” mean that two (poly)peptide or two nucleotide sequences share at least a certain percentage of sequence identity as defined elsewhere in this invention when optimally aligned, preferably over the entire length (at least the shortest sequence in comparison), and when the number of matches is maximized and the number of gaps is minimized, for example, by programs ClustalW (1.83), GAP, or BESTFIT using default parameters. GAP aligns two sequences over their entire length using Needleman and Wunsch full alignment algorithms to maximize the number of matches and minimize the number of gaps. Generally, GAP default parameters are used with a gap generation penalty = 50 (nucleotides) / 8 (proteins) and a gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning protein sequences of the present invention is ClustalW (1) using the Blosum matrix and default settings.83) is (Gap opening penalty: 10; Gap extension penalty: 0.05). Sequence alignment and scoring for the percentage of sequence identity can be determined using a computer program, such as the GCG Wisconsin package, version 10.3 available from Accelrys Inc. (9685 Scranton Road, San Diego, CA 92121-3752 USA), or using open source software, such as EmbossWIN version 2.10.0, using the program “needle” (using the whole Needle-only Bunsch algorithm) or “water” (using the local Smith-Waterman algorithm), using the same parameters as for the GAP, or using default settings (for both “needle” and “water”, and for both protein and DNA alignment, the default gap opening penalty is 10.0 and the default gap extension penalty is 0.5; the default scoring matrix is ​​Blosum62 for protein and DNAFull for DNA). When sequences have substantially different total lengths, it is desirable to use local alignment, such as the Smith-Waterman algorithm. Alternatively, the similarity or identity percentage can be determined by searching a public database using algorithms such as FASTA, BLAST, etc.

[0100] Optionally, in determining the degree of amino acid similarity, those skilled in the art may also consider so-called "conservative" amino acid substitutions, which will be obvious to those skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are provided in the table below.

[0101]

[0102] Alternative conservative amino acid residue substitution class.

[0103]

[0104] Alternative physical and functional classification of amino acid residues.

[0105]

[0106] The term "agent" generally refers to any entity that is not normally present or is not present at a level administered to a cell, tissue, or target. An agent may be a compound or a composition. An agent may be selected from the group consisting of, for example: polynucleotides, polypeptides, small molecules, (bispecific) antigen-binding proteins, e.g., antibodies and functional fragments thereof.

[0107] The term "antigen-binding domain" or "antigen-binding region" refers to a portion of an antigen-binding protein capable of specifically binding to an antigen or epitope. In one embodiment, the antigen-binding region is, for example, an antibody light chain variable region (V L ) and antibody heavy chain variable region (V H It is an immunoglobulin-derived antigen-binding region comprising both. Examples of such antigen-binding regions include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region from a single-domain antibody consisting only of a heavy chain and lacking a light chain, for example, as known from camels, and the antigen-binding site exists on and is formed by a single variable domain (also referred to as an "immunoglobulin single variable domain" or "ISVD"). An example of such an ISVD is the single variable domain of a camel heavy-chain antibody (V), also referred to as a nanobody. HIt includes a single domain (IgNAR domain) derived from a H), a domain antibody (dAb), and a shark antibody. In another embodiment, the antigen-binding region includes a non-immunoglobulin-derived domain capable of specifically binding to an antigen or epitope, such as darpin; affilin; antikalin, etc.

[0108] In this document, the term “antibody” is used in its broadest sense and includes, specifically, full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, insofar as they exhibit the desired biological and / or immunological activity. Various techniques related to the production of antibodies are provided, for example, in the literature [Harlow, et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988)]. Antibodies may be human and / or humanized. A “humanized” form of a non-human (e.g., rodent) antibody is a chimeric antibody containing a minimal sequence derived from a non-human antibody.

[0109] The terms “whole-length antibody,” “intact antibody,” and “total antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of an intrinsic antibody. “Intrinsic antibody” refers to naturally occurring immunoglobulin molecules having various structures. For example, an intrinsic IgG-class antibody is a heterotemeric glycoprotein of about 150,000 daltons composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light chain domain or light chain variable domain, followed by a light chain constant domain (CL), also called the light chain constant domain. The heavy chain of the antibody may be designated as one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or m(IgM), some of which may be further divided into subtypes, e.g., γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of the antibody may be designated as one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0110] "Antibody fragment" comprises a portion of a full-length antibody, for example, its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)s, and Fv (typically V of a single arm of the antibody). H and V L Domain), single-chain Fv(scFv), dsFv, Fd fragment (typically V H and CH1 domain), and dAb (typically V H Domain) Fragment; V H , V L , VH H and V-NAR domains; minibodies, diabodies, triabdies, tetrabodies, and kappabdies (e.g., literature [Ill et al. Protein Eng [Refer to 1997;10: 949-57]); camel IgG; IgNAR; and antibody fragments, and multispecific antibody fragments formed from one or more isolated CDRs or functional paratopes, wherein the isolated CDRs or antigen-binding residues or polypeptides may be assembled or linked together to form functional antibody fragments. For a review of specific antibody fragments, refer to [Hudson et al., Nat Med 9, 129-134 (2003)]. For a review of scFv fragments, for example, [ See [in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NY, pp. 269-315 (1994)]; also see WO 93 / 16185; and U.S. Patents No. 5,571,894 and No. 5,587,458. For the discussion of Fab and F(ab')2 fragments containing rescue receptor-binding epitope residues and having increased in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments having two antigen-binding sites that may be divalent or bispecific, e.g., EP 404,097; WO 1993 / 01161; and reference [Hudson et al., Nat Med 9, 129-134 (2003)]; and refer to the literature [Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993)]. Triabadies and tetraabadies are also described in the literature [Hudson et al., Nat Med 9, 129-134 (2003)]. Various types of antibody fragments are described or reviewed, for example, in the literature [Heiliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136]; WO2005 / 040219, US20050238646, and US20020161201. Antibody fragments are, without limitation, proteolytic digestion of intact antibodies as well as recombinant host cells as described herein (e.g., CHO, E. coli ( E. coli It can be manufactured by various techniques including production by ) or waste paper).

[0111] As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and does not refer to the method by which it is produced. Monoclonal antibodies may be produced using a wide variety of techniques known in the art, including the use of hybridomas, recombinant and phage display technologies, or combinations thereof. For example, monoclonal antibodies may be produced using hybridoma techniques known in the art, including those taught in the literature [Harlow and Lane, “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, NY (1988)]; and literature [Hammerling et al., in: “Monoclonal Antibodies and T-Cell Hybridomas,” Elsevier, NY (1981), pp. 563-681] (both of which are incorporated herein by reference in their entirety).

[0112] As used herein, the term “single-specific” antibody indicates that the antibody portion of a multispecific antigen-binding protein as described herein has one or more antigen-binding sites that each bind to the same epitope of the same antigen. The term “bispecific” means that the antibody portion of a multispecific antigen-binding protein as described herein has at least two antigen-binding sites capable of specifically binding to at least two distinct epitopes. Typically, a bispecific antigen-binding molecule comprises two antigen-binding sites, each of which is specific to a different epitope. In certain embodiments, a bispecific antigen-binding molecule may simultaneously bind to two epitopes, particularly two epitopes expressed on two distinct cells.

[0113] As used in this application, the terms “valence” or “valence” indicate the presence of a specific number of binding sites within an antigen-binding molecule. As such, the terms “divalence,” “sagaci,” and “hexavalence” indicate the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule.

[0114] Antibodies immunologically reactive to a specific antigen can be produced by recombinant methods, such as the selection of a recombinant antibody library in phages or similar vectors (see, e.g., [Huse et al., Science 246:1275-1281 (1989)]; [Ward et al., Nature 341:544-546 (1989)]; and [Vaughan et al., Nature Biotech. 14:309-314 (1996)]), or by the immunization of animals using antigens or DNA encoding antigens. Methods for the production and screening of specific antibodies using hybridoma technology are routine in the art and widely known. In non-limiting examples, mice may be immunized with antigens of interest or cells expressing such antigens. When an immune response is detected—for example, when antibodies specific to the antigen are detected in mouse serum—mouse spleens are collected and splenocytes are isolated. Next, splenocytes are fused with any suitable myeloma cells by a well-known technique. The hybridoma is selected and cloned by limiting dilution. Subsequently, the hybridoma clone is tested against cells secreting antibodies capable of binding to the antigen by a method known in the art. An ascites fluid containing generally high levels of antibodies can be produced by inoculating a mouse with a positive hybridoma clone intraperitoneally.

[0115] Typically, immunoglobulins have heavy and light chains. Each heavy and light chain contains a constant region and a variable region (these regions are also known as "domains"). The variable regions of the light and heavy chains contain four "framework" regions interrupted by three hypervariable regions, also called "complementary-determining regions" or "CDRs." The sequences of the framework regions of different light or heavy chains are relatively conserved within the species. The framework region of an antibody, which is a combined framework region of the constituent light and heavy chains, serves to localize and align the CDRs in three-dimensional space.

[0116] As used herein, the term "hypervariable region" refers to the amino acid residue of an antibody responsible for antigen binding. The hypervariable region generally consists of amino acid residues from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; literature [Kabat et al. 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, USA]) and / or corresponding residues from the "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and residues 26-32 (H1), 53-55 (H2) of the heavy chain variable domain) and 96-101(H3); includes the literature [Chothia and Lesk, J. Mol. Biol 1987;196:901-917]). Typically, the numbering of amino acid residues in this region is performed by the method described in the literature of Kabat et al. As described herein, phrases such as “Kabat position,” “variable domain residue numbering as in Kabat,” and “according to Kabat” refer to this numbering system for a heavy chain variable domain or a light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to the shortening or insertion into the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insert following residue 52 of CDR H2 (residue 52a according to Kavat) and an inserted residue following heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kavat, etc.).The kavat numbering of residues can be determined for a given antibody by aligning them with the "standard" kavat-numbered sequences in the homology regions of the antibody sequence.

[0117] As used herein, the terms “framework” or “FR” residue refer to regions of the antibody variable domain excluding regions defined as CDRs. Each antibody variable domain framework may be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4).

[0118] The term “constant region” as defined herein refers to an antibody-derived constant region encoded by one of the light chain or heavy chain immunoglobulin constant region genes. “Constant light chain” or “light chain constant region” as used herein refers to a region of an antibody encoded by the kappa (CK) or lambda (Cλ) light chain. The constant light chain typically comprises a single domain and, as defined herein, refers to positions 108 through 214 of Cκ or Cλ, numbered according to the EU index (Ibid. [Kabat et al., 1991]).

[0119] As used herein, the terms “constant heavy chain” or “constant heavy chain region” refer to a region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon gene for defining the isotype of the antibody as IgM, IgD, IgG, IgA, or IgE, respectively. In the case of a full-length IgG antibody, the constant heavy chain as defined herein refers to the region from the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, and accordingly includes positions 118 to 447, and the numbering follows the EU index.

[0120] Papain degradation of an intact antibody produces two identical antigen-binding fragments referred to as the “Fab” fragment, each containing a heavy chain variable domain and a light chain variable domain, as well as a light chain constant domain and a heavy chain first constant domain (CH1). Accordingly, the terms “Fab fragment” or “Fab region” as used herein refer to a light chain fragment containing a VL domain and a light chain constant domain (CL), and an antibody fragment containing a VH domain and a heavy chain first constant domain (CH1). Fab may refer to this isolated region, or this region associated with a polypeptide, a multispecific antigen-binding protein, or an antigen-binding region, or any other embodiment as outlined herein. The Fab’ fragment differs from the Fab fragment in the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, which contains one or more cystes derived from the antibody hinge region. Fab’-SH is a Fab’ fragment in which the cysteine ​​residue(s) of the constant domain possess free thiol groups. Pepsin treatment provides an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0121] As used herein, the terms "single-chain Fv" or "scFv" refer to the V of the antibody. H and V L It refers to antibody fragments containing domains, and these domains exist on a single polypeptide chain. Generally, Fv polypeptides are V H and V LIt additionally includes polypeptide linkers between the domains, which enables scFv to form a desired structure for antigen binding. Methods for producing scFv are widely known in the art. For a review of methods for producing scFv, refer to the literature [Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, NY, pp. 269-315 (1994)].

[0122] "Scaffold antigen-binding proteins" are known in the art, and, for example, fibronectin and engineered ankyrin repeat protein (DARPin) have been used as alternative scaffolds for antigen-binding domains, see, for example, the literature [Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009)] and the literature [Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008)]. In one aspect of the present invention, the scaffold antigen-binding protein is CTLA-4 (Evibody), lipocalin (Anticalin), a protein A-derived molecule, e.g., the Z-domain of protein A (Affibody), A-domain (Avimer / Maxibody), serum transferrin (Trans-body ( trans -body)); designed ankyrin repeat protein (DARPin), variable domain of antibody light or heavy chain (single-domain antibody, sdAb), variable domain of antibody heavy chain (nanobody, aVH), V NARFragment, fibronectin (AdNectin), C-type lectin domain (Tetranectin); variable domain of the novel antigen receptor beta-lactamase (V NAR It is selected from the group consisting of fragments), human gamma-crystalline or ubiquitin (Affilin molecule); Kunitz-type domains of human protease inhibitors, microbodies, proteins such as knottin, peptide aptamers, and fibronectin.

[0123] Programmed death-ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein. The binding of PD-L1 to its receptor PD-1 on T cells transmits a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation.

[0124] The ubiquitin E3 ligase gene (GRAIL) (Rnf128), which is associated with anergy in lymphocytes, is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger.

[0125] Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, bilines, retinoids, and lipids. They have a rigid beta-sheet secondary structure with multiple loops at the open ends of a conical structure that can be engineered to bind to different target antigens. Anticalins are 160 to 180 amino acids in size and are derived from lipocalins. For further details, refer to the literature [Biochim Biophys Acta 1482: 337-350 (2000)], US7250297B1, and US20070224633.

[0126] The affibody is a scaffold derived from protein A of Staphylococcus aureus that can be engineered to bind to antigens. The domain consists of a 3-helix bundle of approximately 58 amino acids. The library was generated by randomizing surface residues. For further details, refer to the literature [Protein Eng. Des. Sel. 17, 455-462 (2004)] and EP1641818A1.

[0127] Avimer is a multidomain protein derived from an A-domain scaffold. The domain of approximately 35 amino acids adopts a defined disulfide bond structure. Varieties are generated by the shuffling of natural variations exhibited by the A-domain. For further details, refer to [Nature Biotechnology 23(12), 1556–1561 (2005)] and [Expert Opinion on Investigational Drugs 16(6), 909–917 (June 2007)].

[0128] Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to different target antigens by inserting a peptide sequence within an allowable surface loop. Examples of engineered transferrin scaffolds include transbodies. For further details, refer to the literature [J. Biol. Chem 274, 24066-24073 (1999)].

[0129] The designed ankyrin repeat protein (DARPin) is derived from ankyrin, a protein that mediates the attachment of membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and beta-turns. They can be engineered to bind to different target antigens by randomizing the residues within the first alpha-helice and beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, refer to [J. Mol. Biol. 332, 489-503 (2003)], [PNAS 100(4), 1700-1705 (2003)], [J. Mol. Biol. 369, 1015-1028 (2007)], and US20040132028A1.

[0130] A single-domain antibody is an antibody fragment composed of a single monomeric variable antibody domain. The first single variable domain is the variable domain (nanobody or V) of an antibody heavy chain of camelid origin. H It originated from the H fragment). Additionally, the term single-variable domain antibody refers to the autonomous human heavy chain variable domain (aVH) or V derived from sharks. NAR Includes short stories.

[0131] Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectin consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeat units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich can be engineered so that adnectin specifically recognizes a therapeutic target of interest. For further details, refer to the literature [Protein Eng. Des. Sel. 18, 435-444 (2005)], US20080139791, WO 2005056764, and US6818418B1.

[0132] Peptide aptamers are combinatorial recognition molecules composed of a fixed scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted into an active site. For further details, refer to [Expert Opin. Biol. Ther. 5, 783-797 (2005)].

[0133] The microbody is derived from naturally occurring microproteins of length 25 to 50 amino acids containing 3 to 4 cysteine ​​crosslinks—examples of microproteins include KalataBI, conotoxin, and knottin. The microprotein has a loop that can be engineered to contain up to 25 amino acids without affecting the entire fold of the microprotein. For further details of the engineered notin domain, refer to WO 2008098796.

[0134] As used herein, the terms "Fv," "Fv fragment," or "Fv region" refer to the V of a monoclonal antibody. H and V L It refers to a polypeptide containing a domain.

[0135] As used herein, the term “Fc” or “Fc region” refers to a polypeptide comprising the invariant region of an antibody, excluding the first invariant region immunoglobulin domain. Fc may refer to this isolated region, or this region associated with the Fc polypeptide, as described below. As used herein, “Fc polypeptide” or “Fc-derived polypeptide” means a polypeptide comprising all or part of the Fc region. The Fc polypeptide of the present invention comprises, but is not limited to, antibodies, Fc fusions, and Fc fragments. Additionally, the Fc region according to the present invention comprises variants containing at least one modification that alters (enhances or reduces) Fc-associated effector function. Additionally, the Fc region according to the present invention comprises a chimeric Fc region comprising different parts or domains of different Fc regions, for example, derived from antibodies of different isotypes or species. Therefore, Fc refers to the last two constant-region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant-region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus for these domains. In the case of IgA and IgM, Fc may include a J chain. In the case of IgG, Fc includes the immunoglobulin domains Cγ2 (CH2) and Cγ3 (CH3) and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is typically defined to include residues C226, P230, or A231 at its amino-terminus, and the numbering follows the EU index. The "CH2 domain" of the human IgG Fc region usually extends from the amino acid residue at position approximately 231 to the amino acid residue at position approximately 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain of the present invention may be a native sequence CH2 domain or a variant CH2 domain.The “CH3 domain” comprises a residue stretch at the C-terminus relative to the CH2 domain within the Fc region (i.e., from the amino acid residue at position approximately 341 of IgG to the amino acid residue at position approximately 447). The CH3 region of the present invention may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an “overhang” (“knob”) introduced on one chain thereof and a corresponding “cavity” (“hole”) introduced on the other chain thereof; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Such variant CH3 domains may be used to promote the heteromerization of two unequal antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 of the heavy chain to the carboxyl-terminus. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or invariant region follows the EU numbering system, also known as the EU index, as described in the literature [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991].

[0136] "Knob-into-hole" technology is described, for example, in US Patent No. 5,731,168; US Patent No. 7,695,936; [Ridgway et al., Prot Eng 9, 617-621 (1996)] and [Carter, J Immunol Meth 248, 7-15 (2001)]. Generally, the method comprises introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") at the interface of a second polypeptide so that the protrusion can be positioned within the cavity to promote heterodimer formation and hinder homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and cavity may be produced by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In certain embodiments, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc region, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc region. In further specific embodiments, the subunit of the Fc region containing the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc region containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues forms a disulfide crosslink between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Numbering is from Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed.It is listed in [Public Health Service, National Institutes of Health, Bethesda, MD, 1991].

[0137] "Regions equivalent to the Fc region of immunoglobulin" are intended to include naturally occurring allelic variants of the Fc region of immunoglobulin, and variants having modifications that cause substitution, addition, or deletion but do not substantially reduce the immunoglobulin's ability to mediate effector function (e.g., antibody-dependent cytotoxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulin without substantial loss of biological function. Such variants may be selected according to general rules known in the field to have minimal effect on activity (e.g., see literature [Bowie, JU et al., Science 247:1306-10 (1990)]).

[0138] The term "effector function" refers to biological activity that may be contributed to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0139] "Activated Fc receptors" are Fc receptors that, following binding to the Fc region of an antibody, stimulate receptor-bearing cells to trigger signaling events that perform effector functions. Activated Fc receptors include FcγRIIIa (CD16A), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89). A specific activated Fc receptor is human FcγRIIIa, also referred to as CD16 or CD16A (see UniProt accession number P08637, version 141). In humans, CD16 consists of two isoforms, CD16A and CD16B, encoded by two highly homologous genes. CD16A is a transmembrane protein expressed by lymphocytes and some monocytes, whereas CD16B is attached to the plasma membrane via a GPI anchor and is primarily expressed by neutrophils. Therefore, when CD16 is referred to in relation to expression on NK cells of this invention, it generally means CD16A unless otherwise specified.

[0140] The "variable region" as used herein refers to V constituting the light chain (including κ and λ) and heavy chain immunoglobulin loci, respectively. L (including Vκ and Vλ) and / or V H It refers to a region of an antibody containing one or more Ig domains substantially encoded by any of the genes. Light chain or heavy chain variable region (V L or V H It contains four conserved framework regions (FR) and three hypervariable regions (HVR). For example, see the literature [Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)]. A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0141] As used herein, the term “hypervariable region” or “HVR” refers to each region of the antibody variable domain whose sequence is hypervariable and / or forms a structurally defined loop (“hypervariable loop”). Typically, a native 4-strand antibody contains six HVRs; these are three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). The HVRs generally contain amino acid residues from the hypervariable loop and / or the “complementary determination region” (CDR), the latter having the highest sequence variability and / or being involved in antigen recognition. Exemplary hypervariable loops are present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3) (Reference [Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)]). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24 to 34 of L1, 50 to 56 of L2, 89 to 97 of L3, 31 to 35B of H1, 50 to 65 of H2, and 95 to 102 of H3. (Reference [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)]). The hypervariable region (HVR) is also referred to as the complementarity determining region (CDR), and these terms are used interchangeably herein in relation to the portion of the variable region that forms the antigen-binding region. This specific region is [Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)] and [Chothia et al., J.[Described in Mol. Biol. 196:901-917 (1987)], the definition includes subsets or overlaps of amino acid residues when compared to one another. Nevertheless, the application of any one definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the terms defined and used herein. Appropriate amino acid residues covering the CDR defined by each of the cited references are shown below in Table A for comparison. The exact residue number covering a specific CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a specific CDR by considering the amino acid sequence of the variable region of the antibody.

[0142] [Table A]

[0143] CDR definition 1

[0144]

[0145] Furthermore, Kabat et al. defined a numbering system for variable region sequences applicable to any antibody. Those skilled in the art can explicitly assign this “Kabat numbering” system to any variable region sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system presented in the literature [Kabat et al., US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983)]. Unless otherwise specified, references to the numbering of specific amino acid residue positions within the antibody variable region follow the Kabat numbering system.

[0146] Except for CDR1 in VH, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs also contain "specificity determining residues" or "SDRs," which are residues that come into contact with the antigen. SDRs are contained within a region of the CDR called abbreviated-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDRL2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31 to 34 of L1, 50 to 55 of L2, 89 to 96 of L3, 31 to 35B of H1, 50 to 58 of H2, and 95 to 102 of H3. (See reference [Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)]). Unless otherwise indicated, HVR residues and other residues within the variable domain (e.g., FR residues) are numbered herein according to the above kavat, etc.

[0147] As used herein, with respect to antigen-binding molecules (e.g., antibodies), the term “affinity matured” refers to an antigen-binding molecule derived from a reference antigen-binding molecule, for example by mutation, that binds to the same antigen, preferably to the same epitope as the reference antibody; and having a higher affinity for the antigen than the affinity of the reference antigen-binding molecule. Affinity maturation generally involves modification of one or more amino acid residues within one or more CDRs of the antigen-binding molecule. Typically, the affinity matured antigen-binding molecule binds to the same epitope as the initial reference antigen-binding molecule.

[0148] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and m, respectively.

[0149] The term "specifically binds" refers to the number of different types of antigens or antigenic determinants to which a specific antigen-binding region or antigen-binding protein can bind. The specificity of an antigen-binding protein may be determined based on affinity and / or binding strength. The equilibrium constant (K) for the dissociation of the antigen-binding protein and the antigen. D Affinity, denoted as ), is a measure of the binding strength between an epitope and an antigen-binding site on an antigen-binding protein. Alternatively, affinity is 1 / K D Phosphorus affinity constant (K A It may also be expressed as ). Affinity can be determined in a manner known in itself, depending on the specific combination of the antigen-binding protein of interest and the antigen. Binding strength is understood herein to refer to the strength of binding between a number of binding sites and the target molecule by a larger complex of binders, that is, the binding strength of multivalent bonds. Binding strength relates to both the affinity between the antigen determinant and its antigen-binding site on the antigen-binding protein, and the valence, that is, the number of binding sites present on the antigen-binding protein. On the other hand, affinity refers to a simple monovalent receptor ligand system.

[0150] Typically, the antigen-binding region or the region having affinity for the extracellular portion of the antigen of the bispecific antigen-binding protein of the present invention is about 10 -6 to 10 -12 M or less, preferably 10 -8to 10 -12 Dissociation constant less than or equal to M (K D ) and / or at least 10 -6 M or 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, for example, at least 10 -10 , 10 -11 , 10 -12 It will bind to its target molecule (antigen) with a binding affinity of M or greater. 10 -4 Any K greater than M (i.e., less than 100 μM) D The value is generally considered to indicate non-specific binding. Therefore, the antigen-binding region that "specifically binds" to the antigen can be determined as described herein 10 -4 K less than or equal to M D It is an antigen-binding domain that binds to an antigen at a value. Preferably, the antigen-binding region or the region having affinity for a surface antigen expressed on NK cells of the multispecific antigen-binding protein of the present invention will bind to a target molecule with an affinity of less than 800, 400, 200, 100, 50, 10, or 5 nM, more preferably less than 1 nM, e.g., less than 500, 200, 100, 50, 10, or 5 pM. Various methods for measuring binding affinity are known in the art, and any of these methods may be used for the purposes of the present invention (e.g., the literature [Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds.. Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, NY, (1992, 1993)], and literature[Muller, Meth. Enzymol.[See 92:589-601 (1983)]). Specific exemplary embodiments are described below.

[0151] "K D " or "K D The “value” can be measured by using ELISA as described in the examples herein, or by using surface plasmon resonance assay with BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ, USA) at 25°C with an antigen CM5 chip immobilized in about 10 to 50 reaction units (RU). In summary, the carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. The antigen is diluted to 5 μg / ml (about 0.2 μM) using 10 mM sodium acetate, pH 4.8, and then 5 μl / min Inject at a flow rate to achieve approximately 10 reaction units (RU) of coupled protein. After injection of the antigen, inject 1 M ethanolamine to block unreacted groups. For kinetic measurements, 2x serial dilutions (0.78 nM to 500 nM) of the antibody or Fab are injected into PBS (PBST) containing 0.05% Tween 20 at 25°C at a flow rate of approximately 25 μl / min. By simultaneously fitting association and dissociation sensigrams, the association rate (k) on ) and dissociation rate (k off ) is calculated using a simple 1-to-1 Langmuir coupled model (Viacore evaluation software version 3.2). The equilibrium dissociation constant (K D ) is non k off / k on It is calculated as. For example, refer to the literature [Chen, Y., et al., (1999) J.Mol Biol 293:865-881]. The on-rate is 10 by the above surface plasmon resonance assay.6 M -1 S -1 If the on-speed is exceeded, the on-speed can be determined by using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm passband) of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, as measured by a spectrophotometer, such as a stop-flow spectrophotometer (Aviv Instruments) or an 8000-series SLM-Aminco spectrophotometer with a stirred red cuvette (ThermoSpectronic).

[0152] The terms “humanized antibody” or “humanized immunoglobulin” refer to an immunoglobulin comprising a human framework, at least one, preferably all, complementarity determining regions (CDRs) of non-human antibody origin, and any existing invariant regions are substantially identical to human immunoglobulin invariant regions, i.e., at least about 85%, at least 90%, and at least 95% identical. Thus, possibly excluding the CDRs, all parts of the humanized immunoglobulin are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. Often, framework residues within the human framework regions will be substituted with corresponding residues of CDR donor antibody origin to alter, preferably, improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between the CDR and the framework residues to identify framework residues critical to antigen binding, and by sequence comparison to identify non-conventional framework residues at specific positions. For example, U.S. Patent No. 5,530,101; No. 5,585,089 of Queen et al.; See No. 5,693,761; No. 5,693,762; No. 6,180,370 (each of which is incorporated by reference in its entirety). Antibodies are, for example, for CDR-transplantation (EP 239,400; PCT Publication WO 91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; reference [Padlan, Mol. Immunol., 28:489 498 (1991)]; reference [Studnicka et al., Prot. Eng. 7:805 814 (1994)]; reference [Roguska et al., Proc. Natl. Acad. Sci.It may be humanized using various techniques known in the art, including [91:969 973 (1994)] and chain shuffling (U.S. Patent No. 5,565,332), all of which are incorporated herein by reference in their entirety.

[0153] One class of antigen-binding regions for use in the present invention has an amino acid sequence corresponding to the amino acid sequence of a naturally occurring single variable domain, that is, one or more amino acid residues within the amino acid sequence of said naturally occurring single variable domain sequence are V from a conventional 4-chain antibody of human origin. H It comprises a humanized immunoglobulin single variable domain (ISVD) by replacing one or more amino acid residues appearing at corresponding position(s) within the domain. This can be done in a manner known in itself, such as, for example, [Jones et al. (Nature 321:522-525, 1986)]; [Riechmann et al., (Nature 332:323-329, 1988)]; [Presta (Curr. Op. Struct. Biol. 2:593-596, 1992)], [Vaswani and Hamilton (Ann. Allergy, Asthma and Immunol., 1:105-115 1998)]; [Harris (Biochem. Soc. Transactions, 23:1035-1038, 1995)]; Prior art regarding humanization, including the literature [Hurle and Gross (Curr. Op. Biotech., 5:428-433, 1994)], and, for example, V such as the literature [Vincke et al. (2009, J. Biol. Chem. 284:3273-3284)] HIt will be apparent to those skilled in the art based on certain prior art related to the humanization of H. In other words, it should be noted that such humanized single variable domains of the present invention can be obtained in any suitable manner known in itself, and are therefore not strictly limited to polypeptides obtained using polypeptides containing naturally occurring single variable domains as starting materials.

[0154] "Framework" or "FR" refers to variable domain residues other than the hypervariable domain (HVR) residues. The FRs of the variable domain typically consist of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0155] For the purposes of the present invention, a “recipient human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or the human consensus framework as defined below. A recipient human framework “derived from” the human immunoglobulin framework or the human consensus framework may comprise the same amino acid sequence, or it may comprise amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL recipient human framework is sequence identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0156] As an alternative to humanization, human antibodies may be generated. "Human antibody" means an antibody produced by any of the known standard methods that contains entirely human light and heavy chains as well as an invariant region. For example, transgenic animals (e.g., mice) capable of producing the entire repertoire of human antibodies in the absence of endogenous immunoglobulin production upon immunization are available. For example, it has been described that homozygous deletion of the antibody heavy chain linkage region PH gene in chimeric and germline mutant mice results in the complete inhibition of endogenous antibody production. The delivery of a human germline immunoglobulin gene array to these germline mutant mice will result in the production of human antibodies after immunization. For example, the literature [Jakobovits et al ., Proc. Nat. Acad. Sci. USA, 90:255 1 (1993)]; Literature[Jakobovits et al Refer to ., Nature, 362:255-258 (1993)]. Alternatively, phage display technology (literature [McCafferty et alHuman antibodies and antibody fragments can be produced in vitro from a donor-derived immunoglobulin variable (V) domain gene repertoire using [Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993)]. According to this technique, antibody V domain genes are cloned into frames into major or secondary coat protein genes of filamentous bacteriophages, such as M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Since filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional characteristics of antibodies also results in the selection of genes encoding antibodies that exhibit these characteristics. Thus, phages mimic some characteristics of B cells. Phage display can be performed in various forms; for a review of these, refer, for example, to the literature [Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993)]. Human antibodies can also be generated by SCID mice having their immune system reconstituted with human cells or by in vitro activated B cells. Once the human antibody is obtained, its coding DNA sequence can be isolated, cloned, and introduced into a suitable expression system, i.e., preferably a cell line from a mammal, which subsequently expresses it and releases it into a culture medium from which the antibody can be isolated.

[0157] As used herein, the term "tumor-associated antigen (TAA)" means any antigen associated with cancer, including but not limited to proteins, glycoproteins, gangliosides, carbohydrates, and lipids. Such antigens may be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal matrix, or immune infiltrates. The term TAA explicitly includes homologs of wild-type TAA that result in a different and altered amino acid sequence, i.e., so-called neoantigens, as a result of tumor-specific mutations (which may be patient-specific or shared).

[0158] "Nucleic acid construct" or "nucleic acid vector" is understood herein to mean an artificial nucleic acid molecule resulting from the use of recombinant DNA technology. Thus, while a nucleic acid construct may contain naturally occurring nucleic acid molecules (or parts thereof), the term "nucleic acid construct" does not contain naturally occurring nucleic acid molecules. The terms “expression vector” or “expression construct” refer to nucleic acid molecules capable of causing the expression of a nucleotide sequence or gene in a host cell or host organism compatible with such expression vector or construct. These expression vectors typically include regulatory sequence elements that are operably linked to the nucleotide sequence to be expressed to achieve its expression. These regulatory elements usually include at least a suitable transcriptional regulatory sequence and optionally a 3’ transcription termination signal. Additional elements necessary or helpful to influence expression, such as expression enhancer elements, may also be present. The expression vector may be introduced into a suitable host cell to cause the expression of the coding sequence in an in vitro cell culture of the host cell. While the expression vector will be suitable for replication in the host cell or organism of the present invention, the expression construct will typically be incorporated into the host cell genome to maintain it. Techniques for introducing nucleic acids into cells are widely established in the art, and any suitable technique may be used depending on the specific situation. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, Liposome-mediated transfection and transfection using retroviruses or other viruses, e.g., adenoviruses, AAVs, lentiviruses, or vaccinias may be included. For microorganisms, e.g., bacteria, or cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages. The introduced nucleic acid may be on an extrachromosomal vector within the cell, or the nucleic acid may be incorporated into the host cell's genome.Integration may be facilitated by the inclusion of a sequence within a nucleic acid or vector that promotes recombination with the genome, according to standard techniques. Following the introduction, the nucleic acid may be expressed to produce the encoded fusion protein. In some embodiments, a host cell (which may actually include a transformed cell, but the cell is more likely to be a descendant of a transformed cell) may be cultured in vitro under conditions for the expression of the nucleic acid to produce the encoded fusion protein polypeptide, and when an inducible promoter is used, expression may require the activation of the inducible promoter.

[0159] As used herein, the terms “promoter” or “transcription regulatory sequence” refer to a nucleic acid fragment structurally identified by the presence of any other DNA sequence known to those skilled in the art as functioning to control the transcription of one or more coding sequences, located upstream of the transcription initiation site of the coding sequence in the transcription direction, and comprising, but not limited to, a binding site for DNA-dependent RNA polymerase, a transcription initiation site and a transcription factor binding site, a repressor and activator factor protein binding site, and any other sequence of nucleotides that act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer.

[0160] The term “selectable marker” is familiar to those skilled in the art and is used herein to describe any genetic entity that may be used to select cells or cells containing a selectable marker when expressed. The term “reporter” may be used interchangeably with marker, but is used primarily to refer to visible markers such as green fluorescent protein (GFP). Selectable markers may be dominant, recessive, or bidirectional.

[0161] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is positioned to be in a functional relationship with another nucleic acid sequence. For example, if a transcriptional regulatory sequence influences the transcription of a coding sequence, this sequence is operably linked to the coding sequence. Operatably linked means that the DNA sequences being linked are typically adjacent, and, where it is necessary to link two protein encoding regions, they are adjacent and within a leading frame.

[0162] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule consisting of a chain of amino acids without mention of a specific mode of action, size, three-dimensional structure, or origin.

[0163] The term "signal peptide" (sometimes referred to as a signal sequence) is a short peptide (typically 16 to 30 amino acids in length) located at the N-terminus of most newly synthesized proteins destined for secretion pathways. At the end of a signal peptide, there is typically a stretch of amino acids that is recognized and cleaved by signal peptidases during or after the completion of translocation (from the cytoplasm to the secretion pathway, i.e., the ER) to produce free signal peptides and mature proteins. Signal peptides are highly heterogeneous, and while many prokaryotic and eukaryotic signal peptides are functionally interchangeable between different species, the efficiency of protein secretion can depend on the signal peptide. Suitable signal peptides are generally [referred to in the art, for example, in the literature] It is known from et al. (2004 J. Mol. Biol. 338: 1027-1036)] and literature [von Heijne (1985, J Mol Biol. 184 (1): 99-105)].

[0164] The term “gene” means a DNA fragment containing a region (transcribed region) that is operably linked to a suitable regulatory region (e.g., a promoter) and transcribed into an RNA molecule (e.g., mRNA) in a cell. A gene will typically contain several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region, and a 3’ non-translating sequence (3’ end) containing a polyadenylation site. “Gene expression” refers to the process in which a DNA region operably linked to a suitable regulatory region, particularly a promoter, is transcribed into RNA that is biologically active, that is, can be translated into a biologically active protein or peptide.

[0165] When the term "homology" is used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, it is understood to mean that the nucleic acid or polypeptide molecule is produced in nature by host cells or organisms of the same species, preferably the same variant or strain. If homology to a host cell, the nucleic acid sequence encoding the polypeptide will typically (but not necessarily) be operablely linked to another (heterogeneous) promoter sequence and, where applicable, another (heterogeneous) secreted signal sequence and / or terminator sequence than in that natural environment. It is understood that regulatory sequences, signal sequences, terminator sequences, etc., may also be homology to the host cell. When used to indicate the relationship between two nucleic acid sequences, the term "homology" means that one single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. As discussed below, the degree of hybridization can be influenced by a number of factors, including the amount of identity between sequences and hybridization conditions, such as temperature and salt concentration.

[0166] When used in relation to nucleic acids (DNA or RNA) or proteins, the term “heterogeneous” refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome, or DNA or RNA sequence in which it exists, or that is found at a location or position in the cell, genome, or DNA or RNA sequence that differs from where it is found in nature. A heterogeneous nucleic acid or protein is not endogenous to the cell into which it is introduced, but is obtained from another cell or produced synthetically or recombinantly. Generally, but not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterogeneous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that a person skilled in the art would recognize as heterogeneous or foreign to the cell in which it is expressed is included herein by the term heterogeneous nucleic acid or protein. The term heterogeneous also applies to non-natural combinations of nucleic acid or amino acid sequences, that is, combinations in which at least two of the combined sequences are foreign to each other.

[0167] As used herein, the terms “therapies” and “therapy” may refer to any protocol(s), method(s), and / or agent(s) that may be used for the prevention, treatment, management, or improvement of a disease. A preferred disease is cancer, preferably cancer comprising tumor cells expressing PD-L1. The terms “treat,” “treating,” and “treatment” may refer to a reduction or improvement in the progression, severity, and / or duration of a disease, preferably cancer, preferably cancer comprising tumor cells expressing PD-L1, or a reduction or improvement in one or more symptoms of the disease.

[0168] As used herein, the term “effective amount” refers to an amount of a therapeutic agent as defined herein, i.e., a (bispecific) antigen-binding protein, sufficient to reduce the severity and / or duration of the disease, improve one or more of its symptoms, prevent the progression of the disease, cause regression of the disease, or prevent the occurrence, recurrence, onset, or progression of the disease or one or more of its symptoms, or to enhance or improve the prophylactic and / or therapeutic effect(s) of another therapeutic agent (e.g., another therapeutic agent). Preferably, the disease is cancer, preferably cancer comprising tumor cells expressing PD-L1.

[0169] The medical uses described herein are formulated as antigen-binding proteins for use as medicines for the treatment of the mentioned disease(s), preferably as bispecific antigen-binding proteins, but may be formulated as methods for treating the mentioned disease(s) using (bispecific) antigen-binding proteins as defined herein, (bispecific) antigen-binding proteins as defined herein for use in the manufacture of medicines for treating the mentioned disease(s), and (bispecific) antigen-binding proteins as defined herein for the treatment of the mentioned disease(s) by administration of an effective amount as defined herein. All such medical uses are considered by the present invention.

[0170] Detailed description of the present invention

[0171] The immune system has evolved to identify and eliminate abnormal cells, such as tumor cells. Recognition of these cells is made possible through interactions between peptide-major histocompatibility complexes (MHCs) expressed on cancer cells and T cell receptors (TCRs) on T cells. This process is further significantly regulated by a series of co-stimulatory and co-inhibitory receptors and their ligands known as immune checkpoints. Among these regulators, programmed death ligand 1 (PD-L1; also known as B7-H1 and CD274) and its receptor, PD-1, have emerged as therapeutic targets and have demonstrated significant clinical efficacy in various cancers. However, despite much success, immune checkpoint block therapy has also been associated with specific challenges, such as acquired resistance and harmful side effects.

[0172] E3 ligase-induced targeted protein degradation has emerged as a promising strategy in both oncology and immuno-oncology (I / O), offering unique advantages over traditional blocking antibodies.

[0173] The present invention relates to the concept of using a bispecific antigen-binding protein for the targeted internalization and subsequent degradation of membrane-bound proteins. The bispecific antigen-binding protein of the present invention can simultaneously bind to the RNF128 transmembrane E3 ubiquitin ligase (RNF128) and the extracellular portion of a tumor-associated antigen (TAA), such as PD-L1. Induced proximity (i.e., "forced dimerization") between the desired TAA and the ubiquitin ligase will result in the ubiquitination of the target, followed by its removal from the cell surface and subsequent degradation. For example, as a result, cancer cell growth is impaired. A schematic representation of an exemplary embodiment of the present invention is provided in FIG. 5.

[0174] This approach enables sustained and comprehensive inhibition of disease-associated proteins, potentially minimizing off-target effects and overcoming the limitations associated with traditional inhibitors. In scenarios where the target lacks suitable binding sites for blocking antibodies or exhibits complex structural features, E3 ligase-induced degradation offers an alternative for therapeutic intervention. In indications for immuno-oncology, this innovative approach enables precise immunomodulation by selectively degrading immune checkpoint-associated proteins, such as PD-1 and PD-L1. By leveraging the cell's natural proteolysis mechanisms, targeted proteolysis allows for modulation of control over the tumor microenvironment, offering potential benefits in overcoming resistance mechanisms and establishing a complex balance between anti-tumor immunity and immune inertia.

[0175] The advantages of this approach include at least the following:

[0176] i) The heteroactive molecule of the present invention enables a strong gain in efficacy and requires only less than a stoichiometric amount of the molecule compared to the target molecule when compared to conventional 'occupancy-based' therapeutic agents.

[0177] ii) The necessary specific binding of the two proteins, namely the transmembrane E3 ubiquitin ligase as well as TAA, also reduces potential off-target toxicity. Preferably, a ubiquitin ligase localized to the plasma membrane and exhibiting increased expression in cancer cells will be used.

[0178] iii) Targeted protein degradation results in long-term pharmacodynamic effects compared to standard antibodies due to the time required to synthesize a sufficient amount of new transmembrane proteins.

[0179] iv) Bispecific antigen-binding proteins bind to extracellular protein portions, so they do not need to cross the cell membrane.

[0180] Accordingly, in a first embodiment, the present invention relates to a bispecific antigen-binding protein comprising the following:

[0181] i) a first immunoglobulin monovariable domain (ISVD) that specifically binds to the extracellular portion of RNF128 transmembrane E3 ubiquitin ligase (RNF128); and

[0182] ii) A second ISVD that specifically binds to the extracellular portion of a tumor-associated antigen (TAA).

[0183] The inventors have discovered that the incorporation of a non-blocking antibody into a bispecific antigen-binding protein as described herein enables the complete removal of the target from the cell surface. The use of a non-blocking antibody has several advantages:

[0184] - This enables distinguishing the effects of decomposition versus blocking strategies; and

[0185] - Compared to conventional blocking antibodies, the use of non-blocking PD-L1 binders on bispecific antigen-binding proteins as described herein offers the potential for tissue-specific therapeutic effects (thereby limiting toxicity). When a non-blocking binding arm is used, degradation and functional inactivation of the target will occur only in tissues where both the target and E3 are expressed.

[0186] In one embodiment, at least one of the first and second ISVDs comprises a binding domain of a non-blocking antibody.

[0187] In one embodiment, the immunoglobulin variable region is a single-chain antigen-binding domain, such as scFv, V H Domain, V L A domain, or an immunoglobulin monovariable domain (ISVD), such as dAb, V-NAR domain, or V H It may include or be composed of an H domain. Preferably, the bispecific antigen-binding protein includes a first and a second ISVD.

[0188] The first ISVD of the bispecific antigen-binding protein as described herein specifically binds to the extracellular portion of the RNF128 transmembrane E3 ubiquitin ligase (RNF128). The ubiquitin E3 ligase gene (GRAIL) (Rnf128), which is associated with anergy in lymphocytes, is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger.

[0189] In one embodiment, the first ISVD comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of:

[0190] a) CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 1, CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 3;

[0191] b) CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4; CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5; and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6; and

[0192] c) CDR1 comprising an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 7, CDR2 comprising an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 9.

[0193] A second ISVD of a bispecific antigen-binding protein as described herein specifically binds to TAA. In one embodiment, the antigen-binding region specifically binding to TAA is an antigen-binding region derived from an immunoglobulin or non-immunoglobulin scaffold as defined above. Preferably, the antigen-binding region specifically binding to TAA comprises or consists of at least one immunoglobulin variable domain. More preferably, the antigen-binding region specifically binding to TAA comprises or consists of a Fab specifically binding to TAA or an immunoglobulin single variable domain (ISVD) specifically binding to TAA. In one embodiment, the antigen-binding region specifically binding to TAA can be determined as described herein, 10 -4 K less than or equal to M D It is an antigen-binding region that binds to TAA as a value.

[0194] In one embodiment, the antigen-binding region that specifically binds to TAA comprises, or is composed of, a human or humanized immunoglobulin variable region or an immunoglobulin single variable region as defined herein.

[0195] As used herein, the term tumor-associated antigen (TAA) refers to an antigen that is expressed differently by cancer / tumor cells compared to normal, i.e., non-tumor cells. Alternatively, TAAs may be antigens expressed by non-tumor cells (e.g., immune cells) that possess tumor-promoting effects (e.g., immunosuppressive effects) and can thereby be utilized to target cancer cells. Thus, TAAs may be any antigen that potentially stimulates a tumor-specific immune response. Some of these antigens are encoded by normal cells but are not necessarily expressed, or are expressed at lower levels or less frequently. These antigens can be characterized as antigens that are generally silenced (i.e. not expressed) in normal cells, antigens expressed only at specific stages of differentiation, and transient antigens such as embryonic and fetal antigens. Other TAAs are encoded by fusion proteins resulting from internal deletions or chromosomal translocations, including mutant cell genes, e.g., oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and neo-antigens. Other TAA antigens may be encoded by viral genes, such as those carried on RNA and DNA oncogenes. Other TAAs may be expressed on immune cells that contribute to or mediate tumor-promoting effects, e.g., cells contributing to immune evasion, monocytes, or macrophages, selectively on suppressor T cells, regulatory T cells, or myeloid-derived suppressor cells.

[0196] TAAs are normal cell surface antigens that are typically expressed at abnormal times, overexpressed, or expressed by targeted cell populations. Ideally, target TAAs are expressed only on proliferative cells (e.g., tumor cells) or tumor-promoting cells (e.g., immune cells with immunosuppressive effects), but this is rarely observed in practice. Consequently, in many cases, target antigens are selected based on differential expression between proliferative / diseased tissues and healthy tissues.

[0197] Examples of TAAs include receptor tyrosine kinase-like rare receptor 1 (ROR1), crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47 glycoprotein NMB, CanAg, HER2 (ErbB2 / Neu), Siglec and its members, e.g., CD22 (Siglec2) or CD33 (Siglec3), CD79, CD123, CD138, CD171, CTLA-4 (CD152), PD1, PSCA, L1-CAM, EpCAM, PSMA (prostate-specific membrane antigen), BCMA, TROP2, STEAP1, CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, melanotransferrin, Mud 6, and TMEFF2. Examples of TAAs also include immunoglobulin supergroup (IgSF) proteins, such as cytokine receptors, killer-Ig-like receptors, CD28, and proteins, such as killer-Ig-like receptor 3DL2 (KIR3DL2), B7.1, B7.2, B7-H3, B7-H4, B7-H6, PD-L1, and IL-6 receptors.Examples of TAAs include MAGE, MART-1 / Melan-A, gp100, major histocompatibility complex class I-associated chain A and B polypeptides (MICA and MICB), adenosine deaminase-binding protein (ADAbp), cyclophylline b, colorectal-associated antigen (CRC)-C017-1A / GA733, protein tyrosine kinase 7 (PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1, nectin (e.g., nectin-4), major histocompatibility complex class I-associated chain A and B polypeptides (MICA and MICB), proteins with UL16-binding protein (ULBP), proteins with retinoic acid early transcript-1 (RAET1), carcinogen-embryo antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, CEACAM5, etv6, amll. Prostate-specific antigen (PSA), T-cell receptor / CD3-zeta chain, tumor antigen MAGE- and, e.g., MAGE-A3, tumor antigen GAGE- and, anti-Müllerian hormone type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, ROR1 (receptor tyrosine kinase-like rare receptor 1 or NTRKR1 (EC 2.7.10.Also known as 1), SLAMF7, TRAILR1, TRAILR2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC and, e.g., MUC1 or MUC1-C, VEGF, VEGF receptor, angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR / ERBB1), human EGF-like receptor and receptor, e.g., HER-2 / neu, HER-3 / ERBB3, HER-4 / ERBB4 or a heterodimeric receptor composed of at least one HER subunit, gastrin-releasing peptide receptor antigen, cMET, integrin receptor, a5β3 integrin, a5β1 integrin, α-llbβ3-integrin, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, IL-1 accessory protein, CSF1R (tumor-associated monocytes and macrophages), α-fetoprotein, mesothelin, isoform 2 of claudin-18 (claudin 18.2), folate receptor alpha (FRα, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, α-catenin, β-catenin and γ-catenin, flexin-A1, TNFRSF10B, AXL, EDNRB, OLR1, ADAM12, PLAUR, CCR6, p120ctn, PRAME, NY-ESO-1, cdc27, CDCP1, adenomatous polyposis protein (APC), podrin, connexin 37, Ig-ideotype, p15, gp75, GM2 and GD2 gangliosides, viral products, e.g., human papillomavirus protein, imp-1, P1A, EBV-encoding nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, FcRL5 / FcRH5, Flt3, muc16, muc17, mmp9, FAP, Lewis-Y, EGFRvIII, GPC3, GPRC5D, gpA33, 5T4, SSTR2, CD73, CD25, CD45, and CD133 are additionally included, but this is not intended to be complete.

[0198] Accordingly, in one embodiment, the bispecific antigen-binding protein as described herein comprises a second ISVD that specifically binds to a TAA selected from the group consisting of: PD-L1, PD1, PSCA, L1-CAM, EpCAM, HER2 (ErbB2 / Neu), receptor tyrosine kinase-like rare receptor 1 (ROR1), crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171, CTLA-4 (CD152), PSMA (prostate-specific membrane antigen), BCMA, TROP2, STEAP1, CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, melanotransferrin, Mud 6, TMEFF2, Killer Ig-like receptor, Killer Ig-like receptor 3DL2 (KIR3DL2), B7.1, B7.2, B7-H3, B7-H4, B7-H6, IL-6 receptor, IL-1 accessory protein, MAGE, MART-1 / Melan-A, gp100, MICA, MICB, Adenosine deaminase-binding protein (ADAbp), Cyclophyllin b, Colorectal-associated antigen (CRC)-C017-1A / GA733, Protein tyrosine kinase 7 (PTK7), Receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1, Nectin-4, UL16-binding protein (ULBP), RAET1 protein, Carcinoembryonic antigen (CEA), CEACAM5, etv6, aml1, Prostate-specific antigen (PSA), T-cell Receptor / CD3-zeta chain, MAGE-A3, GAGE-tumor antigen, anti-Müllerian hormone type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, NTRKR1(EC 2.7.10.1), SLAMF7, TRAILR1, TRAILR2, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC1, MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR / ERBB1), HER-3 / ERBB3, HER-4 / ERBB4, heteromeric receptor composed of at least one HER subunit, gastrin-releasing peptide receptor antigen, cMET, CA125, integrin receptor, a5β3 integrin, a5β1 integrin, α-llbβ3-integrin, PDGF alpha receptor, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, IL-1 accessory protein, CSF1R, α-fetal protein, mesothelin (MSLN), Claudin-18 isotype 2 (Claudin 18.2, CLDN18), folate receptor alpha (FRα, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, α-catenin, β-catenin, and γ-catenin, Flexin-A1, TNFRSF10B, AXL, EDNRB, OLR1, ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1, cdc27, CDCP1, Adenomatous Colorectal Polyposis Protein (APC), Podrin, Connexin 37, Ig-Idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, imp-1, P1A, EBV-encoding nuclear antigen (EBNA)-I, brain glycogen Phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, FcRL5 / FcRH5, Flt3, muc16, muc17, mmp9, FAP, Lewis-Y, EGFRvIII, GPC3, GPRC5D, gpA33, 5T4, SSTR2, CD73, CD25, CD45, and CD133.

[0199] In one embodiment, the bispecific antigen-binding protein as described herein comprises a second ISVD obtained from / obtainable from a cytotoxic monoclonal antibody against TAA as known in the art. In one embodiment, at least one antigen-binding region comprises six CDR sequences obtained from / obtainable from at least a monoclonal antibody against TAA as known in the art. In one embodiment, at least one antigen-binding region comprises variable light chain (VL) domain and variable heavy chain (VH) domain sequences obtained from at least a monoclonal antibody against TAA as known in the art.Examples of these monoclonal antibodies against TAAs include the following: trastuzumab (against HER2), pertuzumab (against HER2), margetuximab (against HER2), rituximab (against CD20), tocitumomab (against CD20), ibritumomab (against CD20), obinutuzumab (against CD20), ofatumumab (against CD20), alemtuzumab (against CD52), blinatumomab (against CD19), inebilizumab (against CD19), tafacitamab (against CD19), daratumumab (against CD38), isatuximab (against CD38), polatuzumab (against CD79b), talacotuzumab (against CD123), dinutuximab (against GD2), (against GD2) Nacitamab, Bevacizumab (for VEGF-A), Elotuzumab (for SLAMF7), Enportumab (for Nectin-4), Sacituzumab (for TROP2), Mogamulizumab (for CCR4), Ipilimumab (for CTLA-4), Tremelimumab (for CTLA-4), Durvalumab (for PD-L1), Pidilizumab (for PD-1), Pembrolizumab (for PD-1), Nivolumab (for PD-1), Cemiplimab (for PD-1), Avelumab (for PD-L1), Atezolizumab (for PD-L1), Cosivelimab (for PD-L1), Cetuximab (for EGFR), Necitumumab (for EGFR), Panitumumab (for EGFR), (EGFR Amivantamab (bispecific to cMet), onatuzumab (monovalent to cMet), olaratumab (for PDGFRα), enoblituzumab (for B7-H3), vobramitamab (for B7-H3), zolvetuximab (for Claudin-18 isotype 2), mirvetuximab (for folate receptor alpha, FRα), parletuzumab (for folate receptor alpha, FRα), tisotumab (tissue factor, CD142), rovalfituzumab (for DLL3), ombertamab (for B7-H3), and ramucirumab (for VEGFR2).

[0200] In a further embodiment, the present invention provides a bispecific antigen-binding protein comprising the following:

[0201] i) A first immunoglobulin monovariable domain (ISVD) that specifically binds to the extracellular portion of RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the first ISVD is

[0202] a) a complementary-determining region (CDR)1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3;

[0203] b) CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4, CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6; and c) CDR1 comprising the amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 9.

[0204] A first immunoglobulin single variable domain (ISVD) comprising a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of

[0205] and

[0206] ii) a second ISVD that specifically binds to the extracellular portion of PD-L1, wherein the second ISVD is

[0207] d) a complementary-determining region (CDR) 1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR 2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and a CDR 3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 12;

[0208] e) CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13, CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 15;

[0209] and

[0210] f) CDR1 comprising the amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 18 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 18

[0211] A second ISVD comprising a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from a group consisting of

[0212] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 12.

[0213] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 15.

[0214] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 18 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 18.

[0215] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 12.

[0216] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 15.

[0217] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 18 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 18.

[0218] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 9, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 12.

[0219] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 9, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 15.

[0220] In one embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 9, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 18 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 18.

[0221] In a preferred embodiment, the bispecific antigen-binding protein comprises a first ISVD comprising a CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3, and a CDR1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and It includes a second ISVD comprising a CDR3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 12.

[0222] Preferably, the bispecific antigen-binding protein comprises a first ISVD that specifically binds to the extracellular portion of an RNF128 transmembrane E3 ubiquitin ligase (RNF128), comprising a CDR1 having an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 7, a CDR2 having an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 8, and a CDR3 having an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 9. More preferably, the bispecific antigen-binding protein comprises a first ISVD that specifically binds to the extracellular portion of an RNF128 transmembrane E3 ubiquitin ligase (RNF128), comprising a CDR1 having an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6.More preferably, the bispecific antigen-binding protein comprises a first ISVD that specifically binds to the extracellular portion of an RNF128 transmembrane E3 ubiquitin ligase (RNF128), comprising a CDR1 having an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 3.

[0223] Preferably, the bispecific antigen-binding protein comprises a second ISVD that specifically binds to the extracellular portion of PD-L1, and the second ISVD comprises a CDR1 comprising an amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 18 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 18. More preferably, the bispecific antigen-binding protein comprises a second ISVD that specifically binds to the extracellular portion of PD-L1, wherein the second ISVD comprises a CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 15.More preferably, the bispecific antigen-binding protein comprises a second ISVD that specifically binds to the extracellular portion of PD-L1, wherein the second ISVD comprises a CDR1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 12.

[0224] In one embodiment, a bispecific binding protein as described herein is

[0225] a) Amino acid sequence listed in SEQ ID NO: 19;

[0226] b) the amino acid sequence listed in SEQ ID NO: 20; and

[0227] c) Amino acid sequence listed in SEQ ID NO: 21

[0228] A first ISVD comprising an amino acid sequence selected from the group consisting of, and an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity;

[0229] and

[0230] ii) d) Amino acid sequence listed in SEQ ID NO: 22;

[0231] e) The amino acid sequence listed in SEQ ID NO: 23; and

[0232] f) Amino acid sequence listed in SEQ ID NO: 24

[0233] It includes a second ISVD comprising an amino acid sequence selected from the group consisting of, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to amino acid sequences.

[0234] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 19; and ii) an amino acid sequence described in SEQ ID NO: 22 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0235] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 19; and ii) an amino acid sequence described in SEQ ID NO: 23 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0236] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 19; and ii) an amino acid sequence described in SEQ ID NO: 24 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0237] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 20, and ii) an amino acid sequence described in SEQ ID NO: 22 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0238] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 20; and ii) an amino acid sequence described in SEQ ID NO: 23 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0239] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 20, and ii) an amino acid sequence described in SEQ ID NO: 24 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0240] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 21, and ii) an amino acid sequence described in SEQ ID NO: 22 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0241] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 21; and ii) an amino acid sequence described in SEQ ID NO: 23 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0242] In one embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 21; and ii) an amino acid sequence described in SEQ ID NO: 24 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0243] In a preferred embodiment, the bispecific antigen-binding protein comprises: i) a first ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 19, and ii) an amino acid sequence described in SEQ ID NO: 22 and A second ISVD comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0244] Preferably, the first ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 21. More preferably, the first ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 20.More preferably, the first ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 19.

[0245] Preferably, the second ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 24. More preferably, the second ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 23.More preferably, the second ISVD of the bispecific binding protein as described herein comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 22.

[0246] In one embodiment, the first and second ISVDs of the bispecific binding protein as described herein have their respective heavy chains of the same sequence identity percentage.

[0247] In one embodiment, at least one of the first and second ISVDs is humanized as defined in the above invention.

[0248] In one embodiment, at least the first ISVD is humanized. The first humanized ISVD preferably comprises CDR1, CDR2, and CDR3 selected from the group consisting of:

[0249] a) CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 1, CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 3;

[0250] b) CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4; CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5; and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6; and

[0251] c) CDR1 comprising an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 7, CDR2 comprising an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 9.

[0252] Preferably, the first humanized ISVD comprises a CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3.

[0253] Preferably, the first humanized ISVD comprises a CDR1 having an amino acid sequence described in SEQ ID NO: 1, a CDR2 having an amino acid sequence described in SEQ ID NO: 2, and a CDR3 having an amino acid sequence described in SEQ ID NO: 3.

[0254] Preferably, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of:

[0255] a) Amino acid sequence listed in SEQ ID NO: 114;

[0256] b) Amino acid sequence listed in SEQ ID NO: 115;

[0257] c) the amino acid sequence listed in SEQ ID NO: 116; and

[0258] d) Amino acid sequence listed in SEQ ID NO: 117.

[0259] Preferably, the first humanized ISVD comprises a CDR1 having an amino acid sequence described in SEQ ID NO: 1, a CDR2 having an amino acid sequence described in SEQ ID NO: 2, and a CDR3 having an amino acid sequence described in SEQ ID NO: 3, and comprises an amino acid sequence selected from the group consisting of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least It has an amino acid sequence having 98%, at least 99%, or 100% sequence identity:

[0260] a) Amino acid sequence listed in SEQ ID NO: 114;

[0261] b) Amino acid sequence listed in SEQ ID NO: 115;

[0262] c) the amino acid sequence listed in SEQ ID NO: 116; and

[0263] d) Amino acid sequence listed in SEQ ID NO: 117.

[0264] Preferably, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of:

[0265] a) Amino acid sequence listed in SEQ ID NO: 114;

[0266] b) the amino acid sequence listed in SEQ ID NO: 115; and

[0267] c) Amino acid sequence listed in SEQ ID NO: 117.

[0268] Preferably, the first humanized ISVD comprises a CDR1 having an amino acid sequence described in SEQ ID NO: 1, a CDR2 having an amino acid sequence described in SEQ ID NO: 2, and a CDR3 having an amino acid sequence described in SEQ ID NO: 3, and comprises an amino acid sequence selected from the group consisting of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least It has an amino acid sequence having 98%, at least 99%, or 100% sequence identity:

[0269] a) Amino acid sequence listed in SEQ ID NO: 114;

[0270] b) the amino acid sequence listed in SEQ ID NO: 115; and

[0271] c) Amino acid sequence listed in SEQ ID NO: 117.

[0272] In one embodiment, at least the second ISVD is humanized. The second humanized ISVD is preferably

[0273] d) CDR1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, CDR2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 12;

[0274] e) CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13; CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14; and CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 15; and

[0275] f) CDR1 comprising the amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 18 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 18

[0276] It includes CDR1, CDR2, and CDR3 selected from the group consisting of

[0277] Preferably, the second humanized ISVD comprises a CDR1 comprising an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 12.

[0278] Preferably, the second humanized ISVD comprises a CDR1 having an amino acid sequence described in SEQ ID NO: 10, a CDR2 having an amino acid sequence described in SEQ ID NO: 11, and a CDR3 having an amino acid sequence described in SEQ ID NO: 12.

[0279] Preferably, the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of the following:

[0280] a) Amino acid sequence listed in SEQ ID NO: 107;

[0281] b) Amino acid sequence listed in SEQ ID NO: 108;

[0282] c) the amino acid sequence listed in SEQ ID NO: 110; and

[0283] d) Amino acid sequence listed in SEQ ID NO: 111.

[0284] Preferably, the first humanized ISVD comprises a CDR1 having an amino acid sequence described in SEQ ID NO: 10, a CDR2 having an amino acid sequence described in SEQ ID NO: 12, and a CDR3 having an amino acid sequence described in SEQ ID NO: 13, and an amino acid sequence selected from the group consisting of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, It has an amino acid sequence having at least 98%, at least 99%, or 100% sequence identity:

[0285] a) Amino acid sequence listed in SEQ ID NO: 107;

[0286] b) Amino acid sequence listed in SEQ ID NO: 108;

[0287] c) the amino acid sequence listed in SEQ ID NO: 110; and

[0288] d) Amino acid sequence listed in SEQ ID NO: 111.

[0289] Preferably, the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of the following:

[0290] a) the amino acid sequence listed in SEQ ID NO: 108; and

[0291] b) Amino acid sequence listed in SEQ ID NO: 110.

[0292] Preferably, the first humanized ISVD comprises a CDR1 having an amino acid sequence described in SEQ ID NO: 10, a CDR2 having an amino acid sequence described in SEQ ID NO: 12, and a CDR3 having an amino acid sequence described in SEQ ID NO: 13, and an amino acid sequence selected from the group consisting of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, It has an amino acid sequence having at least 98%, at least 99%, or 100% sequence identity:

[0293] a) the amino acid sequence listed in SEQ ID NO: 108; and

[0294] b) Amino acid sequence listed in SEQ ID NO: 110.

[0295] Preferably, the first and second ISVDs are humanized. In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 114, and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 108. It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0296] In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 114, and the second humanized ISVD comprises an amino acid sequence having at least It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0297] In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 115, and the second humanized ISVD comprises an amino acid sequence having at least It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0298] In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 115, and the second humanized ISVD comprises an amino acid sequence having at least It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0299] In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 116, and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 108. It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0300] In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 116, and the second humanized ISVD comprises an amino acid sequence having at least It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0301] In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 117, and the second humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 108. It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0302] In one embodiment, the first humanized ISVD comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 117, and the second humanized ISVD comprises an amino acid sequence having at least It includes an amino acid sequence having 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0303] In one embodiment, a bispecific antigen-binding protein as described herein is

[0304] i) a first polypeptide comprising a first human Fc fragment and a first ISVD; and

[0305] ii) A second polypeptide comprising a second human Fc fragment and a second ISVD

[0306] It is a bispecific antibody containing,

[0307] Preferably, the antibody does not contain an antibody light chain.

[0308] In one embodiment, each Fc fragment comprises a CH2 domain and a CH3 domain. In one embodiment, the immunoglobulin Fc region comprises at least one of the CH2 and CH3 domains and a hinge region. In one embodiment, the immunoglobulin Fc region comprises or consists of a hinge region and CH2 and CH3 domains.

[0309] In one embodiment, the first and / or second ISVD may be fused to the Fc fragment through a hinge region or through a linker. In the antibody, "hinge" or "hinge region" or "hinge domain" refers to a flexible portion of the heavy chain located between the CH1 domain and the CH2 domain. This is approximately 25 amino acids long and is divided into an "upper hinge," a "middle hinge" or "core hinge," and a "lower hinge." "Hinge subdomain" refers to the upper hinge, the middle (or core) hinge, or the lower hinge. The hinge regions of IgG1, IgG2, IgG3, and IgG4 are well known and described in the art.

[0310] Linker-amino acid sequences suitable for linking various functional domains and regions in multispecific antigen-binding proteins as described herein are known in the art (e.g., reference [Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369]). Linker amino acid sequences may be rigid, but are usually flexible. Flexible linkers are typically applied when the linked domains require some degree of movement or interaction. They generally consist of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and enables the movement of the linked functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing undesirable interactions between the linker and the protein moiety. A preferred flexible linker has a sequence consisting mainly of stretches of Gly and Ser residues ("GS" linker). An example of a desirable (and widely used) flexibility linker is the sequence (GGGGS)n (SEQ ID NO: 46). By adjusting the copy number "n", the length of this GS linker can be optimized to achieve proper separation of functional domains or maintain necessary inter-domain interactions. Specific examples of GS linkers include (GGGGS)4 (SEQ ID NO: 47), GGGSGGG (SEQ ID NO: 48), GGSGGGGSGG (SEQ ID NO: 49), and G. In addition to GS linkers, many other flexibility linkers have been designed for recombinant fusion proteins.These flexibility linkers are also rich in small or polar amino acids, such as Gly and Ser, but may contain additional amino acids to maintain flexibility, such as Thr and Ala, as well as polar amino acids to improve solubility, such as Lys and Glu, as in flexibility linkers KESGSVSSEQLAQFRSLD (SEQ ID NO: 50) and EGKSSGSGSESKST (SEQ ID NO: 51), which are applied for the construction of viable scFvs, for example. Examples of additional flexibility linkers are indicated by SEQ ID NO: 52 to 95. Examples of hard linkers can be found in SEQ ID NO: 96 to 99.

[0311] In one embodiment, a "knob-into-hole" approach is used in which the CH3 domain interface of the antibody Fc region is mutated so that the antibody preferentially forms a heterodimer (additionally including an attached light chain). These mutations create altered charge polarity across the Fc heterodimer interface, thereby promoting the desired Fc heterodimer formation by providing desirable inducing interactions for the co-expression of electrostatically matched Fc chains, whereas adverse repulsive charge interactions inhibit the formation of unwanted Fc homodimers. For example, one heavy chain contains a T366W substitution, and a second heavy chain contains T366S, L368A, and Y407V substitutions, for example, [Ridgway et al (1996) Protein Eng., 9, pp. 617-621; Atwell (1997) J. Mol. Biol., [270, pp. 26-35]; and refer to W02009 / 089004, the disclosures of which are incorporated herein by reference. In another approach, one heavy chain comprises an F405L substitution and a second heavy chain comprises a K409R substitution, e.g., [Labrijn et al. (2013) Proc. Natl. Acad. Sci. USA,Refer to [110, pp. 5145-5150]. In another approach, one heavy chain contains T350V, L351Y, F405A, and Y407V substitutions, and a second heavy chain contains T350V, T366S, K392L, and T394W substitutions, e.g., Von Kreudenstein et al., (2013) mAbs Refer to [5:646-654]. In another approach, one heavy chain contains both K409D and K392D substitutions, and a second heavy chain contains both D399K and E356K substitutions, e.g., [Gunasekaran et al., (2010) J. Biol. Chem. Refer to [285:19637-19646]. In another approach, one heavy chain contains D221E, P228E, and L368E substitutions, and a second heavy chain contains D221R, P228R, and K409R substitutions, for example, [Strop et al., (2012) J. Mol. Biol. Refer to [420:204-219]. In another approach, one heavy chain contains S364H and F405A substitutions and a second heavy chain contains Y349T and T394F substitutions, e.g., [Moore et al., (2011) MAbs Refer to [3: 546-557]. In another approach, one heavy chain may contain an H435R substitution, and a second heavy chain may optionally contain or not contain a substitution, for example, refer to U.S. Patent No. 8,586,713. When such hetero-multimer antibodies have an Fc region derived from human IgG2 or IgG4, the Fc region of these antibodies may be engineered to contain an amino acid modification that allows CD16 binding. In some embodiments, the antibody may contain mammalian antibody-type N-linked glycosylation at residue N297 (Kavat EU numbering).

[0312] Accordingly, in a bispecific antigen-binding protein as described herein in one embodiment, the first Fc fragment and the second Fc fragment comprise different amino acid sequences including a complementary knob-into-hole modification that promotes the association of the first and second polypeptide chains of the Fc region to form a heteromeric Fc region.

[0313] It is understood that when a bispecific antigen-binding protein comprises a heteromeric heavy chain, the “knob-into-hole” technique as described above may be applied, and the CH3 domain of the first chain is modified to have a “protrusion” (“knob”), and the second chain is modified to have a corresponding “cavity” (“hole”).

[0314] In one embodiment, an Fc region variant may be produced by introducing one or more amino acid modifications into the Fc region of the antibody provided herein. The Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0315] In one embodiment, the bispecific antibody as described herein has an effector function, preferably an enhanced effector function.

[0316] In one embodiment, the bispecific antibody as described herein comprises an Fc region having one or more amino acid substitutions that improve ADCC, for example, at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues), or at amino acid 233 to 239 (e.g., see US6737056), or at positions 235, 243, 300, and 396 (e.g., see US10711069).

[0317] In one embodiment, the bispecific antibody as described herein is modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed. If the antibody contains an Fc region, the oligosaccharide attached thereto may be modified. Native antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide generally attached to Asn297 of the CH2 domain of the Fc region by an N-bond. For example, refer to [Wright et al. TIBTECH 15:26-32 (1997)]. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc at the "stem" of the biantennary oligosaccharide structure.

[0318] In one embodiment, modification of the oligosaccharide in the bispecific antibody as described herein may be made to produce an antibody variant having certain improved properties. In one embodiment, an antibody variant is provided having a non-fucosylated oligosaccharide, that is, an oligosaccharide structure lacking a fucose attached (directly or indirectly) to the Fc region. Such a non-fucosylated oligosaccharide (also referred to as an "afucosylated" oligosaccharide) is an N-linked oligosaccharide lacking a fucose residue attached to the first GlcNAc in the stem of the biantenerial oligosaccharide structure, in particular.

[0319] In one embodiment, an antibody variant having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to the native or parental antibody is provided. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry, for example as described in WO 2006082515. Asn297 refers to an asparagine residue located at position about 297 in the Fc region (EU numbering of Fc region residues). These antibodies having an increased non-fucosylated oligosaccharide in the Fc region may have improved FcyRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. For example, refer to WO 2003035835; WO 2003055993. Examples of cell lines capable of producing antibodies having reduced fucosylation include the following:

[0320] ● Lecl3 CHO cells deficient in protein fucosylation (see [Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986)]; WO 2003035835; and WO 2004056312, in particular Example 11)

[0321] ● Knockout cell lines, e.g., alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., see [Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004)]; [Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)]; and WO 2003085107),

[0322] ● Cells in which GDP-fucose synthesis or transporter protein activity is reduced or absent (see, e.g., WO 2003085119, WO 2003084570, WO 2003085118, WO 2003085102).

[0323] In one embodiment, an antibody variant having a bisected oligosaccharide is provided, for example, the biantheranery oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, for example, in the literature [Umana et al., Nat Biotechnol 17, 176-180 (1999)]; the literature [Ferrara et al., Biotechnol Bioeng 93, 851-861 (2006)]; WO9954342; WO 2004065540; and WO 2003011878. An antibody variant having at least one galactose residue in the oligosaccharide attached to the Fc region is also provided. Such antibody variants may have improved CDC function. These antibody variants are described, for example, in WO199730087; WO199858964; and WO199922764.

[0324] In one embodiment, this bispecific antibody has a lack of effector function or a reduced effector function.

[0325] "Silent Fc (Fc-silence) refers to a genetically engineered Fc domain containing a mutation that nullifies the binding of the Fc domain to the Fc receptor (FcγR, FcR) while maintaining the Fc domain's ability to bind to the neonatal Fc receptor (FcRn). Such silencing Fc exhibits an extended half-life as described in the literature [Borrok MJ, et al. J Pharm Sci. 2017].

[0326] In one embodiment, the present invention considers an antibody variant having some, but not all, of the effector functions, which makes said variant a desirable candidate for applications where the antibody's half-life in vivo is important but specific effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful.

[0327] In one embodiment, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcyR binding, for example, substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region.

[0328] In one embodiment, the substitutions are L234A and L235A (LALA). In a specific embodiment, the antibody variant further comprises D265A and / or P329G in the Fc region derived from the human IgG1 Fc region (e.g., the literature [Xu, et al. Cellular Immunology 2000, 200 (1), 16-26] see reference).

[0329] In one embodiment, the substitutions are L234A, L235A, and P329G (LALAPG) in the Fc region derived from the human IgG1 Fc region (see, for example, WO 2012130831).

[0330] In one embodiment, the substitutions are L234A, L235A, and P329S (LALAPS) in the Fc region derived from the human IgG1 Fc region (see, for example, WO 1999051642).

[0331] In one embodiment, the substitutions are L234A, L235A, and G236R (STR) in the Fc region derived from the human IgG1 Fc region (see, for example, WO 2021234402).

[0332] In one embodiment, the substitutions are L234A, L235A, and D265A (LALADA) in the Fc region derived from the human IgG1 Fc region or F234, L235E, and D265A in the Fc region derived from the human IgG4 Fc region (see, for example, WO 2021234402).

[0333] In one embodiment, the antibody may have a modification at the N297 position to reduce or eliminate ADCC activity, such as N297G, N297A, or N297Q (see, for example, WO 2014153063).

[0334] Antibodies having reduced effector function include antibodies having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6737056). These Fc mutants include Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant having substitutions of residues 265 and 297 to alanine (US 7332581). Specific antibody variants having enhanced or reduced binding to specific selected FcRs are described (e.g., US 6737056; WO 2004056312, and reference [Shields et al., J Biol. Chem. 9(2): 6591-6604(2001)]). In some of these cases, the antibody lacks an effector function.

[0335] In one embodiment, modifications are made in the Fc region resulting in modified (i.e. improved or reduced) Clq binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in US6194551, WO9951642, and in the literature [Idusogie et al. J Immunol. 164: 4178-4184 (2000)]. Additionally, for other examples of Fc region variants, refer to the literature [Duncan & Winter, Nature 322:738-40 (1988)], the literature [Damelang et al., Frontiers in Immunology 2024, vol 14], and WO 1988007089 and WO9429351.

[0336] In vitro and / or in vivo cytotoxicity assays may be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that antibodies lack FcyR binding (and consequently likely lack ADCC activity) but retain FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcyRIII, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells [ Ravetch and Kinet It is summarized in Table 3 and on page 464 of Annu. Rev. Immunol. 9:457-492 (1991).

[0337] Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in WO 1988004936. Alternatively, non-radioactive assay methods may be used (e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, California, USA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin, USA)). Effector cells useful for these assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example, in animal models such as those disclosed in the literature [Clynes et al. Proc. Nat. Acad. Sci. USA 95:652-656 (1998)]. A Clq binding assay may also be performed to confirm that the antibody cannot bind to Clq and consequently lacks CDC activity. For example, refer to the Clq and C3c binding ELISAs in WO 2006029879 and WO 2005100402. To evaluate complement activation, a CDC assay may be performed (for example, refer to [Gazzano-Santoro et al., J Immunol. Methods 202:163 (1996)]; [Cragg, MS et al., Blood 101:1045-1052 (2003)]; and [Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)]).

[0338] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus, are described in US20050014934. These antibodies comprise an Fc region having one or more internal substitutions that improve the binding of the Fc region to FcRn. These Fc variants include those having substitutions in one or more of the Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution of Fc region residue 434 (e.g., US7371826; see WO 2002060919). Some mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L (see, e.g., US7365168) and M252Y / S254T / T256E + H433K / N434F (see, e.g., WO 2019147973), have been shown to increase binding affinity to the neonatal Fc receptor (FcRn) and the half-life of IgG1 in vivo. This may further extend the serum half-life of the Fc-containing molecule.

[0339] In one embodiment, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 253, and / or 310, and / or 435 (EU numbering of residues) of the Fc-region. In a specific embodiment, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435.

[0340] In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from the human IgG1 Fc region (see, e.g., Grevys, A, et al., J. Immunol. 194 (2015) 5497-5508).

[0341] In one embodiment, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, a substitution at the 310, and / or 433, and / or 436 positions (EU numbering of residues) of the Fc region.

[0342] In one embodiment, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433, and 436.

[0343] In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from the human IgG1 Fc region (see, for example, WO 2014177460).

[0344] In one embodiment, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, for example, substitutions at positions 252, and / or 254, and / or 35-256 (EU numbering of residues) of the Fc region.

[0345] In one embodiment, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256.

[0346] In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from the human IgG1 Fc region. For example, refer to the teachings of WO 2002060919 or WO 2013165690.

[0347] In one embodiment, the substitutions are M428L and N434S in the Fc region derived from the human IgG1 Fc region. For example, refer to the teaching of EP3138853B1.

[0348] Residues in the Fc region critical to mouse Fc-mouse FcRn interactions were identified by localized mutagenesis (e.g., see [Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180]). Residues I253, H310, H433, N434, and H435 (EU index numbering) are involved in the interaction (see [Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533]; [Firan, M., et al., Int. Immunol. 13 (2001) 993]; [Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542]). Residues I253, H310, and H435 have been identified as important for the interaction between human Fc and rat FcRn (Reference [Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819]). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are critical for the interaction (Reference [Firan, M., et al., Int. Immunol. 13 (2001) 993]; Reference [Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604]). Reference [Yeung, YA, et al. In [J. Immunol. 182 (2009) 7667-7671], various mutants of residues 248 to 259, 301 to 317, 376 to 382, ​​and 424 to 437 were reported and investigated. Determination of FcRn binding and in vivo clearance / half-life can be performed using methods known in the art (e.g., see Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013120929).

[0349] Bispecific antigen-binding proteins as described herein may have one or more biological activities, including, for example, antigen binding, endocytosis, lysosomal degradation, tumor cell death, and induction of cytokine secretion.

[0350] In one embodiment, the bispecific antigen-binding protein as described herein has at least one biological activity selected from the following:

[0351] a) Bispecific antigen-binding proteins induce endocytosis and lysosomal degradation of PD-L1 when in contact with cells expressing PD-L1 and RNF128; and

[0352] b) When a bispecific antigen-binding protein is added to a co-culture of tumor cells and human PBMCs, it induces at least one of the secretion of cytokines and the death of tumor cells expressing PD-L1 and RNF128, wherein the secreted cytokines include at least one of IL-2 and interferon γ.

[0353] In a further aspect, the present invention relates to a pharmaceutical composition comprising a bispecific antigen-binding protein as described herein and a pharmaceutically acceptable carrier (excipient). The pharmaceutically acceptable carrier, such as an adjuvant or vehicle, is intended to administer the polypeptide to a subject. The pharmaceutical composition may be used in the therapeutic method described below by administering an effective amount of the composition to a subject in need thereof. As used herein, the term “subject” refers to any animal classified as a mammal, including but not limited to primates and humans. The subject is preferably a male or female human of any age or race.

[0354] As used herein, the term “pharmaceuticalally acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, absorption retardants, etc. that are compatible with drug administration (e.g., literature [“Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7 th edition, 2012, www.pharmpress.com ] See reference). The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, their use in the composition is considered. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulin; Hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn 2+- Protein complex); and / or nonionic surfactant, such as Tween™, PLURONICS™, or polyethylene glycol (PEG).

[0355] Complementary active compounds may also be incorporated into the pharmaceutical composition of the present invention. Accordingly, in certain embodiments, the pharmaceutical composition of the present invention may also contain one or more active compounds, preferably those having complementary activities that do not adversely affect each other, if necessary for the specific indication being treated. For example, it may be desirable to additionally provide chemotherapy agents, cytokines, analgesics, thrombolytics, or immunomodulators, such as immunosuppressants or immunostimulators. The effective amount of these other active agents depends particularly on the amount of polypeptide of the present invention present in the pharmaceutical composition, the type of disease or disorder, or the treatment.

[0356] In one embodiment, the polypeptide of the present invention is prepared using a controlled-release formulation comprising a carrier, such as an implant, and a microencapsulated delivery system, such as a liposome, to protect the compound against rapid removal from the body. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid may be used. Methods for preparing such formulations will be obvious to those skilled in the art. Liposome suspensions comprising targeted liposomes may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, such as those described in US 4,522,811 or WO2010 / 095940.

[0357] The administration route of the polypeptide of the present invention may be parenteral. As used herein, the term "parenteral" includes intravenous, intra-arterial, intralymphatic, intraperitoneal, intramuscular, or subcutaneous. Intravenous or intramuscular forms of parenteral administration are preferred. "Systemic administration" refers to intravenous, intraperitoneal, and intramuscular administration. The amount of polypeptide required for a therapeutic or prophylactic effect will depend on the selected polypeptide, the nature and severity of the disease being treated, and the patient. Additionally, the polypeptide may be appropriately administered, for example, by pulse infusion while reducing the polypeptide dose. Preferably, administration is provided by injection, most preferably intravenous, intramuscular, or subcutaneous injection, depending on whether the administration is short-term or chronic.

[0358] Accordingly, in certain embodiments, the pharmaceutical composition of the present invention may be in a form suitable for parenteral administration, such as a sterile solution, suspension, or lyophilized product in a suitable unit dosage form. A pharmaceutical composition suitable for injectable use comprises a sterile aqueous solution (if water-soluble) or dispersion and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, NJ, USA), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy injectability exists. It must be stable under manufacturing and storage conditions and preserved against the action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, pharmaceutically acceptable polyols such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by coating, such as by the use of lecithin; by maintaining the required particle size in the case of a dispersion; and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be desirable to include an isotonic agent, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition.

[0359] Extended absorption of the injectable composition may be achieved by including an absorption-delaying agent, for example, aluminum monostearate and gelatin, in the composition.

[0360] A sterile injectable solution can be prepared by incorporating a required amount of an active compound (e.g., a polypeptide of the present invention) into a suitable solvent, together with one or a combination thereof of the components listed above as needed, and then filtering and sterilizing. Generally, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other necessary components from the components listed above. For a sterile powder for the preparation of a sterile injectable solution, a preferred preparation method is vacuum drying and freeze-drying to yield a powder of any additional desired component in addition to the active component from a pre-sterile-filtered solution.

[0361] In certain embodiments, the pharmaceutical composition is administered via an intravenous (IV), intramuscular (IM), or subcutaneous (SC) route. Suitable excipients, such as fillers, buffers, or surfactants, may be used. The formulations mentioned are well known in the art, and, for example, the literature ["Remington: The Science and Practice of Pharmacy" (Ed. Allen, LV 22nd edition, 2012, www.pharmpress.com As described in more detail in various sources including ]), a composition that can be administered parenterally will be prepared using a standard method for preparing a composition.

[0362] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate pharmaceutical compositions, i.e., parenteral compositions, into unit dosage forms. As used herein, unit dosage forms refer to physically separated units suitable as unit dosages for a subject to treatment; each unit contains a predetermined amount of the active compound (polypeptide of the present invention) calculated to produce the desired therapeutic effect, together with the necessary pharmaceutical carrier. Details regarding the dosage unit forms of the present invention are determined by and directly depend on the unique characteristics of the active compound, the specific therapeutic effect to be achieved, and the limitations inherent in the field of formulating such active compound for the treatment of the individual.

[0363] Generally, for the prevention and / or treatment of the diseases and disorders mentioned herein, and depending on the specific disease or disorder to be treated and its severity, the efficacy of the specific polypeptide of the invention to be used, the specific route of administration, and the specific pharmaceutical formulation or composition used, the polypeptide of the invention will generally be administered continuously (e.g., by infusion), as a single daily dose or as multiple divided doses over a day, in a range of about 0.001 to 1,000 mg / kg (body weight) / day, preferably about 0.01 to about 100 mg / kg (body weight) / day, most preferably about 0.05 to 10 mg / kg (body weight) / day, e.g., about 1, 10, 100, or 1,000 micrograms / kg (body weight) / day. A clinician will generally be able to determine a suitable daily dose based on the factors mentioned herein. It will also be apparent that in certain cases, a clinician may choose to deviate from these amounts, for example, based on the factors cited above and their expert judgment. The pharmaceutical composition may be contained in a container, pack, or dispenser along with administration instructions.

[0364] In another aspect, a bispecific antigen-binding protein as described herein is provided for use as a medicine. In one embodiment, the multispecific antigen-binding is used as a protein as described herein, used as an active ingredient, component, or substance in a medicine.

[0365] In one embodiment, the present invention relates to the use of a multispecific antigen-binding protein as described herein for producing a pharmaceutical preparation for the treatment, prevention, or diagnosis of a disease in a subject requiring treatment, prevention, or diagnosis of a disease, for example, a pharmaceutical preparation comprising a bispecific antigen-binding protein as an active ingredient.

[0366] In one embodiment, the present invention relates to a pharmaceutical formulation comprising a bispecific antigen-binding protein as described herein, or a multispecific antigen-binding protein as an active ingredient, for use in the treatment, prevention, or diagnosis of a disease in subjects requiring treatment, prevention, or diagnosis of a disease.

[0367] In one embodiment, the present invention relates to a method for treating a disease in a subject requiring treatment of the disease, said method comprising the step of administering to the subject a (effective amount) bispecific antigen-binding protein as described herein, or a pharmaceutical preparation comprising a bispecific antigen-binding protein as an active ingredient.

[0368] Diseases treated, prevented, or diagnosed using bispecific antigen-binding proteins may be cancer, infectious diseases, inflammatory diseases, or autoimmune diseases.

[0369] In one embodiment, the disease treated, prevented, or diagnosed using a bispecific antigen-binding protein is cancer, for example, cancer as described below.

[0370] In one embodiment, the cancer to be treated, prevented, or diagnosed using a bispecific antigen-binding protein is a cancer comprising tumor cells expressing PD-L1.

[0371] In one embodiment, the cancer is a solid tumor or a blood cancer. In one embodiment, the solid tumor is colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung carcinoma, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin cancer or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin lymphoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumor, bladder cancer, cancer of the kidney or ureter, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, vertebral axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, It is selected from the group consisting of T-cell lymphoma, environmentally induced cancer, combinations of the above cancers, and metastatic lesions of the above cancers.

[0372] In one embodiment, the blood cancer is selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell pre-lymphocytic leukemia, blastocystic plasmacytic dendritic neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hair cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytic dendritic neoplasm, Waldenström macroglobulinemia or pre-leukemia.

[0373] In one embodiment, the bispecific antigen-binding protein as described herein may be used as a monotherapy (i.e., without other therapeutic agents). In another embodiment, the bispecific antigen-binding protein as described herein may be used in combination therapy.

[0374] In one embodiment, the bispecific antigen-binding protein as described herein is used in combination with another immunotherapy, e.g., cellular immunotherapy. Thus, the bispecific antigen-binding protein may be used in combination with the adoptive transfer of immune cells, including the adoptive transfer of T cells, e.g., CAR T cells or NK cells. The NK cells may be enriched or proliferated, for example, by methods known in the art, or may be in vitro NK cells as described herein.

[0375] In one embodiment, a bispecific antigen-binding protein as described herein may be used in combination therapy with one or more other therapeutic agents. Additional therapeutic agents or agents will be administered in amounts and therapeutic regimens typically used for the agent in monotherapy for the specific disease or condition being treated. Such therapeutic agents include, but are not limited to, anticancer agents and chemotherapy agents when used in the treatment of cancer. Exemplary therapeutic agents that may be used as part of combination therapy in cancer treatment include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, genostatin, cetrorelix, letrozole, raltitrexid, daunorubicin, padrozole, potemustine, timalfacine, sobuzoxan, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumid, elliptinium acetate, ketanserin, doxyfluridine, etretinate, isotretinoin, streptozin, nimustine, vindecin flutamide, derogenyl, butosin, It includes carmofur, razoxic acid, sizophyllan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, fisibanil, levamisole, tenifoside, improsulfan, enositabine, risulide, oxymetholone, tamoxifen, progesterone, mepitiostan, epithiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diptitox, interleukin-2, and luteinizing hormone-releasing factor.

[0376] An additional class of agents that can be used as part of a combination therapy in cancer treatment is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. Another agent that may be used as part of a combination therapy for treating cancer is a cytotoxic monoclonal antibody against TAA, comprising the following cytotoxic monoclonal antibodies against TAA known in the art: trastuzumab (against HER2), pertuzumab (against HER2), rituximab (against CD20), tocitumomab (against CD20), ibritumomab (against CD20), obinutuzumab (against CD20), ofatumumab (against CD20), alemtuzumab (against CD52), blinatumomab (against CD19), inbilizumab (against CD19), tafacitamab (against CD19), daratumumab (against CD38), isatuximab (against CD38), polatuzumab (against CD79b), dinutuximab (against GD2). Nacitamab (for GD2), Bevacizumab (for VEGF-A), Elotuzumab (for SLAMF7), Enfortumab (for Nectin-4), Saxituzumab (for TROP2), Mogamulizumab (for CCR4), Ipilimumab (for CTLA-4), Tremelimumab (for CTLA-4), Durvalumab (for PD1), Pidilizumab (for PD-1), Pembrolizumab (for PD-1), Nivolumab (for PD-1), Cemiplimab (for PD-1), Avelumab (for PD-L1), Cetuximab (for EGFR), Necitumumab (for EGFR), Panitumumab (for EGFR), Olaratumab (for PDGFRα), and (for VEGFR2) Ramucirumab.

[0377] In some embodiments, administration of a bispecific antigen-binding protein and other therapeutic agents may produce an additive or synergistic effect on immunity and / or therapeutic efficacy.

[0378] In one embodiment, a bispecific antigen-binding protein as described herein is used as at least one of neoadjuvant therapy and adjuvant therapy in addition to primary therapy, for example, including surgery and / or radiation therapy. As neoadjuvant therapy, the bispecific antigen-binding protein is administered before primary treatment to reduce tumor size (so that less extensive surgery and / or radiation therapy is required), to kill spread cancer cells (e.g., micrometastatic disease), or to reduce the risk of tumor cells spreading after surgery. As adjuvant therapy, the bispecific antigen-binding protein is administered after primary treatment, for example, to treat minimal residual disease (to destroy remaining cancer cells). The use of the bispecific antigen-binding protein as neoadjuvant therapy and / or adjuvant therapy lowers the recurrence rate. In neoadjuvant therapy and / or adjuvant therapy, the bispecific antigen-binding protein may be used as a monotherapy or in combination therapy as described above.

[0379] In one embodiment, the present invention relates to a nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of a bispecific antigen-binding protein as described herein. The nucleotide sequence encoding such a polypeptide chain preferably encodes a signal peptide operably linked to the polypeptide chain. A nucleic acid molecule comprising one or more of the nucleotide sequences encoding the polypeptide chain further preferably comprises a regulatory element for (or to aid in) the expression of the polypeptide chain in a suitable host cell, wherein the regulatory element is operably linked to the nucleotide sequence.

[0380] In one embodiment, the present invention relates to a host cell comprising a nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of a bispecific antigen-binding protein as described herein. In one embodiment, the host cell is an isolated cell or a cultured cell. Among the host cells that may be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes are Gram-negative or Gram-positive organisms, for example, Escherichia coli ( Escherichia coli It includes ) or bacilli. Suitable yeast cells are Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and Pikia Pastoris( Pichia pastorisIt includes ). Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include COS-1, COS-7 monkey kidney cell lines (Gluzman et al., 1981, Cell 23:175), L cells, HEK 293 cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, BHK cell lines, e.g., BHK21, BSC-1, Hep G2, 653, SP2 / 0, and CVI / EBNA cell lines derived from the African green monkey kidney cell line CVI as described in McMahan et al. (1991, EMBO J. 10: 2821). The host cell may be any suitable species or organism capable of producing N-linked glycosylated polypeptides, for example, human or rodent IgG-type mammalian host cells capable of producing N-linked glycosylation. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in the literature [Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985)]. Host cells containing the nucleic acid molecules of the present invention may be cultured under conditions that promote the expression of polypeptides.

[0381] Accordingly, in another aspect, the present invention relates to a method for producing a bispecific antigen-binding protein as described herein. The method preferably comprises the step of culturing a host cell as described above so that one or more nucleotide sequences are expressed and a bispecific antigen-binding protein is produced. The method preferably comprises the step of culturing a host cell comprising one or more nucleotide sequences encoding a polypeptide chain of a bispecific antigen-binding protein. The host cell is preferably cultured under conditions that facilitate the expression of one or more polypeptide chains.

[0382] The method may further include a step of recovering a bispecific antigen-binding protein. The bispecific antigen-binding protein may be recovered by conventional protein purification procedures, including, for example, using streptavidin / biotin, for example, protein A-cephalosporin chromatography, gel electrophoresis, dialysis, size exclusion chromatography or affinity chromatography (see, for example, [Low et al., 2007, J. Chromatography B, 848:48-63]; [Shukla et al., 2007, J. Chromatography B, 848:28-39]), ion-exchange chromatography on a strong or weak cation or anion medium, or hydrophobic interaction chromatography.

[0383] In a further embodiment, the present invention relates to a method for producing a pharmaceutical composition comprising a bispecific antigen-binding protein as described herein, the method comprising: a) producing a bispecific antigen-binding protein in a method as defined above; and b) formulating the bispecific antigen-binding protein with a pharmaceutically acceptable carrier as defined above to obtain a pharmaceutical composition.

[0384] In another aspect, the present invention relates to a novel specific RNF128 binding protein. The RNF128 binding protein according to the present invention is described in more detail herein when described as being part of the dual-specific antigen-binding protein of the present invention. However, the RNF128 binding protein as described herein is not limited to being part of the dual-specific antigen-binding protein. The RNF128 binding protein according to the present invention comprises a first ISVD as defined herein. Preferably, the RNF128 binding protein comprises an ISVD that specifically binds to the extracellular portion of RNF128, and the first ISVD comprises a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of:

[0385] a) CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 1, CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 3;

[0386] b) CDR1 comprising an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4; CDR2 comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 5; and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 6; and

[0387] c) CDR1 comprising an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 7, CDR2 comprising an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 9.

[0388] Preferably, the immunoglobulin single variable domain of the RNF128 binding protein comprises a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein CDR1 comprises an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1, CDR2 comprises an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2, and CDR3 comprises an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3.

[0389] Preferably, the immunoglobulin monovariable domain of the RNF128 binding protein comprises a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of:

[0390] a) CDR1 comprising the amino acid sequence described in SEQ ID NO: 1, CDR2 comprising the amino acid sequence described in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 3;

[0391] b) CDR1 comprising the amino acid sequence described in SEQ ID NO: 4, CDR2 comprising the amino acid sequence described in SEQ ID NO: 5, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 6; and

[0392] c) CDR1 comprising the amino acid sequence described in SEQ ID NO: 7, CDR2 comprising the amino acid sequence described in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 9.

[0393] Preferably, the immunoglobulin single variable domain of the RNF128 binding protein comprises a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein CDR1 comprises the amino acid sequence described in SEQ ID NO: 1, CDR2 comprises the amino acid sequence described in SEQ ID NO: 2, and CDR3 comprises the amino acid sequence described in SEQ ID NO: 3.

[0394] The ISVD of the RNF128 binding protein is preferably a) the amino acid sequence described in SEQ ID NO: 19, b) the amino acid sequence described in SEQ ID NO: 20; and c) an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO: 21.

[0395] Preferably, the ISVD of the RNF128 binding protein preferably comprises an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 19.

[0396] The ISVD of the specific RNF128 binding protein is preferably a humanized ISVD. Preferably, the humanized ISVD comprises a CDR1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 3. Preferably, the humanized ISVD comprises a CDR1 comprising an amino acid sequence described in SEQ ID NO: 1, a CDR2 comprising an amino acid sequence described in SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence described in SEQ ID NO: 3.

[0397] The humanized ISVD preferably comprises an amino acid sequence selected from the group consisting of the following and an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity:

[0398] a) Amino acid sequence listed in SEQ ID NO: 114;

[0399] b) Amino acid sequence listed in SEQ ID NO: 115;

[0400] c) the amino acid sequence listed in SEQ ID NO: 116; and

[0401] d) Amino acid sequence listed in SEQ ID NO: 117.

[0402] Preferably, the first humanized ISVD comprises a CDR1 having an amino acid sequence described in SEQ ID NO: 1, a CDR2 having an amino acid sequence described in SEQ ID NO: 2, and a CDR3 having an amino acid sequence described in SEQ ID NO: 3, and comprises an amino acid sequence selected from the group consisting of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least It has an amino acid sequence having 98%, at least 99%, or 100% sequence identity:

[0403] a) Amino acid sequence listed in SEQ ID NO: 114;

[0404] b) Amino acid sequence listed in SEQ ID NO: 115;

[0405] c) the amino acid sequence listed in SEQ ID NO: 116; and

[0406] d) Amino acid sequence listed in SEQ ID NO: 117.

[0407] The RNF128 binding protein may be an antibody, and the antibody comprises a first polypeptide comprising a first human Fc fragment and a (first) ISVD as defined herein. The antibody further preferably comprises a second polypeptide comprising a second human Fc fragment and an additional ISVD. The additional ISVD may be the same as the (first) ISVD included in the first polypeptide or may be a different ISVD. Preferably, the antibody does not contain an antibody light chain. Preferably, the Fc fragment comprises a CH2 domain and a CH3 domain as defined herein.

[0408] In one embodiment, the present invention relates to a pharmaceutical composition as described herein, wherein the pharmaceutical composition comprises a specific RNF128 binding protein as described herein. The pharmaceutical composition and / or the specific RNF128 binding protein may be intended for use as a medicine as described herein. The pharmaceutical composition and / or the specific RNF128 binding protein may be intended for use in the treatment of cancer, preferably cancer comprising tumor cells expressing RNF128 as described herein.

[0409] In one embodiment, the present invention relates to a nucleic acid molecule as described herein, wherein the nucleic acid molecule comprises one or more nucleotide sequences encoding a specific RNF128 binding protein, preferably, one or more nucleotide sequences are operably linked to a regulatory sequence as described herein for the expression of the RNF128 binding protein in a host cell. The present invention also relates to a host cell as described herein, wherein the host cell comprises said nucleic acid molecule.

[0410] In one embodiment, the present invention further relates to a method as described herein for the production of a specific RNF128 binding protein, said method comprising the step of culturing a host cell under conditions suitable for the expression of one or more nucleotide sequences and the production of a specific RNF128 binding protein. Optionally, said method further comprises one or more steps of recovering the specific RNF128 binding protein and formulating the protein into a pharmaceutically acceptable carrier.

[0411] The present invention has been described above with reference to a number of exemplary embodiments as illustrated in the drawings and embodiments. Modifications of some parts or elements and alternative implementations are possible and are included within the scope of protection as defined in the appended claims. Brief explanation of the drawing

[0412] Fig. 1 . Characterization of PD-L1 binding VHH (a)Detection of binding of newly generated myc-tagged anti-PD-L1 VHH (1B8, 1G7, 2F3) to overexpressed FLAG-tagged PD-L1 on the cell surface of HEK293T cells using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHH for 16 hours (h). One representative image is shown for the NT condition treated with VHH for non-specific binding. Images were taken with a 20x lens. NT = non-transfected control, (b) Detection of binding of newly generated myc-tagged anti-PD-L1 VHH (1B8, 1G7, 2F3) to HEK293T cells transfected with FLAG-PD-L1 and treated with 50 nM VHH for 16 hours. VHH-myc was detected using an Alexa 488-conjugated anti-myc antibody, and mean fluorescence intensity (MFI) at the cell surface was determined by flow cytometry. An IgG control antibody was used as a negative control. Normalized MFI levels for untreated control cells are presented. (c) Detection of binding of newly generated myc-tagged anti-PD-L1 VHH (1B8, 1G7, 2F3) to endogenous PD-L1 on the surface of MDA-MB-231 cells after 16 hours of treatment with 50 nM anti-PD-L1 VHH. VHH was detected with an anti-myc antibody. Normalized MFI levels for untreated control cells are shown and presented. And (d). The ability of the newly generated anti-PD-L1 VHH to block PD1 / PD-L1 pathway activity was analyzed using the Raji-PD-L1 / Jurkat luciferase reporter assay (Invivogen). A PD-L1 blocking antibody (clone #2340D, R&D Systems) was used as a positive control. 10 µM anti-PD-L1 VHH was added to co-cultures of Raji and Jurkat cells for 6 hours. Luciferase activity reflects the level of pathway activation. Luciferase activity was normalized to control Raji Null cells. Fig. 2Characterization of PD-L1 binding VHH (a) Detection of the binding of a newly generated set of myc-tagged anti-PD-L1 VHH to overexpressed FLAG-tagged PD-L1 on the cell surface of HEK293T cells using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHH for 16 hours. Images were taken with a 20x lens. A representative image of the background binding of VHH on NT cells is presented in Fig. 1a. NT = non-transfected control. (b) Heatmap of the relative binding of anti-PD-L1 VHH to cells overexpressing PD-L1 (HEK293T) or expressing endogenous PD-L1 (MDA-MB-231), as evaluated by flow cytometry, and (c) PD-L1 pathway blocking ability analyzed using the Raji-PD-L1 / Jurkat reporter assay (Invivogen). A PD-L1 blocking antibody (clone #2340D, R&D Systems) was used as a positive control. Co-cultures of Raji and Jurkat cells were incubated with VHH for 6 hours. Pathway inhibition was evaluated by measuring luciferase activity. Luciferase activity was normalized to control Raji Null cells. Fig. 3 Characterization of RNF43-bound VHH Detection of binding of newly generated (a) FLAG- or (b) V5-tagged anti-RNF43 VHH to overexpressed myc-tagged RNF128 on the cell surface of HEK293T cells using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHH for 16 hours in the presence of 10 nM bafilomycin A1 to block lysosomal degradation and increase the signal for RNF128. Images were taken with a 20x lens. NT = non-transfected control. Fig. 4 Characterization of RNF128-bound VHH Detection of binding of newly generated (a) FLAG- or (b) V5-tagged anti-RNF128 VHH to overexpressed myc-tagged RNF128 on the cell surface of HEK293T cells using immunofluorescence confocal microscopy. Cells were treated with 50 nM VHH for 16 hours in the presence of 10 nM bafilomycin A1 to block lysosomal degradation and increase the signal for RNF128. Images were taken with a 20x lens. NT = non-transfected control. Fig. 5. Schematic diagram of how SureTAC works The SureTAC molecule binds simultaneously to PD-L1 and membrane-bound E3 ligase. After binding, the E3 ligase ubiquitines PD-L1, inducing its internalization and subsequent lysosome degradation. Fig. 6 Schematic diagram of a heterobispecific SureTAC molecule using a knob-in-hole strategy. Bispecific SureTAC assembled using the "Knob-in-Hol" heteromerization strategy. VHH for PD-L1 and E3 ligases is fused to the Knob-in-Hol variant. Fig. 7 Surface removal and degradation of SureTAC-induced endogenous PD-L1 (a) Levels of endogenous PD-L1 expression on the surface of colorectal cancer cells (HT29) pre-treated with 1 ng / ml IFNγ for 1.5 days followed by treatment with 10 nM of the indicated SureTAC for 16 hours, (b) Representative histogram of PD-L1 staining in flow cytometry data, (c) Evaluation of PD-L1 protein levels by Western blotting. HT29 cells were treated with the indicated SureTAC (10 nM) for 16 hours. An actin-loading control is shown. (d) HT29 cells were pre-treated with 1 ng / ml IFNγ for 1.5 days followed by treatment with 10 nM or 100 nM of the indicated SureTAC for 16 hours. PD-L1 levels are expressed as mean fluorescence intensity (MFI) and normalized to PD-L1 levels in untreated cells. (e) Mean fluorescence intensity (MFI) of PD-L1 levels on the cell surface of HT29 cells treated with the indicated dose range of SureTAC (nM) for 16 hours after treatment with 1 ng / mL IFNγ for 1.5 days, as determined by flow cytometry; (f) Levels of SureTAC binding (V5 staining) in HT29 cells treated with the indicated dose range of SureTAC (nM) for 16 hours after treatment with 1 ng / mL IFNγ for 1.5 days, as determined by flow cytometry; and (g) Timelapse montage of PD-L1 levels in mouse colorectal cancer cell line MC38 treated with 1 ng / mL INFy 24 hours prior to the start of the experiment. PD-L1 was visualized with a PE-labeled anti-PD-L1 antibody (1:1000; BD Biosciences) using 1 hour of incubation prior to imaging. Excess antibodies were washed away before the start of the experiment. The indicated SureTAC (10 nM) was added at the start of the imaging montage. The time-lapse is displayed with 25-minute (min) jumps per frame display. Fig. 8 Sustained efficacy and efficiency of SureTAC compared to existing blocking antibodies (a) Six cell lines of various cancer origins were treated with 1 ng / mL INFy for 1.5 days, followed by treatment with 10 nM of the indicated SureTAC for 16 hours. PD-L1 surface levels are expressed as mean fluorescence intensity (MFI). PD-L1 levels for each cell line were normalized to untreated control samples. Significance was assessed by 2-way ANOVA analysis, and data were corrected using Dunnet multiple comparison analysis. mRNA expression levels of RNF43 and RNF128 were evaluated by qPRC. (b) PD-L1 surface levels expressed as mean fluorescence intensity (MFI). Mouse colorectal cancer cells (MC38) were treated with 10 nM of control SureTAC and PD-L1-targeted SureTAC containing RNF128 for 16 hours. Levels were assessed using flow cytometry at the indicated time after washing-removal of the SureTAC reagent and normalized to untreated samples. (c) PD-L1 surface levels determined by flow cytometry and normalized to untreated control samples. MC38 cells were treated with 10 nM, 1 nM, and 0.1 nM controls or PD-L1-targeted SureTAC for the indicated times, and the medium was not changed for 96 hours. (d) Evaluation of PD-L1 surface levels in HT29 cells pretreated with 1 ng / mL IFγN. When treated with SureTAC containing different RNF128 VHH (4E2; RNF128 VHH newly identified in this work and B8; previously described RNF128 VHH) compared to SureTAC containing an Atz-based PD-L1 blocking binding arm and SureTAC containing the newly identified 1B8.2 PD-L1 blocking VHH. PD-L1 surface levels were determined by flow cytometry and normalized to untreated control samples. and (e) V5 levels as MFI. The binding of SureTAC to the surface of HT29 cells was evaluated by V5 staining and flow cytometry. Fig. 9 Functional effects of ST in mixed lymphocyte response (a) IL2 and (b) IFNγ cytokine secretion in hPBMCs activated by SEB (50 ng / mL) and incubated with the indicated ST (5 nM, 10 nM, or 50 nM) for 66 hours. Plots represent the concentrations of IL2 or IFNγ released in pg / mL. (c) Bar graph representing the percentage of LDH release normalized to the maximum release (100%). HT29 colorectal cancer cells were pre-incubated with the indicated ST (10 nM and 50 nM) for 4 hours, then hPBMCs were added to the co-culture (non-3:1 effector:tumor cells), and tumor cell lysis was evaluated by LDH secretion according to the manufacturer's protocol (ThermoFisher Scientific). and (d) Graph representing the mean of two separate experiments using different hPBMC donors. HT29 colorectal cancer cells were pre-incubated with labeled ST (10 nM and 50 nM) for 4 hours, then hPBMCs were added to the co-culture (non-3:1 effector:tumor cells), and tumor cell lysis was evaluated by LDH secretion according to the manufacturer's protocol (ThermoFisher Scientific). Fig. 10: PK studies and in vivo efficacy studies for PD-L1 / RNF128 SureTAC. (a) The half-life of SureTac 04 (ST04) PD-L1 (2F3) x RNF128 (4E2) is similar to that of monoclonal antagonist PD-L1 antibodies. Serum concentrations of ST04 PD-L1 (2F3) x RNF128 (4E2), ST45 PD-L1 (2F3) x GFP, ST78 RNF128 (4E2) x GFP, and ST74 (GFPxGFP) were analyzed over time (h) after a single intravenous injection of the indicated 10 mg / kg SureTAC in female C57BL / 6JRj mice. Mice were injected with the indicated SureTAC at the same time point (n=20 mice per group), and blood was sampled three times from each mouse (5 mice per time point). Time points before administration, 15 minutes, 30 minutes, 1 hour, 2 hours, 1 day, 2 days, 3 days, 5 days, 10 days, and 14 days. The half-life of SureTAC was calculated by determining serum antibody concentrations; summarized in the table. (b) and (c) PD-L1 / RNF128 SureTAC inhibits tumor proliferation in a dose-dependent manner. (b) MC38 mouse colorectal cancer cells were injected subcutaneously into C57BL / 6 mice. Tumor volume 80 to 100 mm³ 3 Mice were included at random time intervals. Mice (n=10 per group) were treated with the indicated concentrations of control ST74 GFP x GFP or ST04 PD-L1 (2F3) x RNF128 (4E2). The established tumor size endpoint was 1200 mm². 3 was. Graph calculation nonlinear regression curve fit (y=100) (c) The Kaplan-Meier plot shows the treatment group described in (b), and the loss of survival is indicated when the mouse reaches the set evaluation variable. Fig. 11:(a) Alignment of 2F3 (PD-L1 coupled) and 4E2 (RNF128 coupled) to the most similar human germline framework and presented humanized versions (top: SEQ ID NO: 22, 100, 106-108, 101, 109-111, 103, 112, 102 and 113; bottom: SEQ ID NO: 104, 114-117, 103, 118, 102 and 119), (b) Modeling of the PD-L1 x RNF128 bispecific SureTAC. (c) and (d) PD-L1(c) and RNF128( d Wireframe overlays of six different humanized versions of the binder. The humanized version in (a) was produced as a fusion for the Fc portion of human IgG1 and tested by ELISA for equilibrium binding to antigens PD-L1 (e) and RNF128 (f). Specific details for implementing the invention

[0413] Examples

[0414] ● Example 1

[0415] Generation of dual-specific antigen-binding proteins, a unique VHH panel

[0416] To investigate proximity-induced PD-L1 endocytosis and lysosomal degradation at the endogenous level, the inventors used the No-In-Hole (KiH) strategy to generate a bispecific antibody (SureTAC) having one arm binding to PD-L1 and another arm binding to E3 ligase (RNF128, RNF43, RNF130, or RNF167). To generate SureTAC, various single-strand antibodies (VHH; using llama immunization) against PD-L1, RNF43, and RNF128 were generated. These VHHs were incorporated into the KiH format to generate a heterogeneous bispecific SureTAC antibody ( Fig. 6 ). As an example, the inventors describe herein the development and characterization of Rama anti-PD-L1 and anti-RNF128 human / mouse cross-reactive antibodies.

[0417] Human and mouse RNF128 proteins were produced in HEK293 cells fused to a hexahistidine tag and isolated by immobilized metal affinity chromatography (IMAC). Two llamas were primed with 50 μg of human and mouse RNF128 and then boosted with 50 μg of mouse RNF128. Peripheral blood lymphocytes were isolated, and mRNA was extracted from them. RNA was transcribed into cDNA using reverse transcriptase and random hexamers. IG-H cDNA fragments (both conventional and heavy chains) were amplified using primers that annealed the IGH leader sequence region and the CH2 region. VHH-specific fragments were excised, purified from an agarose gel, and used as templates for nested PCR with cloning primers. The obtained fragment was cloned into a phagemid vector as a fusion with M13gpD after the amber stop codon, ligated, and transformed into TG1 E. coli amber suppressor cells (K-12 supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5, (rK-mK-) F' [traD36 proAB+ lacIq lacZΔM15]) by electroporation. The bacteriophage was produced by infecting the helper phage VCSM13 and precipitated using PEG-NaCl according to standard methods. The antigen was coated onto MaxiSorp plates at concentrations of 5, 0.5, and 0.05 μg / ml. After two rounds of panning, individual clones were selected, and the reactivity of the peripheral cytoplasmic extract was evaluated in a standard ELISA using human or mouse antigens as targets. A significant proportion of these clones exhibit cross-reactivity to mouse and human RNF128, whereas a smaller subset of clones exhibits specificity only to human RNF128. These clones were sequenced by Sanger sequencing. Different clusters of VHH molecules were sequence annotated, and various antibody paratopes were selected. The selected clones were BL21 E.Secreted VHH produced in E. coli (E. coli B dcm ompT hsdS(rB-mB-) gal) and tagged at the C-terminal HIS was purified by immobilized metal affinity chromatography (IMAC). The concentration of VHH was measured, and the equilibrium constant KD was determined by ELISA. Twenty specific anti-RNF128 VHHs were identified, and for 12 of them, binding to RNF128-overexpressing cells was shown via immunofluorescence (IF) (Fig. 4).

[0418] Seven distinct binders exhibited KDs of 0.5 to 30 nM for humans and 10 to 500 nM for mouse RNF128. Epitope binning was performed using SPR technology, and two epitope bins were identified. Five binders (O4A5, O4E2, O4H4, O4D7, and L5H4) were selected for further evaluation.

[0419] [Table 1]

[0420]

[0421] VHH binders for human and mouse PD-L1 were isolated in a similar manner using commercial recombinant human PD-L1 (R&D Systems Catalog No.: 9049-B7) and mouse PD-L1 (R&D Systems Catalog No.: 9048-B7). Eighteen specific anti-PD-L1 VHHs were identified, with affinities ranging from 0.02 to 10 nM (less than 10 nM) for human PD-L1 and cross-reactivity for mouse PD-L1 (KD 8 to 50 nM).

[0422] The functional analysis is based on the inventors' PD-L1 downstream signaling of 2 blockers (O1G8 and O1B8.2) and 16 non-blockersIt was shown that [it] was generated. Epitope binning was performed using SPR technology, and three binders were selected for further evaluation (O2F3, O1G7, O1B8.2). These VHHs bind to specific epitopes on PD-L1. 1B8.2 is a blocker, and 2F3 and 1G7 are non-blockers.

[0423] [Table 2]

[0424]

[0425] Development and Characterization of PD-L1 and RNF128 Cross-linking Bispecific SureTAC Antibodies

[0426] DNA encoding a VHH variable domain antibody generated by gene synthesis and fused to the hinge-Fc region of human immunoglobulin. A linker sequence was optionally used. Typically, human IgG1 or IgG4 sequences were used.

[0427] The synthesized gene fragments were inserted into mammalian expression vectors containing corresponding heavy or light chain invariant domains. The species and isotypes included human IgG1 and IgG4. For analytical purposes, selective C-terminal tags (e.g., His-tag, V5-tag, myc-tag, FLAG-tag) were fused to the antibody sequences. Recombinant antibodies were produced by transiently transfecting Expi293 cells with mammalian expression vectors encoding the antibody heavy chains. Bispecific antibodies were generated using the Knob-Into-Hole technique (WO 1996027011). Knob-Into-Hole (KiH) refers to a set of heterodimerization mutations within the CH3 domain of the IgG Fc portion. Thus, it generates a Knob chain that specifically heterodimerizes with the Hall chain. The heavy chains were encoded on individual vectors and transfected using a 1:1 ratio. Homomeric by-products were removed by purifying the antibody from the cell culture supernatant via protein-A affinity chromatography followed by a single polishing step based on hydroxyapatite, ion exchange, hydrophobic interactions, or size exclusion. Homomeric by-products were typically less than 15% in the consumed medium and were efficiently removed in downstream processing. The backbone typically contained mutations intended to reduce effector function (e.g., L234A, L235A, P329G, or P329S and / or N297G). Optionally, the Fc portion contained half-life extension mutations, such as M252Y, S254T, and T256E (WO 2002060919, WO 2013165690) or M428L and N434S (EP3138853B1).

[0428] Typical Fc-fusion partners used are provided in Table 3.

[0429] [Table 3]

[0430] Hinge and Fc sequences

[0431]

[0432] Either the PD-L1 binder or the RNF128 binder can be fused to the knob or hole Fc portion without any difference in functionality. Table 4 lists some VHH-Fc fusions evaluated using human IgG1-based Fc with the knockout mutant set KO1 (LALAP) combined with the knob or hole mutation.

[0433] [Table 4]

[0434]

[0435] Transient expression levels were found to be typically about 200 mg / L, resulting in more than 100 mg / L of purified heterodimer products. All selected VHH moieties had a Tm melting temperature greater than 65°C, typically greater than 80°C. The bispecific SureTAC molecules maintained the affinity of the selected individual VHH molecules (1 to 2 mM for the selected PD-L1 binder and 2 to 7 mM for the RNF128 binder). The bispecific SureTAC molecules were stable in standard buffer for more than three freeze / thaw cycles and maintained monodispersity as determined by analytical size exclusion chromatography and binding characteristics as determined by ELISA experiments.

[0436] PD-L1 / RNF128 SureTAC potently induces PD-L1 endocytosis and lysosomal degradation.

[0437] To evaluate the degradation agent activity of SureTAC induced against endogenous PD-L1, the inventors used the colorectal cancer cell line HT29 expressing PD-L1 and each of four selected E3 ligases (Fig. 8a), and after mining an online database of E3 expression to study expression in healthy and tumor tissues (TCGA) (Fig. 10a), HT29 cells were treated with 1 ng / ml IFNγ for 36 hours to induce high levels of PD-L1 expression on the cell surface, and then the cells were treated with 10 nM SureTAC for 16 hours. SureTAC binding to PD-L1 (VHH: 2F3) and RNF128 (VHH: 4E2, 4A5, and 4H4) induced potent removal of PD-L1 from the cell surface, as shown by flow cytometry (Figs. 7a and 7b), total protein levels (Fig. 7c), and real-time imaging (Fig. 7g). PD-L1 cell surface removal is already visible after 2 hours of SureTAC treatment (Fig. 8c). PD-L1 / RN128 SureTAC using a previously reported RNF128 binder (B8, described in WO 2020139950) was unable to remove PD-L1 from the cell surface, indicating that the inventors' RNF128 binder is surprisingly successful in degradation activity (Figs. 8d and 8e). Additionally, the three different PD-L1 binders characterized by the inventors (blocking: 1B8.2 and non-blocking: 2F3, 1G7) performed equally well in the down-regulation of PD-L1 when combined with the optimal RNF128 binder (4E2) (Figs. 7a and 7c). With these results, the inventors have shown for the first time that non-blocking binders can be used in a membrane E3 degrader strategy to completely eradicate cell surface expression. Using non-blocking PD-L1 binders maintains tissue-specific PD-L1 degradation potential and lowers overall toxicity compared to a blocking strategy.

[0438] Related features of PD-L1 / RNF128 SureTAC

[0439] Treatment of cells with 10 nM PD-L1 / RNF128 SureTAC removed PD-L1 from the cell surface of colorectal cancer cells within 2 hours, and surface levels remained undetectable for up to 1 week (end of experiment), indicating a sustained response (Fig. 8c).

[0440] - A dose of 1 nM PD-L1 / RNF128 SureTAC was sufficient to remove PD-L1 from the cell surface of colorectal cells within 24 hours, and surface levels remained undetectable for up to 1 week (end of experiment), indicating a sustained response (Fig. 8c).

[0441] - The dose of 0.1 nM PD-L1 / RNF128 SureTAC induced up to 50% PD-L1 down-regulation, which was maintained for up to 1 week (end of experiment), indicating a sustained response (Fig. 8c).

[0442] - The inventors investigated the degradation agent activity of the inventors' most optimal PD-L1 / RNF128 SureTAC on a panel of cell lines expressing various mRNA levels of RNF128 (Fig. 8a). Only the cell lines in which RNF128 expression was detected exhibited PD-L1 cell surface removal and degradation (Fig. 8a). Two cell lines that did not exhibit detectable RNF128 expression remained non-reactive. Thus, these results demonstrate that the degradation agent activity of the inventors' SureTAC reagent depends on endogenous E3 ligase expression.

[0443] - The inventors treated HT29 cells with PD-L1 / RNF128 SureTAC for 16 hours to completely remove PD-L1 levels from the cell surface. After washing all SureTAC reagents from the medium, the cells were monitored for the recovery of PD-L1 levels on the cell surface. The inventors observed that (partial) recovery of PD-L1 levels took more than 24 hours (PD-L1 levels returned to 50% after 24 hours and to approximately 60% after 72 hours; to date, the inventors have not observed complete recovery of PD-L1 surface levels in their assay), which indicates that SureTAC-induced PD-L1 degradation may have a prolonged pharmacological effect (Fig. 8b).

[0444] Comparison of E3

[0445] SureTAC using the inventors' optimal RNF128 binding arms (4E2, 4A5, and 4H4) outperformed all other SureTACs using binders for RNF43, RNF130, or RNF167 at a dose of 10 nM (Figs. 7a, 7b, and 7c). Additionally, referring to Fig. 10b, the PD-L1 down-regulation by RNF128 was much more pronounced than the effect of RNF43.

[0446] In PD-L1 / RNF130 SureTAC, increasing the concentration to 100 nM increased the performance of this PD-L1 degrading agent (Fig. 7d). RNF43 has previously been used in the form of a heterospecific antibody for the degradation of PD-L1 (Publication [James Wells, and Garcia]). However, in the case of the inventors, only PD-L1 / RNF43 SureTAC achieves up to 50% PD-L1 degradation (Figs. 7a, 7b, 7c, 7e, 7f, and 8a).

[0447] PD-L1 / RNF128 SureTAC-mediated PD-L1 degradation induces cytokine secretion and tumor cell death.

[0448] To evaluate the functional effects of SureTAC-mediated PD-L1 cell surface clearance and degradation, PD-L1 (1G7 / 2F3) / RNF128 (4E2) SureTAC was added to mixed lymphocyte reactants at concentrations of 5 nM, 10 nM, and 50 nM. Treatment with atezolizumab (Atz; benchmark PD-L1 blocker) at similar concentrations was used as a positive control. Treatment with PD-L1 / RNF128 SureTAC induced the secretion of the cytokine IL-2 to levels similar to those of Atz, whereas IFNγ secretion was significantly enhanced by SureTAC treatment compared to Atz at all tested concentrations (Figs. 9a and 9b). These results indicate that PD-L1 / RNF128 SureTAC potently induces T cell activation to levels similar to or greater than those of the benchmark antibody Atz.

[0449] Next, the functional effects of PD-L1 / RNF128 SureTAC treatment were evaluated in a tumor lysis assay. In this setup, human colorectal cancer cells (HT29) were co-cultured with human PBMCs and treated with or without 10 nM or 50 nM control antibodies, Atz, or PD-L1 (1G7 / 2F3) / RNF128 (4E2) SureTAC. In this assay, PD-L1 / RNF128 SureTAC induced up to 70% tumor death (LDH release) at concentrations of 10 nM and 50 nM, whereas Atz-induced death reached a maximum of 50% at 50 nM (Figs. 9c and 9d). These results indicate that PD-L1 targeting by PD-L1 / RNF128 SureTAC outperforms Atz for tumor cell death in this experimental setting.

[0450] conclusion

[0451] Combinations of membrane E3 ligases can be successfully used to induce the elimination of various transmembrane targets. PD-L1-targeting dual-specific binders significantly downregulate the surface levels of PD-L1 and enhance tumor cell death. By using non-blocking PD-L1 binders and tissue or cancer-specific E3 ligases, a reduction in toxicity is achieved, while a reduction in resistance can be achieved by not utilizing the active site of PD-L1. Finally, the pharmacological window is enhanced by completely eliminating the PD-L1 protein instead of blocking it.

[0452] ● Example 2

[0453] In vivo efficacy study

[0454] MC38 mouse colorectal cancer cells were injected subcutaneously (sc) into C57BL / 6JRj (female) mice. Tumor volume 80 to 100 mm³ 3 Upon reaching [value], mice were randomly assigned to a group. MC38 tumor-bearing mice were treated every 3 days with the indicated concentrations of control ST74 GFP x GFP (n=10) or ST04 PD-L1 (2F3) x RNF128 (4E2) (n=10 per concentration), and the compounds were injected intraperitoneally (ip). Tumor volume measurements were performed every 3 days (on the same day as treatment). Mice were placed at approximately 1200 mm² 3 It was sacrificed from the established tumor size evaluation variable. At each time point, the tumor volume was non-linear regression curve ( Formula: Y=Y0*exp(k*x), Y0=100 It was calculated in pits, and y was set to 100 (including mice in the experiment is 100 mm 3 (It was performed at the average tumor size).

[0455] conclusion

[0456] In vivo efficacy studies show that PD-L1-targeted SureTAC ST04 PD-L1 (2F3) x RNF128 (4E2) inhibits in vivo tumor growth of MC38 (mouse colorectal cancer) tumors injected subcutaneously at a dose of 10 mg / kg compared to the control SureTAC GFP x GFP, which does not impair tumor growth.

[0457] ● Example 3

[0458] Humanization of PD-L1 x RNF128 bispecific SureTAC.

[0459] SureTAC ST04 consists of the PD-L1 binder-Fc(KO) fusion O2F3-Fc-KO1-HOLE (SEQ ID NO: 43) and the RNF128 binder-Fc(KO) fusion O4E2-Fc-KO1-KNOB (SEQ ID NO: 39). A 3D model of the fusion protein was constructed based on predictions from AlphaFold 2-Multimer running on a local server [Jumper et al. 2021 "Highly accurate protein structure prediction with AlphaFold", Nature 596(7873); 583-589]. The model was enhanced using Protean3D and PyMol3 software and is illustrated in Fig. 11b (space-filled representation).

[0460] Using BLAST software, the inventors probed a local database containing human germline V-regions extracted from V-BASE and IMGT. For O2F3, both IGHV3-48 (SEQ ID NO: 100) and VH3-13374 (SEQ ID NO: 101) were retained, and for O4E2, IGHV3-23*01 (SEQ ID NO: 104) showed high homology. For both sequences, two different pre-humanized framework sequences were also tested: one containing an engineered additional disulfide binding to stabilize CDR2 (preHum_ds, SEQ ID NO: 103), and [Vincke et al., 2009: "General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold", Journal of Biological Chemistry 284(5); General recipient framework for camelid antibodies as described in [3273-3284] (preHum-Nb, SEQ ID NO: 102). Figure 11a shows the alignment of O2F3 and O4E2 to the selected human germline framework and pre-humanized general recipient frameworks FR1-FR2 and FR3, as well as different versions of humanization sequences. The consensus sequence from SEQ ID NO: 105 was used for FR4. The humanized sequences and their respective SEQ ID NOs are listed in Tables 7 and 8 below.

[0461] The proposed humanized sequences were structure-predicted as described above and aligned with the original sequences using Protean3D. These models are shown as wireframe overlays in Fig. 11c for O2F3 and Fig. 11d for O4E2. The root mean square deviation (RMSD) from the camelid sequences was taken as an indicator for a good model, and sequences with an RMSD exceeding 1.5 angstroms were discarded. The sequences were also compared for "humanity" calculations as described in the literature [Abhinandan et al. 2007: "Analysing the degree of humanness of antibody sequences", J Mol Biol 369(3); 852-62], which essentially calculates the Z-score from the standard deviation of mean sequence identity (see Tables 5 and 6 below).

[0462] [Table 5]

[0463] Humanization of PD-L1 binder O2F3

[0464]

[0465] [Table 6]

[0466] Humanization of RNF128 binder O4E2

[0467]

[0468] Humanized binders were produced in HEK293 cells using transient transfection as single-arm VHH-Fc(KO) fusions, purified, and tested for equilibrium binding to each antigen by ELISA. Figures 11e and 11f show the respective dilution curves, while the calculated KD values ​​are shown in Tables 5 and 6.

[0469] conclusion

[0470] In the case of O2F3 (PD-L1 binder), the conservation of camelid sequences at the site where human VH interacts with the VL domain is important in both selected human frameworks, but it was found that the conservation of threonine at position 62 within SEQ ID NO: 22 is more relevant to correct folding and activity. None of the universal pre-humanized frameworks provided active antibody function. V2.1 (SEQ ID NO: 108) and V4.1 (SEQ ID NO: 110) provided KD values ​​close to camelid antibodies with an acceptable level of humanization (both 0.36 nM vs. 0.22 nM).

[0471] O4E2 (RNF128 binder) already demonstrated a high degree of humanity. Some variants not only increased the equilibrium constant but also dramatically increased humanity. V2 (SEQ ID NO: 115) specifically increased humanity to +0.8 (compared to +0.1) and increased binding (KD 0.79 vs. 1.05). V3.2 (SEQ ID NO: 117) is of particular interest because it preserved binding while having a high humanization score. As can be recognized from Figure 11f, most variants using the IGHV3-23*01 framework provided similar results. V1 (SEQ ID NO: 114), which achieves a high humanity score through simple CDR transplantation, also performs well. However, in the case of O4E2, the pre-humanized framework did not provide similar antibody activity, indicating that not all frameworks will provide the described results.

[0472] [Table 7]

[0473] Humanized PD-L1 binder amino acid sequence

[0474]

[0475] [Table 8]

[0476] Humanized RNF128 binder amino acid sequence

[0477]

Claims

Claim 1 As a bispecific antigen-binding protein, i) a first immunoglobulin monovariable domain (ISVD) that specifically binds to the extracellular portion of RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the first ISVD comprises: a) a complementation-determining region (CDR) 1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1; a CDR 2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2; and a CDR 3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3; b) an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 4 CDR1 comprising an amino acid sequence having the amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one amino acid difference(s) from the amino acid sequence of SEQ ID NO: 6;and c) a combination of complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 7, a CDR2 having an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 8, and a CDR3 having an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 9; and ii) a second ISVD that specifically binds to the extracellular portion of PD-L1, wherein the second ISVD comprises d) an amino acid sequence described in SEQ ID NO: 10 or an amino acid sequence of SEQ ID NO: 10 and two or one amino acid Complementary-Determining Region (CDR) 1 comprising an amino acid sequence having difference(s), CDR 2 comprising an amino acid sequence described in SEQ ID NO: 11 or an amino acid sequence having 2 or 1 amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 11, and CDR 3 comprising an amino acid sequence described in SEQ ID NO: 12 or an amino acid sequence having 2 or 1 amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 12;e) CDR1 comprising an amino acid sequence described in SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 13, CDR2 comprising an amino acid sequence described in SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 14, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 15; and f) CDR1 comprising an amino acid sequence described in SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 16, CDR2 comprising an amino acid sequence described in SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 17, and on SEQ ID NO: 18 A bispecific antigen-binding protein comprising a second ISVD comprising a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of CDR3s comprising an amino acid sequence having two or one difference(s) from the described amino acid sequence or the amino acid sequence of SEQ ID NO:

18. Claim 2 In claim 1, i) the first ISVD is a) the amino acid sequence described in SEQ ID NO: 19; b) the amino acid sequence described in SEQ ID NO: 20; and c) an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with respect to an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO: 21; ii) the second ISVD comprises d) the amino acid sequence listed in SEQ ID NO: 22; e) SEQ A bispecific antigen-binding protein comprising an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequence described in SEQ ID NO:

24. Claim 3 In claim 1 or 2, at least one of the first and second ISVDs is a humanized, bispecific antigen-binding protein. Claim 4 In paragraph 3, i) the first humanized ISVD is a) the amino acid sequence described in SEQ ID NO: 114; b) the amino acid sequence described in SEQ ID NO: 115; c) the amino acid sequence described in SEQ ID NO: 116; and d) an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with respect to an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO: 117; and ii) the second humanized ISVD comprises e) the amino acid listed in SEQ ID NO: 108 A sequence; and f) a bispecific antigen-binding protein comprising at least one of an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to an amino acid sequence selected from the group consisting of the amino acid sequences listed in SEQ ID NO:

110. Claim 5 A bispecific antigen-binding protein according to claim 4, wherein i) the first humanized ISVD comprises an amino acid sequence having at least one of SEQ ID NO: 114, 115, 116 and 117; and ii) the second humanized ISVD comprises an amino acid sequence having at least one of SEQ ID NO: 108 and 110. Claim 6 In any one of claims 1 to 5, the bispecific antigen-binding protein is a bispecific antibody comprising i) a first polypeptide comprising a first human Fc fragment and a first ISVD; and ii) a second polypeptide comprising a second human Fc fragment and a second ISVD, and preferably, the antibody is a bispecific antigen-binding protein that does not include an antibody light chain. Claim 7 In claim 6, each Fc fragment is a bispecific antigen-binding protein comprising a CH2 domain and a CH3 domain. Claim 8 A bispecific antigen-binding protein according to claim 4 or 5, wherein the first Fc fragment and the second Fc fragment comprise different amino acid sequences including a complementary knob-into-hole modification that promotes the association of the first and second polypeptide chains of the Fc region to form a heteromeric Fc region. Claim 9 In any one of claims 1 to 8, the bispecific antigen-binding protein has at least one biological activity selected from the following: a) the bispecific antigen-binding protein induces endocytosis of PD-L1 and lysosomal degradation of PD-L1 when in contact with cells expressing PD-L1 and RNF128; and b) the bispecific antigen-binding protein induces at least one of secretion of cytokines and death of tumor cells expressing PD-L1 and RNF128 when added to a co-culture of tumor cells and human PBMCs, wherein the secreted cytokines include at least one of IL-2 and interferon γ. Claim 10 A pharmaceutical composition comprising a bispecific antigen-binding protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier. Claim 11 A bispecific antigen-binding protein according to any one of claims 1 to 9 for use as a medicine, or a pharmaceutical composition according to claim 10. Claim 12 A bispecific antigen-binding protein according to any one of claims 1 to 9 for use in the treatment of cancer, preferably including tumor cells expressing PD-L1, or a pharmaceutical composition according to claim 10. Claim 13 A nucleic acid molecule comprising one or more nucleotide sequences encoding at least one polypeptide chain of a bispecific antigen-binding protein according to any one of claims 1 to 9, wherein, preferably, one or more nucleotide sequences are operably linked to a regulatory sequence for the expression of one or more polypeptide chains in a host cell. Claim 14 A host cell containing a nucleic acid molecule according to paragraph 13. Claim 15 A method for producing a bispecific antigen-binding protein according to any one of claims 1 to 9, comprising the step of culturing a host cell according to claim 14 such that one or more nucleotide sequences are expressed and a bispecific antigen-binding protein is produced. Claim 16 A method according to claim 15, further comprising the step of recovering a bispecific antigen-binding protein, and optionally, the step of formulating the bispecific antigen-binding protein using a pharmaceutically acceptable carrier. Claim 17 An RNF128 binding protein comprising an immunoglobulin monovariable domain (ISVD) that specifically binds to the extracellular portion of RNF128 transmembrane E3 ubiquitin ligase (RNF128), wherein the ISVD comprises: a) a complementation-determining region (CDR) 1 comprising an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 1; a CDR 2 comprising an amino acid sequence described in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 2; and a CDR 3 comprising an amino acid sequence described in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO: 3; and b) an amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence having two or one amino acid difference(s) with respect to the amino acid sequence of SEQ ID NO:

4. CDR1, comprising an amino acid sequence described in SEQ ID NO: 5 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising an amino acid sequence described in SEQ ID NO: 6 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 6;and c) a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of an amino acid sequence described in SEQ ID NO: 7 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 7, a CDR2 having an amino acid sequence described in SEQ ID NO: 8 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 8, and a CDR3 having an amino acid sequence described in SEQ ID NO: 9 or an amino acid sequence having two or one difference(s) from the amino acid sequence of SEQ ID NO: 9, and a combination of CDR1, CDR2, and CDR3. Claim 18 In claim 17, the immunoglobulin monovariable domain comprises a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 selected from the group consisting of a) a complementary-determining region (CDR)1 comprising the amino acid sequence described in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence described in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence described in SEQ ID NO: 3; b) a CDR1 comprising the amino acid sequence described in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence described in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence described in SEQ ID NO: 6; and c) a combination of complementary-determining regions (CDRs) CDR1, CDR2, and CDR3 comprising the amino acid sequence described in SEQ ID NO: 9, wherein the combination comprises a combination of CDR1, CDR2, and CDR3 comprising the amino acid sequence described in SEQ ID NO:

9. Claim 19 In claim 17 or 18, the RNF128 binding protein comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from the group consisting of a) the amino acid sequence described in SEQ ID NO: 19; b) the amino acid sequence described in SEQ ID NO: 20; and c) the amino acid sequence described in SEQ ID NO:

21. Claim 20 In claim 19, the ISVD is an RNF128 binding protein comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence described in SEQ ID NO:

19. Claim 21 In any one of paragraphs 17 through 19, ISVD is a humanized RNF128 binding protein. Claim 22 In claim 21, the ISVD comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from the group consisting of a) the amino acid sequence described in SEQ ID NO: 114; b) the amino acid sequence described in SEQ ID NO: 115; c) the amino acid sequence described in SEQ ID NO: 116; and d) the amino acid sequence described in SEQ ID NO: 117.

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