Anti-ROR1 / anti--4-1BB antibodies and uses thereof

Abstract

Provided herein are bispecific and multispecific antibodies that target tumor antigens and 4-1BB, the Fc region of which has a retained or enhanced effector function; in particular, the multispecific antibodies have binding specificity for human ROR1 protein and human 4-1BB protein. Also provided herein are methods of treating cancer using the antibodies or fragments thereof.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of PCT Application No. PCT / CN2023 / 117286, filed September 6, 2023, the contents of which are incorporated herein in their entirety by this reference. BACKGROUND

[0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the receptor tyrosine kinase family with a molecular weight of 106 kDa. Structurally, the extracellular domain of the ROR1 receptor is composed of three distinct domains: an immunoglobulin-like domain distal to the membrane; a Kringle domain proximal to the membrane; and a Frizzled domain in between. ROR1 is expressed during embryonic and fetal development and controls cell polarity, cell migration, and neurite growth, among others. Its expression gradually decreases as the developmental process progresses. ROR1 is almost not expressed in adults, it is transiently expressed during B-cell development, and it is reported to be minimally expressed in adipocytes. Although ROR1 expression is tightly regulated in normal adult tissues, high levels of expression are observed in both hematological and solid tumors. ROR1 is normally expressed during early development but is activated by tumor-specific mechanisms in adults and can contribute to disease progression.

[0003] The ligands of ROR1 are believed to be wnt5a and NKX1-2. Wnt5a has been shown to bind the Frizzled domain in the extracellular portion of ROR1 and has been shown to regulate NF-κΒ activation and proliferation of normal and lung tumor cell lines in transfected cells. Binding of NKX1-2 to ROR1 has been shown to play a role in the survival of lung cancer cell lines through both kinase-dependent and non-kinase-dependent mechanisms. ROR1 has been shown to interact with EGFR through the Kringle domain and this interaction modulates signaling pathways that control apoptosis in lung cancer cell lines. While ROR1 expression does correlate with a poorer prognosis for ovarian cancer, the association between ROR1 expression and clinical stage or shortened survival has not been demonstrated for lung cancer. Furthermore, although ROR1 siRNA knockdown of lung tumor cell lines in vitro resulted in decreased cell viability, there is no evidence that targeting ROR1 on primary lung cancer cells results in increased cell death.

[0004] 4-1BB (CD137 or TNFRSF9) is a transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) receptor superfamily that was initially identified as an inducible costimulatory receptor expressed on activated T cells. Current understanding of 4-1BB indicates that its expression is generally activation-dependent and is found on a wide variety of immune cell subsets, including activated NK and NKT cells, regulatory T cells, dendritic cells (DCs, including follicular DCs), stimulated mast cells, differentiating myeloid cells, monocytes, neutrophils, eosinophils, and activated B cells. In addition, expression of 4-1BB has been demonstrated on tumor vascular endothelium and atherosclerotic endothelium. The ligand for 4-1BB stimulation (4-1BBL) is expressed on activated antigen-presenting cells (APCs), myeloid progenitors, and hematopoietic stem cells.

[0005] Upon engagement of the B cell receptor, 4-1BB on activated normal human B cells interacts with its ligand to stimulate cell proliferation and enhance survival. 4-1BB is not detectable on the surface of naive T cells, but its expression increases upon cell activation. When 4-1BB is activated, the pro-survival members of the TNFR-associated factor (TRAF) family, TRAF1 and TRAF2, are recruited to the cytoplasmic tail of 4-1BB, leading to downstream activation of NFkB and the mitogen-activated protein (MAP) kinase cascade, including Erk, Jnk, and p38 MAP kinases.

[0006] The specific expression of ROR1 on cancer cells and 4-1BB on immune cells indicates that these two targets can serve as potential cancer targets for antibody therapy. SUMMARY

[0007] The present disclosure provides multispecific antibodies having binding specificity for human Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) protein and 4-1BB protein. These antibodies and fragments are useful for treating diseases and disorders such as cancer.

[0008] In one aspect of the disclosure, a multispecific antibody is provided, comprising: (1) a first antibody portion that specifically binds to a tumor-associated antigen (TAA), and (2) a second antibody portion that specifically binds to human 4-1BB protein.

[0009] In certain embodiments, the multispecific antibody further comprises an Fc domain with retained or enhanced effector function.

[0010] In certain embodiments, the second antibody portion is an sdAb.

[0011] In certain embodiments, the second antibody moiety comprises HCDR1, HCDR2, and HCDR3, which are SNCMG (SEQ ID NO: 47), VICTGGGSPSYADSVKG (SEQ ID NO: 48), DLLRAGTPLSSYEFNY (SEQ ID NO: 49), respectively.

[0012] In certain embodiments, the second antibody moiety comprises the amino acid sequence of SEQ ID NO: 9, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0013] In certain embodiments, the Fc domain is derived from any one selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

[0014] In certain embodiments, the Fc domain is derived from IgGl.

[0015] In certain embodiments, the Fc domain comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 52 and 140-143.

[0016] In certain embodiments, the first antibody moiety is a Fab’ fused to the N-terminus of the IgG Fc domain, and the second antibody moiety is a sdAb fused to the C-terminus of the IgG Fc domain.

[0017] In certain embodiments, the second antibody moiety is fused to the IgG Fc domain via a linker.

[0018] In certain embodiments, the multispecific antibody further comprises a third antibody moiety that specifically binds a second tumor-associated antigen.

[0019] In certain embodiments, the first antibody moiety is a Fab’ fused to the N-terminus of the IgG Fc domain, the second antibody moiety is a sdAb fused to the C-terminus of the IgG Fc domain, and the third antibody moiety is a scFv fused to the N-terminus of the first antibody moiety.

[0020] In certain embodiments, the second antibody moiety is fused to the IgG Fc domain via a linker, and the third antibody moiety is fused to the first antibody moiety via a linker.

[0021] In certain embodiments, the first antibody moiety is a Fab’ fused to the N-terminus of an IgG Fc domain, the second antibody moiety is a sdAb fused to the C-terminus of the IgG Fc domain, the third antibody moiety is a scFv fused to the N-terminus of an IgG heavy chain constant region (IgG’(CH)), and the second antibody moiety is a sdAb fused to the C-terminus of the IgG’(CH), wherein the IgG’(CH) comprises a paired IgG Fc domain and forms a heterodimer with the IgG Fc domain.

[0022] In certain embodiments, the second antibody moiety is fused to the IgG1 Fc domain via a linker, and the second antibody moiety is fused to the IgG’(CH) via a linker.

[0023] In certain embodiments, the first antibody moiety and the third antibody moiety specifically bind to human Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) protein, but bind to different epitopes.

[0024] In certain embodiments, the first antibody moiety or the third antibody moiety comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the following: (a) HCDR1: SYAMS (SEQ ID NO: 10) or RYAMS (SEQ ID NO: 11), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12) or SISSGGNTYYPDTVKGR (SEQ ID NO: 13) or SISSGGTRYYPDTVKGR (SEQ ID NO: 14), HCDR3: DSYLYYGSSLYYAMDY (SEQ ID NO: 15), DALYYGGSLYYAMDY (SEQ ID NO: 16) or DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDG (SEQ ID NO: 18), RENRLVDA (SEQ ID NO: 19) or RANRLVDA (SEQ ID NO: 75), and LCDR3: LQYDEFPYT (SEQ ID NO: 20); (b) HCDR1: TYVMH (SEQ ID NO: 21) or NYVMH (SEQ ID NO: 22), HCDR2: YINPYNGGIRYNEKFKG (SEQ ID NO: 23), YINPYNGVIRYNEKFKG (SEQ ID NO: 24) or YINPYSGGIRYNEKFKG (SEQ ID NO: 24), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), RERGVYYGMSE (SEQ ID NO: 27) or RERGVTAGMDE (SEQ ID NO: 28), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29) or KSSQSLLHSNDKTYLN (SEQ ID NO: 30), LCDR2: LVSKLESG (SEQ ID NO: 31) or LVSKLSSG (SEQ ID NO: 32), and LCDR3: LQATYFPYT (SEQ ID NO: 33) or YQATYFPYT (SEQ ID NO: 34); (c) HCDR1: DYWMH (SEQ ID NO: 35), HCDR2: AIDTSDSSTRNNQKFKG (SEQ ID NO: 36), HCDR3: GARTGTGFGY (SEQ ID NO: 37), LCDR1: KSSQSLLHINGKTYLN (SEQ ID NO: 38), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATHFPYT (SEQ ID NO: 40); and (d) HCDR1: SYGVH (SEQ ID NO: 41), HCDR2: VIWAGGHTNYNSDLMSR (SEQ ID NO: 42), HCDR3: RSIYGDYALDY (SEQ ID NO: 43), LCDR1: VTYMH (SEQ ID NO: 44), LCDR2: DISKLASG (SEQ ID NO: 45), and LCDR3: QQWNYPLMT (SEQ ID NO: 46).

[0025] In certain embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody portion or of the third antibody portion are: (a) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12), HCDR3: DSLYYGSSLYYAMDY (SEQ ID NO: 15), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDG (SEQ ID NO: 18), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), (b) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDA (SEQ ID NO: 75), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), (c) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12), HCDR3: DALYYGGSLYYAMDY (SEQ ID NO: 16), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDA (SEQ ID NO: 75), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), (d) HCDR1: RYAMS (SEQ ID NO: 11), HCDR2: SISSGGNTYYPDTVKGR (SEQ ID NO: 13), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RENRLVDA (SEQ ID NO: 19), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), or (e) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTRYYPDTVKGR (SEQ ID NO: 14), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RENRLVDA (SEQ ID NO: 19), and LCDR3: LQYDEFPYT (SEQ ID NO: 20).

[0026] In certain embodiments, the first antibody moiety or the third antibody moiety comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 2; (b) a VH comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 58, and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59; (c) a VH comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59; (d) a VH comprising the amino acid sequence of SEQ ID NO: 63, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 63, and VL comprising the amino acid sequence of SEQ ID NO: 64, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 64; or (e) a VH comprising the amino acid sequence of SEQ ID NO: 65, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 65, and VL comprising the amino acid sequence of SEQ ID NO: 64, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 64.

[0027] In certain embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody moiety or the third antibody moiety are: (a) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGGIRYNEKFKG (SEQ ID NO: 23), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATYFPYT (SEQ ID NO: 33); (b) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGVIRYNEKFKG (SEQ ID NO: 24), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLSSG (SEQ ID NO: 32), and LCDR3: LQATYFPYT (SEQ ID NO: 33); (c) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGGIRYNEKFKG (SEQ ID NO: 23), HCDR3: RERGVYYGMSE (SEQ ID NO: 27), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: YQATYFPYT (SEQ ID NO: 34); (d) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO: 25), RERGVYYGMSE (SEQ ID NO: 27), LCDR1: KSSQSLLHSNDKTYLN (SEQ ID NO: 30), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: YQATYFPYT (SEQ ID NO: 34); (e) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO: 25), HCDR3: RERGVTAGMDE (SEQ ID NO: 28), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATYFPYT (SEQ ID NO: 33); or (f) HCDR1: NYVMH (SEQ ID NO: 22), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO: 25), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATYFPYT (SEQ ID NO: 33).

[0028] In certain embodiments, the first antibody moiety or the third antibody moiety comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 3, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 3, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 4; (b) a VH comprising the amino acid sequence of SEQ ID NO: 60, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 60, and a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61; (c) a VH comprising the amino acid sequence of SEQ ID NO: 66, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 66, and a VL comprising the amino acid sequence of SEQ ID NO: 67, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 67; (d) a VH comprising the amino acid sequence of SEQ ID NO: 68, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 68, and a VL comprising the amino acid sequence of SEQ ID NO: 69, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 69; (e) a VH comprising the amino acid sequence of SEQ ID NO: 70, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 70, and a VL comprising the amino acid sequence of SEQ ID NO: 71, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 71; (f) a VH comprising the amino acid sequence of SEQ ID NO: 72, or a peptide with at least 90% (or at least 95%, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 72, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 61; (g) VH, comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 73, and VL, comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 61; or (h) VH, comprising the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 77.

[0029] In some embodiments, the first antibody portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 6.

[0030] In some embodiments, the first antibody portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0031] In some implementations, the first antibody portion comprises (a) VH, comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 1, and VL, which contains the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 2; (b) VH, comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 58, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 59; (c) VH, comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 62, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 59; (d) VH, comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 63, and VL, comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 64; or (e) VH, comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 65, and VL, which contains the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 64; And the third antibody portion contains (a) VH, comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 3, and VL, which contains the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 4; (b) VH, comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 60, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 61; (c) VH, comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 66, and VL, which contains the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 67; (d) VH, comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 68, and VL, which contains the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 69; (e) VH, comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 70, and VL, which contains the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 71; (f) VH, comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 72, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 61; (g) VH, comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 73, and VL, comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 61; or (h) VH, comprising the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 77.

[0032] In some implementations, the third antibody portion includes (a) VH, comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 1, and VL, which contains the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 2; (b) VH, comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 58, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 59; (c) VH, comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 62, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 59; (d) VH, comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 63, and VL, comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 64; or (e) VH, comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 65, and VL, which contains the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 64; And the first antibody portion contains (a) VH, comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 3, and VL, which contains the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 4; (b) VH, comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 60, and VL, which contains the amino acid sequence of SEQ ID NO:61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO:61; (c) VH, comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 66, and VL, which contains the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 67; (d) VH, comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 68, and VL, which contains the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 69; (e) VH, comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 70, and VL, which contains the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 71; (f) VH, comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 72, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 61; (g) VH, comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 73, and VL, comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 61; or (h) VH, comprising the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% (or at least 95%, at least 98%) sequence identity with the amino acid sequence of SEQ ID NO: 77.

[0033] In some embodiments, the adapter comprises the amino acid sequence of SEQ ID NO: 56.

[0034] In some implementations, the first antibody portion and the third antibody portion pair up via the Fc region to form a heterodimer.

[0035] In some embodiments, (a) the first antibody portion comprises an IgG heavy chain constant region (IgG(CH)) containing the amino acid sequence of SEQ ID NO: 51, and the third antibody portion comprises IgG'(CH) containing the amino acid sequence of SEQ ID NO: 53; or (b) the first antibody portion comprises IgG(CH) containing the amino acid sequence of SEQ ID NO: 55, and the third antibody portion comprises IgG'(CH) containing the amino acid sequence of SEQ ID NO: 54.

[0036] In one aspect, this disclosure provides a composition comprising the multispecific antibody and a pharmaceutically acceptable carrier provided herein.

[0037] In one aspect, this disclosure provides an isolated cell containing one or more polynucleotides that encode the multispecific antibodies provided herein.

[0038] In one aspect, this disclosure provides a polynucleotide that encodes one or more chains of the multispecific antibody provided herein.

[0039] In one aspect, this disclosure provides a method of treating cancer in a patient in need, comprising administering to the patient a multispecific antibody provided herein. In some embodiments, the cancer is selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0040] In some implementations, the method includes administering a treatment for the cancer to the patient.

[0041] In some embodiments, the method further includes administering a therapy to the patient to treat the cancer. In some embodiments, the therapy is selected from immunotherapy, chemotherapy, and radiotherapy.

[0042] In some implementations, the cancer is selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. Attached Figure Description

[0043] Figures 1A-1C The bispecific (A) and bi-paratopic (BC) forms of anti-ROR1 / anti-4-1BB antibodies are shown.

[0044] Figures 2A-2B The cell-binding activity of the bispecific anti-ROR1 / anti-4-1BB antibody, as measured by FACS, is shown.

[0045] Figures 3A-3C This demonstrates ROR1-dependent 4-1BB activation of the bispecific anti-ROR1 / anti-4-1BB antibody in reporter gene assays.

[0046] Figures 4A-4C The epitope binning of anti-ROR1 antibodies is shown. A. Epitope binning experimental procedure; B. Epitope binning results for 3C5 and 8F5, respectively.

[0047] Figures 5A-5C This demonstrates ROR1-dependent 4-1BB activation of the dual complementary anti-ROR1 / anti-4-1BB antibody in reporter gene assays.

[0048] Figures 6A-6D show (AB) the cell-binding activity of anti-ROR1 mAb to ROR1 as measured by FACS before and after humanization, and (CD) the ROR1-dependent 4-1BB activation in reporter gene assays of bispecific anti-ROR1 / anti-4-1BB antibodies before and after humanization.

[0049] Figures 7A-7F show (AC) cell binding activity of bispecific and bicomplementary anti-ROR1 / anti-4-1BB antibodies as measured by FACS; and (DF) ROR1-dependent 4-1BB activation of bispecific and bicomplementary anti-ROR1 / anti-4-1BB antibodies in reporter gene assays using ROR1-positive and ROR1-negative tumor cell lines.

[0050] Figures 8A-8C SPR results for humanized bispecific and bicomplementary anti-ROR1 / anti-4-1BB antibodies are shown.

[0051] Figures 9A-9B This study demonstrates the in vivo potency of humanized bispecific and bicomplementary anti-ROR1 / anti-4-1BB antibodies. (A) Study design; (B) In vivo antitumor potency in 4-1BB knock-in mice.

[0052] Figure 10A-10E indicates ROR1-dependent 4-1BB activation of the affinity-matured bispecific anti-ROR1 / anti-4-1BB antibody in reporter gene assays.

[0053] Figures 11A-11C This demonstrates ROR1-dependent 4-1BB activation of the affinity-matured bicomponent anti-ROR1 / anti-4-1BB antibody in reporter gene assays.

[0054] Figures 12A-12B This demonstrates ROR1-dependent 4-1BB-induced cytokine release from affinity-matured bispecific and bicomplementary anti-ROR1 / anti-4-1BB antibodies during reporter gene assays.

[0055] Figures 13A-13C This study demonstrates the in vivo potency of the affinity-matured dual complementary anti-ROR1 / anti-4-1BB antibodies. (A) Study design; (B) In vivo antitumor potency in hROR1-expressing MC38 cell-inoculated 4-1BB knock-in C57 mice; (C) Summary of tumor growth inhibition (TGI) in each treatment group.

[0056] Figures 14A-14B This study demonstrates tumor suppression in hu4-1BB mice after treatment with the CLDN6 x4-1BB BsAb disclosed herein. Detailed Implementation Definitions

[0057] It should be noted that the terms "an" or "a type" refer to one or more of the entities; for example, "an antibody" should be understood to mean one or more antibodies. Therefore, the terms "an" (or "a type"), "one or more," and "at least one" are used interchangeably in this document.

[0058] As used herein, "antibody" or "antigen-binding moiety" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any of its antigen-binding fragments or single chains. Therefore, the term "antibody" includes any molecule containing a protein or peptide that comprises at least a portion of an immunoglobulin molecule and has the biological activity of binding an antigen. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR) or its ligand-binding moiety of the heavy or light chain, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region or any portion thereof, or at least a portion of a binding protein.

[0059] Full-length antibodies consist of two heavy chains and two light chains. Variable regions of the light and heavy chains are responsible for antigen binding. These variable domains can be referred to as "VH" and "VL," respectively. Each variable region typically contains three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody and antigen-binding fragments disclosed herein can be defined or determined according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three core-residue junctions (CDRs) of the heavy or light chain are interspersed between flanking segments called framework regions (FRs), which are more conserved than CDRs and form a scaffold to support the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified into different classes based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes can be further subdivided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0060] As used herein, the term "hapten" refers to the association of an immunoglobulin heavy chain with an immunoglobulin light chain. Those skilled in the art will understand that a hapten can comprise fragments of itself and can also have an antigen-binding domain composed of a single variable domain, such as variable domains derived from camels (camelidae).

[0061] As used herein, the term "single-chain half antibody" refers to a single-chain polypeptide comprising a VL domain, an optional CL domain, a tether, a VH domain, an optional CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the relative positions of these domains from the N-terminus to the C-terminus are as follows: VL-tether-VH-hinge-CH2-CH3, VL-tether-VH-partial hinge-CH2-CH3, VL-tether-VH-hinge variant-CH2-CH3, or VL-CL-tether-VH-CH1-hinge-CH2-CH3.

[0062] The term "single-domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) can confer antigen binding. In other words, a single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include antibodies derived from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks) and antibodies derived from human and mouse antibodies via recombinant methods (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett (1994) 339:285-290; WO00 / 29004; WO 02 / 051870). When the sdAb contains only the heavy chain, it can be used interchangeably with "VHH" or "single-heavy-chain variable domain antibody" or "nanobody".

[0063] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody (such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc.). Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, specigelmers, and diaboses. The term "antibody fragment" also includes any synthetic or genetically engineered protein capable of forming a complex by binding to a specific antigen, similar to an antibody.

[0064] In the context of antibodies, "Fab" refers to a monovalent antigen-binding fragment of an antibody, which consists of a light chain (variable region and constant region) bound to the variable region and the first constant region of a heavy chain via disulfide bonds. Fab can be obtained by digesting the antibody with papain at residues near the N-terminus of the disulfide bonds between the heavy chains in the hinge region.

[0065] "Fab" refers to a Fab fragment that includes a portion of the hinge region. It can be obtained by digesting an antibody with pepsin at residues near the C-terminus of the disulfide bond between the heavy chains in the hinge region, and therefore contains a small number of residues (including one or more cysteine ​​residues) in the hinge region, unlike Fab.

[0066] "F(ab)2" refers to the dimer of Fab', which contains two light chains and a portion of two heavy chains.

[0067] "Single-chain variable fragment" or "scFv" refers to the variable region (V) of the heavy chain of immunoglobulins. H ) and the variable region (V) of the light chain L These are fusion proteins. In some respects, these regions are linked by short linker peptides of 10 to 25 amino acids. For flexibility, the linker may be enriched with glycine, and for solubility, it may be enriched with serine or threonine. The linker can also carry V... H N-terminus and V L The C-terminus is linked, or vice versa. Despite the removal of the constant region and the introduction of the adaptor, this protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent 5,892,019.

[0068] The term antibody encompasses a wide range of biochemically distinguishable classes of polypeptides. Those skilled in the art will understand that heavy chains can be classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and within these classes are further subclasses (e.g., γ1-γ4). The nature of this chain determines the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specificity. Given this disclosure, those skilled in the art can readily identify modifications of each of these classes and isotypes, and therefore these modifications are within the scope of this disclosure. All immunoglobulin classes are explicitly within the scope of this disclosure, and the following discussion generally pertains to IgG class immunoglobulin molecules. In the case of IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually connected by disulfide bonds in a "Y" configuration, where the light chain starts from the opening of the "Y" shape, extends along the variable region, and encloses the heavy chain.

[0069] The antibodies, antigen-binding moieties, variants, or derivatives disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-derived antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies of the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0070] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, light and heavy chains are covalently bonded to each other, while the "tail" regions of the two heavy chains are bonded to each other via covalent disulfide bonds or non-covalent bonds, regardless of whether the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration branch to the C-terminus at the bottom of each chain.

[0071] Both the light and heavy chains are divided into regions that are structurally and functionally homologous. The terms "constant" and "variable" are used according to function. In this regard, it should be understood that the variable domains (VK) of the light chain and the variable domains (VH) of the heavy chain determine antigen recognition and specificity. Conversely, the constant domains (CK) of the light chain and the constant domains (CH1, CH2, or CH3) of the heavy chain confer important biological properties such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of constant domains increases with their distance from the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0072] As described above, the variable region enables antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VK and VH domains, or complementarity-determining regions (CDRs), forms the variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites at the ends of each arm of the Y-chain. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) for each of the VH and VK chains. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins, the complete immunoglobulin molecule may consist only of heavy chains, without light chains. See, for example, Hamers-Casterman. et al. , Nature 363:446-448 (1993).

[0073] In naturally occurring antibodies, the six “complementarity-determining regions” or “CDRs” present in each antigen-binding domain are short, discontinuous amino acid sequences that are specifically localized to form the antigen-binding domain when the antibody presents its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain (called “framework” regions) exhibit less intermolecular variability. Framework regions primarily adopt a β-sheet conformation, and CDRs form cyclic structures connecting these β-sheets and, in some cases, form part of a β-sheet. Thus, the framework regions, by forming a scaffold, position the CDRs in the correct orientation via interchain non-covalent interactions. The antigen-binding domain formed by the localized CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, those skilled in the art can readily identify the amino acids containing the CDRs and framework regions, as they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al ., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol ., 196:901-917 (1987)).

[0074] If a term is used and / or has two or more definitions in the art, its definition as used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe discontinuous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. This specific region has been described by Kabat. et al ., US Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983) and Chothia et al ., J. MoI. Biol The description in 196:901-917 (1987), the entire contents of which are incorporated herein by reference. The CDR definition according to Kabat and Chothia includes overlapping amino acid residues or subsets of amino acid residues when compared with each other. However, the application of either definition to refer to a CDR of an antibody or a variant thereof is within the scope of the terminology defined and used herein. The appropriate amino acid residues covering the CDRs defined in the above-cited literature are listed in the table below for comparison. The exact residue numbering covering a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR based on the amino acid sequence of the variable region of an antibody.

[0075] Kabat et al. also defined a numbering system applicable to variable domain sequences of any antibody. Those skilled in the art can readily apply this "Kabat numbering" system to any variable domain sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat number" refers to Kabat... et al The numbering system proposed in US Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0076] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately the 31st amino acid (i.e., approximately 9 residues after the first cysteine ​​residue), comprises approximately 5-7 amino acids, and terminates at the next tryptophan residue. CDR-H2 begins at the 15th residue after the termination of CDR-H1, comprises approximately 16-19 amino acids, and terminates at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the termination of CDR-H2; comprises 3-25 amino acids; and terminates at the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately the 24th residue (i.e., after the cysteine ​​residue); comprises approximately 10-17 residues; and terminates at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the termination of CDR-L1 and comprises approximately 7 residues. CDR-L3 begins at approximately the 33rd residue after the termination of CDR-L2 (i.e., after the cysteine ​​residue); it consists of approximately 7-11 residues and terminates at sequence F or WGXG, where X is any amino acid.

[0077] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, rodent, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be derived from cartilaginous fish (e.g., from sharks).

[0078] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in this disclosure may comprise a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain containing a CH3 domain. Furthermore, an antibody for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified so that their amino acid sequences differ from those of naturally occurring immunoglobulin molecules.

[0079] The heavy chain constant regions of antibodies disclosed in this paper can be derived from different immunoglobulin molecules. For example, the heavy chain constant regions of peptides can contain components derived from IgG.l The molecule contains the CH1 domain and a hinge region derived from the IgG3 molecule. In another example, the heavy chain constant region may contain a portion derived from IgG. l The molecule is partially derived from the hinge region of the IgG3 molecule. In another example, the heavy chain portion may contain components partially derived from IgG. l The molecule is also partially derived from the chimeric hinge of the IgG4 molecule.

[0080] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant κ domain or a constant λ domain.

[0081] "Light chain-heavy chain pair" refers to a set of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0082] As previously mentioned, the subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (closest to the N-terminus) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the N-terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0083] The "CH1 domain" (also known as the "C1" domain of "H1") typically extends from about the 118th amino acid to about the 215th amino acid (EU numbering system).

[0084] As used herein, the term "hinge region" includes the heavy chain portion of the molecule that connects the CH1 and CH2 domains, corresponding in IgG to the regions Glu216 to Pro230 of human IgG1 (EU numbering system) (Burton, Molec. Immunol. 22:161-206 (1985)). The hinge region of other IgG isotypes can be compared with the IgG1 sequence by placing the first and last cysteine ​​residues forming the inter-heavy chain SS bond in the same position. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain (Roux). et. al. , J. Immunol 161:4083 (1998)).

[0085] As used herein, the term "CH2 domain" includes portions of the heavy chain molecule, for example, extending from approximately residue 244 to residue 360 ​​of the antibody using a conventional numbering scheme (residues 244-360, Kabat numbering system; residues 231-340, EU numbering system; see Kabat). et. al. (US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983)). The unique feature of the CH2 domain is that it does not pair tightly with other domains. Instead, in the intact natural IgG molecule, two N-linked branched sugar chains are inserted between the two CH2 domains. It is well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, containing approximately 108 residues.

[0086] The “CH3 domain” (also known as the “C3 domain”) contains a segment of residues from the C-terminus of the CH2 domain in the Fc region (i.e., from approximately amino acid residue 341 of the antibody sequence to the C-terminus (usually amino acid residue 446 or 447 of IgG, EU numbering system)).

[0087] In this document, the terms “Fc region,” “Fc domain,” or “crystallizable fragment region” are used to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid of the heavy chain encoding the antibody. Thus, compositions of complete antibodies can comprise groups of antibodies with all K447 residues removed, groups of antibodies without K447 residues removed, and groups of antibodies containing and without K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0088] As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or disulfide bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 (positions 226 or 229, EU numbering system) corresponding to positions using the Kabat numbering system.

[0089] As used herein, the term "chimeric antibody" should be understood to mean any antibody in which the immunoreactive region or site is derived from or obtained from a first species, and the constant region (which, according to this disclosure, may be complete, partial, or modified) is derived from a second species. In some embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate), while the constant region is derived from a human.

[0090] The term “humanized antibody” is used herein to describe an antibody comprising heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but wherein at least a portion of the VH and / or VL sequences has been modified to be more “human-like,” i.e., more similar to human germline variable sequences. A “humanized antibody” is an antibody or a variant, derivative, analog, or fragment thereof that binds immunospecifically to an antigen of interest and comprises a frame (FR) region having substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR means a CDR having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical amino acid sequence to the CDR of a non-human antibody. Humanized antibodies comprise substantially all of at least one and typically two variable domains (Fab, Fab', F(ab')2, Fv), wherein all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulins (i.e., donor antibodies), and all or substantially all frame regions are frame regions of human immunoglobulin common sequences. In one embodiment, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. In some embodiments, the humanized antibody comprises at least a variable domain of a light chain and a heavy chain. The antibody may also comprise CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In a specific embodiment, the humanized antibody comprises only a humanized light chain variable domain and / or a humanized heavy chain variable domain.

[0091] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen that binds to an antibody or antibody moiety. If two antibodies or antibody moiety exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0092] "Specific binding" or "specific to" generally refers to an antibody binding to an epitope through its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to it more easily through its antigen-binding domain than to a random, unrelated epitope. In this paper, the term "specificity" is used to define the relative affinity of an antibody for a given epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with higher specificity than to related epitope "D."

[0093] As used herein, the term "treat" refers to therapeutic actions and preventative or protective measures aimed at preventing or slowing (mitigating) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to prolonged survival compared to expected survival without treatment. Individuals requiring treatment include those who already have a condition or disease, those susceptible to a condition or disease, or those seeking to prevent a condition or disease.

[0094] "Subject," "individual," "animal," "patient," or "mammal" refers to any subject requiring diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, livestock, farm animals, and zoo, racing, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0095] As used herein, phrases such as “patient in need of treatment” or “subject in need of treatment” include subjects who would benefit from the administration of the antibodies or compositions disclosed herein (e.g., for detection, for diagnostic procedures and / or for treatment), such as mammalian subjects. Anti-ROR1 antigen binding site

[0096] This disclosure provides a multispecific antibody containing one or more antigen-binding moieties that are specific for binding to the human ROR1 protein. Mouse clones 3C5, 8F2, 10C9, and 9C9 were selected for constructing the multispecific antibody.

[0097] According to one embodiment of this disclosure, an antigen-binding moiety is provided, comprising heavy chain and light chain variable domains having CDR regions. The CDRs are summarized in Table 1A below (Kabat numbers). Table 1A. CDR sequence of the anti-ROR1 antigen binding region

[0098] The variable regions are summarized in Table 1B below. Table 1B. Variable region sequence of the anti-ROR1 antigen binding site

[0099] In some embodiments, VH CDR1, CDR2, and CDR3 are selected from any set of VH CDR1, CDR2, and CDR3 shown in Table 1, and VL CDR1, CDR2, and CDR3 are selected from any set of VL CDR1, CDR2, and CDR3 shown in Table 1. In some embodiments, VH CDR1, CDR2, and CDR3, as well as VL CDR1, CDR2, and CDR3, are selected from those derived from the same antigen-binding moiety.

[0100] In some embodiments, at least one, two, three, four, five, or six of the above-mentioned VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 are modified by adding, deleting, or substituting one, two, or three amino acids or a combination thereof.

[0101] Depending on the specific implementation, the antibody is humanized. Humanized forms of non-human (e.g., mouse) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) containing a minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibodies) in which residues constituting the complementarity-determining region (CDR) of the receptor are replaced by residues of the CDR from a non-human species (donor antibody) (such as mouse, rat, or rabbit) with the desired specificity, affinity, and capability. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not found in the receptor antibody or in the introduced CDR or framework sequence. Generally, humanized antibodies will contain at least one, and typically substantially all, of two variable domains, where all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of the human immunoglobulin common sequence. Humanized antibodies preferably also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)).

[0102] Methods for humanizing nonhuman antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are commonly referred to as importing residues and are usually derived from the importing variable domain. Humanization can be performed essentially as Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567), in which a portion far less than the complete human variable domain has been replaced by a corresponding sequence from a nonhuman species. In practice, humanized antibodies are usually human antibodies, with some CDR residues and possibly some FR residues replaced by residues at similar sites in rodent antibodies.

[0103] In some implementations, the CDR residues undergo post-translational modification (PTM) during humanization. For example, the CDR3 in the heavy chain variable region of 3C5 can be mutated from DSLYYGSSLYYAMDY (SEQ ID NO: 15) to D. A LYYGSSLYYAMDY (SEQ ID NO: 74). In some embodiments, the CDR2 of the light chain variable region of 3C5 can mutate from RANRLVDG (SEQ ID NO: 18) to RANRLVD. A (SEQ ID NO: 75). 3C5 CDR in the PTM of the humanization process

[0104] The humanization variable regions are summarized in Table 1C. Table 1C. Variable region sequences of humanized 3C5 and 8F5

[0105] In some embodiments, the antibody and its fragments are humanized and contain a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 58.

[0106] In some embodiments, the antibody and its fragments are humanized and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 59.

[0107] In some embodiments, the antibody and its fragments are humanized and contain a heavy chain variable region and a light chain variable region, the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 58 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 59.

[0108] In some embodiments, the antibody and its fragments are humanized and contain a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 60.

[0109] In some embodiments, the antibody and its fragments are humanized and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 61.

[0110] In some embodiments, the antibody and its fragments are humanized and contain a heavy chain variable region and a light chain variable region, the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 60 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 61.

[0111] Antibodies can be produced through an affinity maturation process, in which modified antibodies are generated that have increased affinity for antigens compared to unmodified parental antibodies. Affinity-matured antibodies can be produced by methods known in the art, for example, Marks et al., Rio / Technology 10:779-783 (1992); Barbas et al. ProcNat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0112] The CDR variants of 3C5 and 8F2 after affinity maturation and removal of potential deamidated amino acids are listed in Table 1D below (Kabat number). Table 1D. Affinity-mature CDR variants of 3C5 and 8F2 3C5 affinity maturity (Kabat CDR with underline) 8F2 affinity maturity (Kabat CDR with underline) 8F2 removes potentially deamidated amino acids (Kabat CDR underlined).

[0113] Examples of affinity-matured anti-ROR1 antibodies or fragments thereof include the following CDRs: (a) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12), HCDR3: DALYYGGSLYYAMDY (SEQ ID NO: 16), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDA (SEQ ID NO: 75), and LCDR3: LQYDEFPYT (SEQ ID NO: 20); (b) HCDR1: RYAMS (SEQ ID NO: 11), HCDR2: SISSGGNTYYPDTVKGR (SEQ ID NO: 13), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RENRLVDA (SEQ ID NO: 19), and LCDR3: LQYDEFPYT (SEQ ID NO: 20); or (c) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTRYYPDTVKGR (SEQ ID NO: 14), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RENRLVDA (SEQ ID NO: 19), and LCDR3: LQYDEFPYT (SEQ ID NO: 20).

[0114] Examples of other affinity-matured anti-ROR1 antibodies or fragments thereof include the following CDRs: (a) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGVIRYNEKFKG (SEQ ID NO:24), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO:29), LCDR2: LVSKLSSG (SEQ ID NO: 32), and LCDR3: LQATYFPYT (SEQ ID NO: 33); (b) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGGIRYNEKFKG (SEQ ID NO:23), HCDR3: RERGVYYGMSE (SEQ ID NO: 27), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO:29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: YQATYFPYT (SEQ ID NO: 34); (c) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO:25), HCDR3: RERGVTAGMDE (SEQ ID NO: 28), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO:29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATYFPYT (SEQ ID NO: 33); (d) HCDR1: NYVMH (SEQ ID NO: 22), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO:25), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO:29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATYFPYT (SEQ ID NO: 33); or (e) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO: 25), RERGVYYGMSE (SEQ ID NO: 27), LCDR1: KSSQSLLHSNDKTYLN (SEQ ID NO: 30), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: YQATYFPYT (SEQ ID NO: 34).

[0115] The variable regions of 3C5 and 8F2, which have undergone affinity maturation and removal of potential deamidating amino acids, are listed in Table 1E below. Table 1. Variable regions and CDRs of affinity maturation for E.3C5 and 8F2

[0116] In some embodiments, the antibody and its fragments are humanized and affinity-matured, and contain a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 62-63 and 65, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with an amino acid sequence selected from SEQ ID NO: 62-63 and 65.

[0117] In some embodiments, the antibody and its fragments are humanized and affinity-matured, and contain a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 59 and 64, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with an amino acid sequence selected from SEQ ID NO: 59 and 64.

[0118] In some embodiments, the antibody and its fragments are humanized and affinity-matured, and contain a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 66, 68, 70 and 72-73, or a peptide having at least 90%, at least 95%, or at least 98% sequence identity with an amino acid sequence selected from SEQ ID NO: 66, 68, 70 and 72-73.

[0119] In some embodiments, the antibody and its fragments are humanized and affinity-matured, and contain a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 61, 67, 69 and 71, or a peptide having at least 90%, at least 95% or at least 98% sequence identity with an amino acid sequence selected from SEQ ID NO: 61, 67, 69 and 71.

[0120] One or more disulfide bonds are introduced into the variable region. In some embodiments of this disclosure, a pair of cysteine ​​residues are introduced into the heavy chain variable region and the light chain variable region, respectively. A G44C amino acid substitution is introduced in SEQ ID NO: 70 (SEQ ID NO: 76), and a Q105C amino acid substitution is introduced in SEQ ID NO: 71 (SEQ ID NO: 77), respectively, to form a disulfide bond.

[0121] In various embodiments, this disclosure provides an antibody or antigen-binding fragment thereof that is specific to human ROR1 protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3.

[0122] In one embodiment, HCDR1 comprises the amino acid sequence of SEQ ID NO: 35, HCDR2 comprises the amino acid sequence of SEQ ID NO: 36, HCDR3 comprises the amino acid sequence of SEQ ID NO: 37, LCDR1 comprises the amino acid sequence of SEQ ID NO: 38, LCDR2 comprises the amino acid sequence of SEQ ID NO: 31, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 5, and VL comprises the amino acid sequence of SEQ ID NO: 6.

[0123] In one embodiment, HCDR1 comprises the amino acid sequence of SEQ ID NO: 41, HCDR2 comprises the amino acid sequence of SEQ ID NO: 42, HCDR3 comprises the amino acid sequence of SEQ ID NO: 43, LCDR1 comprises the amino acid sequence of SEQ ID NO: 44, LCDR2 comprises the amino acid sequence of SEQ ID NO: 45, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 8. Anti-4-1BB antibody

[0124] This disclosure provides a multispecific antibody containing one or more antigen-binding moieties that have binding specificity to human 4-1BB protein.

[0125] According to one embodiment of this disclosure, an antigen-binding portion is provided, comprising a single-domain antibody (sdAb) having a CDR region. CDRs are summarized in Table 2A below (Kabat numbers). Exemplary single-chain variable domains are shown in Table 2B. Table 2A. CDR sequences of the anti-4-1BB antigen binding site Table 2B. Exemplary single-chain variable domains of anti-4-1BB antigen binding sites

[0126] In some embodiments, at least one, two, three, four, five, or six of the above-mentioned VH CDR1, CDR2, and CDR3 are modified by adding, deleting, or substituting one, two, or three amino acids or a combination thereof.

[0127] In one embodiment, HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 9, or a peptide having at least 90% (or at least 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 9.

[0128] The CDR, heavy chain variable region, light chain variable region, or single heavy chain variable domain disclosed herein can be further modified. In some embodiments, the modified heavy chain variable region, light chain variable region, or single heavy chain variable domain retains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity and is still able to bind to the target site.

[0129] In some embodiments, the modification is a substitution at no more than one hot spot location in each CDR. In some embodiments, the modification is a substitution at one, two, or three such hot spot locations. In one embodiment, the modification is a substitution at one of the hot spot locations. In some embodiments, such substitution is a conservative substitution.

[0130] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin peptides are preferably replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid sequence may be replaced with a structurally similar sequence that differs in the order and / or composition of the side chain family members.

[0131] The table below provides non-limiting examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates that two amino acids can be conservedly substituted. Amino acid similarity matrix Conservative amino acid substitution

[0132] Those skilled in the art will also understand that the antibodies disclosed herein can be modified to differ in amino acid sequence from the naturally occurring binding polypeptide from which they originate. For example, a polypeptide or amino acid sequence derived from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity with the starting sequence, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. Multispecific and dual complementary site antibodies

[0133] The antibodies described herein are multispecific antibodies, such as bispecific antibodies. As used herein, the term "multispecific antibody" refers to an antibody that contains an antigen-binding domain having multi-epitope specificity (i.e., the ability to bind to two or more different epitopes on one molecule or to bind to epitopes on two or more different molecules).

[0134] In some embodiments, a multispecific antibody is a monoclonal antibody (such as a bispecific antibody) that has binding specificity to at least two different antigen-binding sites. In some embodiments, the first and second antigen-binding domains of a multispecific antibody can bind to two epitopes within the same molecule (intramolecular binding). For example, the first and second antigen-binding domains of a multispecific antibody can bind to two different epitopes on the same ROR1 molecule. In some embodiments, the two different epitopes bound by the multispecific antibody are epitopes that are not typically bound simultaneously by a single monospecific antibody (e.g., a conventional antibody or an immunoglobulin monovariable domain). In some embodiments, the first and second antigen-binding domains of a multispecific antibody can bind to epitopes located in two different molecules (intermolecular binding). For example, the first antigen-binding domain of a multispecific antibody can bind to one epitope on a ROR1 molecule, while the second antigen-binding domain of the multispecific antibody can bind to another epitope on a different ROR1 molecule, thereby cross-linking the two molecules.

[0135] In some embodiments, the multispecific antibodies provided herein may be bispecific antibodies. As used herein, the term "bispecific antibody" refers to a multispecific antibody containing an antigen-binding domain capable of binding to two different epitopes on one molecule or capable of binding to epitopes on two different molecules. Bispecific antibodies may also be referred to herein as having "bispecificity" or "bispecific". Exemplary bispecific antibodies may bind to ROR1 and any other antigen. In some embodiments, one binding specificity is against ROR1, and the other against 4-1BB. In some embodiments, a bispecific antibody may bind to two different epitopes on the same ROR1 molecule. In some embodiments, a bispecific antibody may bind to two different epitopes on two different ROR1 molecules. Bispecific antibodies may also be used to target cytotoxic agents to cells expressing ROR1.

[0136] Techniques for preparing multispecific antibodies include, but are not limited to: recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al, EMBO J. 10: 3655 (1991)), and mortar engineering (see, for example, U.S. Patent No. 5,731,168, WO2009 / 089004, US2009 / 0182127, US2011 / 0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, and Kontermann (2005) Acta Pharmacol. Sin., 26: 1-9).

[0137] This disclosure provides anti-ROR1 antigen-binding moieties and anti-4-1BB antigen-binding moieties as bispecific or multispecific antibodies. By simultaneously targeting the ROR1 antigen to increase 4-1BB clustering, activation of the 4-1BB pathway can be enhanced. Designs targeting the dual complementary antigen sites of ROR1 can even induce stronger 4-1BB activation.

[0138] In some embodiments, the anti-ROR1 antigen-binding moiety provided herein is in the form of a full-length antibody / IgG, and the anti-4-1BB antigen-binding moiety provided herein is in the form of a single-domain antibody (sdAb) / VHH / nanobody.

[0139] In some embodiments, VHH is fused to the N-terminus or C-terminus of IgG. In some embodiments, VHH is fused to the N-terminus of the variable region of the IgG heavy chain. In some embodiments, VHH is fused to the C-terminus of the constant region of the IgG heavy chain (IgG(CH)).

[0140] In some embodiments, the multispecific antibody provided herein includes a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, VH(ROR-1)-IgG(CH)-VHH(4-1BB), and the second polypeptide comprises, from the N-terminus to the C-terminus, VL(ROR-1)-IgG light chain constant region (IgG(CL)). The first polypeptide and the second polypeptide are paired via VH(ROR-1)-VL(ROR-1) pairing.

[0141] In some embodiments, the multispecific antibody provided herein comprises two first polypeptides and two second polypeptides. The first polypeptides, from N-terminus to C-terminus, comprise: VH(ROR-1)-IgG(CH)-VHH(4-1BB), and the second polypeptides, from N-terminus to C-terminus, comprise: VL(ROR-1)-IgG light chain constant region (IgG(CL)) (see Figure 1A). The first and second polypeptides are paired via VH(ROR-1)-VL(ROR-1) pairing. The two first polypeptides can be paired via IgG(CH) pairing.

[0142] This disclosure also provides two or more anti-ROR1 antigen-binding moieties as dual complementary sites. Dual complementary site antibodies are a subset of bispecific antibodies that recognize two different epitopes on the same antigen.

[0143] In some embodiments, the anti-ROR1 antigen-binding moiety provided herein includes a first anti-ROR1 antigen-binding moiety and a second anti-ROR1 antigen-binding moiety. The first anti-ROR1 antigen-binding moiety has an epitope on the ROR1 antigen that is different from that of the second anti-ROR1 antigen-binding moiety.

[0144] In some embodiments, the first and second anti-ROR1 antigen-binding moieties provided herein are in the form of full-length antibody / IgG, respectively, and the anti-4-1BB antigen-binding moieties provided herein are in the form of single-domain antibody (sdAb) / VHH / nanobody. In some embodiments, the first anti-ROR1 antigen-binding moieties comprise a pair of VH and VL, and the second anti-ROR1 antigen-binding moieties comprise a pair of VH and VL. The paired VH and VL are fused to the IgG constant region (IgG(CH+CL)).

[0145] In some embodiments, the anti-ROR1 antigen-binding moieties provided herein are in both IgG and scFv forms, and the anti-4-1BB antigen-binding moieties provided herein are in the form of single-domain antibody (sdAb) / VHH / nanobody. In some embodiments, the first anti-ROR1 antigen-binding moieties comprise a pair of VH and VL, and the second anti-ROR1 antigen-binding moieties comprise scFv. The VH and VL pair of the first anti-ROR1 antigen-binding moieties are fused to the IgG constant region, wherein the VH is fused to the IgG heavy chain constant region (IgG(CH)) and the VL is fused to the IgG light chain constant region (IgG(CL)). The scFv of the second anti-ROR1 antigen-binding moieties is fused to the VH of the first anti-ROR1 antigen-binding moieties, or fused to another IgG heavy chain constant region (IgG'(CH)).

[0146] In some implementations, IgG(CH) is different from IgG'(CH). In other implementations, IgG(CH) is the same as IgG'(CH).

[0147] In some embodiments, the scFv of the second anti-ROR1 antigen-binding moiety comprises, from the N-terminus to the C-terminus: VL-linker chain-VH. The scFv is fused to the VH of the first anti-ROR1 antigen-binding moiety via its VH (scFv-IgG(CH)) or IgG'(CH)(scFv-IgG'(CH)). The scFv-IgG(CH) form comprises, from the N-terminus to the C-terminus: (VL-linker chain-VH)-(VH-CH1-hinge-CH2-CH3). The IgG Fc domain provided herein may comprise CH2-CH3 or hinge-CH2-CH3. In some embodiments of this disclosure, the IgG Fc domain extends from the Cys226 position to its C-terminus (EU numbering system).

[0148] The scFv-IgG'(CH) form contains, from the N-terminus to the C-terminus: (VL-linker-VH)-hinge-CH2-CH3, (VL-linker-VH)-partial hinge-CH2-CH3, or (VL-linker-VH)-hinge variant-CH2-CH3. IgG'(CH) may contain hinge-CH2-CH3, partial hinge-CH2-CH3, or hinge variant-CH2-CH3.

[0149] The hinge variant may contain a two-amino acid residue linker AA before the hinge region starting with “EPKSC” (SEQ ID NO:138), in which the C residue is mutated to S because there is no light chain to form a disulfide bond with it.

[0150] In some embodiments, the scFv of the second anti-ROR1 antigen-binding portion comprises, from N-terminus to C-terminus: VH-linker chain-VL. The scFv is fused to the VH of the first anti-ROR1 antigen-binding portion via its VL (scFv-IgG(CH)) or IgG'(CH)(scFv-IgG'(CH)). The scFv-IgG(CH) form comprises, from N-terminus to C-terminus: (VH-linker chain-VL)-(VH-CH1-hinge-CH2-CH3). The scFv-IgG'(CH) form comprises, from N-terminus to C-terminus: (VH-linker chain-VL)-hinge-CH2-CH3, (VH-linker chain-VL)-partial hinge-CH2-CH3, or (VH-linker chain-VL)-variant hinge-CH2-CH3. IgG'(CH) may comprise hinge-CH2-CH3, partial hinge-CH2-CH3, or hinge variant-CH2-CH3.

[0151] In some implementations, the single-domain antibody (sdAb) / VHH of the anti-4-1BB antigen-binding portion is fused to the C-terminus of IgG (CH) or IgG' (CH).

[0152] In some embodiments, the multispecific antibody provided herein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus: scFv(2 nd ROR-1)-VH(1 st ROR-1)-IgG(CH)-VHH(4-1BB), the second polypeptide contains from the N-terminus to the C-terminus: VL(1 st ROR-1)-IgG(CL) (see Figure 1C). The first polypeptide is transmitted via VH(1) st ROR-1)-VL(1 st ROR-1 pairing with the second polypeptide. The two first polypeptides can pair via IgG(CH) pairing, or more specifically via Fc region pairing.

[0153] In some embodiments, the multispecific antibody provided herein comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus: VH(1 st ROR-1)-IgG(CH)-VHH(4-1BB), the second polypeptide contains from the N-terminus to the C-terminus: VL(1 st ROR-1)-IgG(CL), the third polypeptide comprising from the N-terminus to the C-terminus: scFv(2 nd ROR-1)-IgG'(CH)-VHH(4-1BB) (see Figure 1B). The first polypeptide passes through VH(1) st ROR-1)-VL(1 stROR-1) pairs with the second polypeptide, and the first polypeptide pairs with the third polypeptide via IgG(CH)-IgG'(CH) pairing. Heterodimer

[0154] Normally, two identical Fc regions form homodimers. However, by mutating one or both single strands through means such as kilohms (KIH), disulfide bonds (-SS-), or through hydrophobic, electrostatic, hydrophilic interactions, or by increasing flexibility, two different Fc regions can form heterodimers. IgG(CH)-IgG'(CH) pairing, or alternatively, Fc region pairing, can be achieved through kilohms (KIH), hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or by increasing flexibility to form heterodimers.

[0155] In some implementations, the Fc domains provided herein contain a knob mutation, and the paired Fc domains contain one or more mortar mutations, or vice versa.

[0156] As used herein, the term "mortar and pestle" or "KIH" technology refers to a technique that guides the pairing of two peptides in vitro or in vivo by introducing a protrusion (mortar) into one peptide and a groove (socket) into the other peptide at the interface where two peptides interact. For example, KIHs have been introduced into the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of antibodies (see, for example, US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, Zhu et al, 1997, Protein Science 6:781-788, and WO2012 / 106587). In some embodiments, KIHs drive the pairing of two different heavy chains during the preparation of multispecific antibodies. For example, multispecific antibodies having KIHs in their Fc regions may also include a single variable domain linked to each Fc region, or different heavy chain variable domains paired with similar or different light chain variable domains. KIH technology can also be used to pair two different receptor extracellular domains or any other polypeptide sequence containing different target recognition sequences (e.g., including affibody, peptibody and other Fc fusions).

[0157] As used herein, the term "pallet mutation" refers to a mutation that introduces a protrusion (pallet) into the polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a mortar mutation.

[0158] As used herein, the term "groove mutation" refers to a mutation that introduces a groove (groove) into the polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a groove mutation.

[0159] In some embodiments, the acetabular mutation in the IgG1 constant region includes T366W (EU number). In some embodiments, the acetabular mutation in the IgG1 constant region includes one or more mutations selected from T366S, L368A, and Y407V (EU number). In some embodiments, the acetabular mutation in the IgG1 constant region includes T366S, L368A, and Y407V (EU number). In some embodiments, the acetabular mutation in the IgG1 constant region is S354C and T366W (EU number). The acetabular mutation in the IgG1 constant region includes one or more mutations selected from Y349C, T366S, L368A, and Y407V.

[0160] Multispecific antibodies can also be prepared by: engineering antibody Fc heterodimer molecules using electrostatic guiding effects (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al, Science, 229: 81 (1985)); generating bispecific antibodies using leucine zippers (see, for example, Kostelny et al, J. Immunol, 148(5): 1547-1553 (1992)); preparing bispecific antibody fragments using “dual antibody” technology (see, for example, Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, for example, Gruber et al, J. Immunol, 152:5368). (1994)); and the preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147: 60 (1991).

[0161] In some embodiments, IgG(CH) comprises CH1, a hinge region, CH2, and CH3. In some embodiments, IgG(CH) comprises the amino acid sequence of SEQ ID NO: 50. Two IgG(CH) molecules can form a homodimer.

[0162] In some embodiments, IgG(CH) comprises the amino acid sequence of SEQ ID NO: 51 having an S237C / T249W (corresponding to the EU number S354C / T366W) substitution, or the amino acid sequence of SEQ ID NO: 55 having an S237C / T249W and M311L / N317S (LS mutation, corresponding to the EU number M428L / N434S, which prolongs the half-life of the antibody in vivo) substitution.

[0163] In some embodiments, IgG(CH) comprises CH1, a hinge region, and an Fc domain. The Fc domain of this disclosure comprises the amino acid sequence of SEQ ID NO: 141 having an S237C / T249W substitution (corresponding to the EU numbered mutation S354C / T366W), or the amino acid sequence of SEQ ID NO: 142 having S237C / T249W and M311L / N317S (LS mutation, corresponding to the EU numbered M428L / N434S, which prolongs the half-life of the antibody in vivo) substitutions.

[0164] In some embodiments, IgG'(CH) includes CH1, a hinge region, CH2, and CH3. In some embodiments, IgG'(CH) is missing a portion of CH1, missing CH1, or missing CH1 plus a portion of the hinge region. In some embodiments, IgG'(CH) includes a portion of CH1, a hinge region, CH2, and CH3. In some embodiments, IgG'(CH) includes a hinge region, CH2, and CH3.

[0165] In some implementations, IgG'(CH) includes hinge region variants, CH2, and CH3. Hinge region variants can be defined using "EPKS". C The hinge region starting with (SEQ ID NO: 138) contains a short linker AA with two amino acid residues, where the C residue is mutated to S because there is no light chain to form a disulfide bond with it.

[0166] In some embodiments, IgG'(CH) comprises the amino acid sequence of SEQ ID NO: 53 having a substitution of Y136C / T153S / L155A / Y194V (corresponding to the EU numbered mutation Y349C / T366S / L368A / Y407V). In some embodiments, IgG'(CH) comprises the amino acid sequence of SEQ ID NO: 54 having substitutions of Y136C / T153S / L155A / Y194V and M215L / N221S (an LS mutation corresponding to the EU numbered M428L / N434S, which prolongs the antibody's half-life in vivo).

[0167] In some embodiments, IgG'(CH) includes a hinge region variant and a paired Fc domain. In some embodiments, the paired Fc domain of this disclosure comprises the amino acid sequence of SEQ ID NO: 143 with a Y136C / T153S / L155A / Y194V substitution (corresponding to the EU-numbered acetamipelago mutation Y349C / T366S / L368A / Y407V). In some embodiments, IgG'(CH) comprises the amino acid sequence of SEQ ID NO: 52 with a Y136C / T153S / L155A / Y194V and an M215L / N221S substitution (an LS mutation, corresponding to the EU-numbered M428L / N434S, which prolongs the antibody's half-life in vivo). Table 2C. IgG1 Fc region sequence

[0168] IgG(CH) and IgG'(CH) can form heterodimers. The Fc domain and paired Fc domains can also form heterodimers.

[0169] In some embodiments, IgG(CH) and IgG'(CH) form a mortise and tenon joint (KIH): (1) IgG(CH) of SEQ ID NO: 51 (or a variant having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it) may be paired with IgG'(CH) of SEQ ID NO: 53 (or a variant having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it); or (2) IgG(CH) of SEQ ID NO: 55 (or a variant having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it) may be paired with IgG'(CH) of SEQ ID NO: 53. Pair 54 of IgG'(CH) (or with a variant that has at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity).

[0170] In some embodiments, the Fc domain and the paired Fc domain form a mortar and pestle (KIH): (1) the Fc domain of SEQ ID NO: 141 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) may be paired with the paired Fc domain of SEQ ID NO: 143 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity); or (2) the Fc domain of SEQ ID NO: 142 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) may be paired with the paired Fc domain of SEQ ID NO: 52 is paired with the Fc domain (or with a variant that has at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity).

[0171] In some embodiments, the first or second antibody portion includes an Fc region (also referred to herein as an "Fc fragment"). In some embodiments, the Fc region is an Fc domain, i.e., an Fc region with some or all of the effector functions, including, for example, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In some embodiments, the Fc domain is derived from IgG1 or IgG3.

[0172] In some embodiments, one or more amino acid modifications may be introduced into the Fc domain to create Fc domain variants. Fc domain variants may contain human Fc domain sequences (e.g., derived from the human IgG1, IgG2, IgG3, or IgG4 Fc regions) with amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, Fc domain variants alter one or more functional and / or pharmacokinetic properties of the antibody. Effect function

[0173] The Fc region can also be engineered to enhance or eliminate effector function. IgG antibodies can induce direct antitumor effects by generating indirect antitumor effects through Fc-mediated effector functions (recruiting other immune cells or killing mechanisms). As used herein, “effector function” or “antibody effector function” refers to the biological activity attributable to the binding of the antibody Fc region to its effector, such as the C1 complex and the Fc receptor (FcγRIIa or FcγRIIIa)). Exemplary effector functions include: complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the antibody Fc region to the Fc receptor on effector cells; and antibody-dependent cell-mediated phagocytosis (ADCP), in which nonspecific cytotoxic cells expressing the Fcγ receptor (FcγR) recognize the antibody bound to the target cell, subsequently leading to the phagocytosis of the target cell.

[0174] In some implementations, the Fc region provided herein preserves or enhances effector functionality, such as ADCC and / or CDC.

[0175] Of the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc effector functions. However, because IgG1 has the longest half-life and is more stable than IgG3, most therapeutic antibodies with Fc-mediated functions are IgG1 isotypes.

[0176] Both IgG2 and IgG4 isoforms exhibit significantly lower binding affinity for FcγR. Recent evidence suggests that the IgG2 isoform is not entirely devoid of effector function, while the IgG4 isoform can undergo Fab arm exchange in vivo, leading to bispecific antibodies and off-target effects.

[0177] In some embodiments, the Fc domain is derived from human IgG1. In some embodiments, the Fc domain derived from human IgG1 does not contain the L234A mutation and / or the L235A mutation. In some embodiments, the Fc domain derived from human IgG1 contains the L234A mutation and / or the L235A mutation. In some embodiments, the Fc domain is derived from human IgG3. In some embodiments, the Fc domain is derived from human IgG2 or IgG4. In some embodiments, the Fc domain is derived from human IgG4. In some embodiments, the Fc domain derived from human IgG4 contains the S228P, F234A, and / or L235A mutation. In some embodiments, the Fc domain derived from human IgG4 does not contain the S228P, F234A, and / or L235A mutation.

[0178] In some implementations, the Fc domain is modified to alter (i.e., enhance or weaken) C1q binding and / or complement-dependent cytotoxicity (CDC), as in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie. et. al. J. Immunol. As stated in 164: 4178-4184 (2000).

[0179] In some implementations, the multispecific construct comprises a variant Fc domain containing one or more amino acid substitutions that alter the half-life and / or change the binding to the neonatal Fc receptor (FcRn). This results in an extended half-life and improved binding to the neonatal Fc receptor (FcRn) (responsible for transferring maternal IgG to the fetus). et. al. , J. Immunol. 117:587 (1976) and Kim et. al. , J. Immunol. Antibodies binding to 24:249 (1994) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions that alter the binding of the Fc region to FcRn. Such Fc variants include variants in which substitution occurs at one or more residues in the Fc region, such as substitution at residue 434 of the Fc region (US Patent No. 7,371,826).

[0180] In vitro and / or in vivo cytotoxicity assays can be performed to analyze CDC and / or ADCC activity in the Fc region. For example, Fc receptor (FcR) binding assays can be performed to determine whether the antibody has FcγR binding (and therefore may have ADCC activity) and / or retains FcRn binding ability. The main cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet. Annu. Rev. Immunol. Table 2 on page 464 of 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I). et. al. Proc. Nat’l Acad. Sci. USA et. al. 83:7059-7063 (1986)) and Hellstrom, I Proc. Nat’l Acad. , Sci. USA et. al. 82:1499-1502 (1985); 5,821,337 (See Bruggemann, M.) J. Exp. ,Med. et. al. Proc. Nat’l Acad. Sci. USA 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTI of flow cytometry). TM Non-radioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox96 ® Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Optionally or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as Clynes. et. al. As disclosed in 95:652-656 (1998). A C1q binding assay can also be performed to confirm whether the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro). J. Immunol. , Methods et. al. 202:163 (1996); Cragg, MS Blood , Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, et. al. 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB). Int’l. , Immunol. Figure 10A 18(12):1759-1769 (2006)).

[0181] Different antibody domains can be fused via one or more linkers. For example, the fusion of scFv with VH or the fusion of VHH with IgG (CH) are both achieved through linkers. Therefore, the specific forms of multispecific antibodies presented in this article include... (1) The first polypeptide, which comprises from the N-terminus to the C-terminus: VH(1 st ROR-1)-IgG(CH)-linker-VHH(4-1BB), and a second polypeptide comprising, from N-terminus to C-terminus: VL(1 st ROR-1)-IgG(CL); (2) The first polypeptide, which comprises from the N-terminus to the C-terminus: scFv(2 nd ROR-1)-Connector-VH(1 st ROR-1)-IgG(CH)-linker-VHH(4-1BB), and a second polypeptide comprising, from N-terminus to C-terminus: VL(1 st ROR-1)-IgG(CL); (3) The first polypeptide, which comprises from the N-terminus to the C-terminus: VH(1 st ROR-1)-IgG(CH)-linker-VHH(4-1BB), the second polypeptide, which contains VL(1) from the N-terminus to the C-terminus. st ROR-1)-IgG(CL), and a third polypeptide, which includes scFv(2) from the N-terminus to the C-terminus. nd ROR-1)-IgG'(CH)-linker-VHH(4-1BB); and (4) The first polypeptide, which comprises from the N-terminus to the C-terminus: VH(1 st ROR-1)-IgG(CH)-linker-VHH(4-1BB), the second polypeptide, which contains VL(1) from the N-terminus to the C-terminus. st ROR-1)-IgG(CL), and a third polypeptide, which includes scFv(2) from the N-terminus to the C-terminus. nd ROR-1)-linker-IgG'(CH)-linker-VHH(4-1BB). connector

[0182] The linker strand in the scFv can be the same as the adapter. Adapters within the scope of this disclosure can be characterized based on their amino acid composition, length, rigidity, and secondary structure. Adapters within the scope of this disclosure separate one functional polypeptide from another and allow each domain to fold correctly and function. In this way, adapters can be customized for specific functional polypeptides and other functional polypeptides. According to one aspect, the functional independence of the structural domains from the fused (heterologous) domains is maximized by using appropriate adapters to limit steric interference between domains during bacterial cell export and assembly. According to one aspect, longer and more flexible (GGGGS)n-type adapters are exemplary, where n is an integer from 1 to 20 (SEQ ID NO: 39). According to another aspect, cellular stress is minimized by limiting the total length of the fusion protein. Longer adapter sequences and higher induction levels can stress the cell's biosynthetic mechanisms, inhibit cell growth, and in extreme cases, lead to cell lysis.

[0183] The linkers within the scope of this disclosure facilitate the functioning of antibody domains. The linkers within the scope of this disclosure allow for efficient protein processing and export via bacterial curli secretion mechanisms and provide appropriate space and physicochemical separation for the corresponding antibody domains to preserve their respective functions.

[0184] Linkers within the scope of this disclosure include amino acid residues. These amino acid residues can be any naturally occurring amino acid residue. They can also be synthetic amino acids known to those skilled in the art. Representative amino acids that can be used as linkers include glycine, alanine, valine, leucine, isoleucine, serine, cysteine, selenocysteine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine, and glutamine.

[0185] According to one aspect, the linker length can be any length that a cell (such as a bacterial cell) can express when linking the antibody domain. According to one aspect, the linker sequence is a polypeptide sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48 or more amino acids. In some embodiments, the linker sequence comprises about 3 amino acids to about 50 amino acids. In some embodiments, the linker sequence comprises about 3 amino acids to about 40 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 30 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 25 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 24 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 23 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 22 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 21 amino acids. In some embodiments, the linker sequence comprises about 5 amino acids to about 20 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 19 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 18 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 17 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 16 amino acids. In some embodiments, the adapter sequence comprises about 5 amino acids to about 15 amino acids. In some embodiments, the adapter sequence of this disclosure has 15 amino acids.

[0186] In some embodiments, the adapter sequence comprises a flexible polypeptide, such as a polypeptide without rigid secondary and / or tertiary structures. In some embodiments, the adapter sequence comprises glycine and serine residues. In some embodiments, at least 50% of the amino acids contained in the adapter sequence are glycine or serine residues, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more. In some embodiments, the adapter sequence consists of glycine and serine residues. In some embodiments, the adapter sequence of this disclosure has the amino acid sequence of SEQ ID NO: 56.

[0187] In some embodiments, the antibody comprises an amino acid sequence or one or more portions that are not normally associated with the antibody naturally. Exemplary modifications will be described in more detail below. For example, the antibodies of this disclosure may comprise a flexible linker sequence or may be modified to add a functional portion (e.g., a PEG, drug, toxin, or marker).

[0188] The antibodies, variants, or derivatives disclosed herein include modified derivatives, i.e., those covalently linked to the antibody by any type of molecule, provided that such covalent linking does not impede the binding of the antibody to the epitope. For example, but not as a limitation, the antibody may be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. Any of these numerous chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, the antibody may contain one or more non-classical amino acids.

[0189] In some implementations, antibodies can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, or PEG.

[0190] Antibodies can be conjugated or fused to therapeutic agents, which may include detectable markers (such as radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutics or diagnostics, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radiolabels, combinations thereof, and other such substances known in the art. Polynucleotides encoding antibodies and methods for preparing antibodies

[0191] This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding antibodies, variants, or derivatives thereof. The polynucleotides of this disclosure may encode the entire heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on different polynucleotide molecules. Furthermore, the polynucleotides of this disclosure may encode portions of the heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant, or derivative thereof, either on the same polynucleotide molecule or on different polynucleotide molecules.

[0192] Humanized antibodies can be designed to minimize adverse immune responses to rodent anti-human antibodies, which limit the duration and effectiveness of therapeutic applications of those portions in human recipients. Humanized antibodies may have one or more amino acid residues introduced from a non-human source. These non-human residues are often referred to as “import” residues and are typically derived from the variable domain. Humanization can be achieved by replacing the corresponding sequence of a human antibody with a hypervariable region sequence. Thus, such “humanized” antibodies are chimeric antibodies in which a portion far less than the complete human variable domain has been replaced by a corresponding sequence from a non-human species. See, for example, U.S. Patent No. 4,816,567, the contents of which are incorporated herein by reference. Humanized antibodies may be human antibodies in which some of the hypervariable region residues, and possibly some FR residues, are replaced with residues from similar sites in rodent antibodies. The humanization or engineering of the antibodies of the present invention can be performed using any known method, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567. Treatment

[0193] As described herein, the antibodies, variants, or derivatives disclosed herein can be used for certain therapeutic applications.

[0194] This disclosure also relates to antibody-based therapies involving the administration of the antibodies of this disclosure to a patient (such as an animal, mammal, or human) to treat one or more of the diseases or conditions described herein. Therapeutic compounds of this disclosure include, but are not limited to, the antibodies of this disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the antibodies of this disclosure (including variants and derivatives thereof as described herein).

[0195] In some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method includes administering an effective amount of the antibody disclosed herein to the patient.

[0196] In some embodiments, the use of the antibodies disclosed herein in the preparation of medicaments for treating cancer in patients in need is provided.

[0197] In some implementations, antibodies of this disclosure are provided for the treatment of cancer in patients in need.

[0198] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0199] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed, and / or prognosed by the antibodies, variants, or derivatives disclosed herein, including but not limited to the progression and / or metastasis of malignancies and related conditions such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including medulloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, and chondrosarcoma). Osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymal carcinoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0200] The specific dosage and treatment regimen for any given patient depends on a variety of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of these factors by healthcare professionals is a standard skill in the field. Dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined using principles of pharmacology and pharmacokinetics well known in the art.

[0201] Methods of administration of antibodies or their variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through an epithelial or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be co-administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides disclosed herein can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., via powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0202] As used in this article, the term "parenteral" refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0203] Administration can be systemic or localized. Furthermore, it may be necessary to introduce the antibodies of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be assisted by an intraventricular catheter (e.g., connected to a reservoir, such as the Ommaya reservoir). Pulmonary administration may also be used, for example, by using an inhaler or nebulizer and formulating with an aerosol.

[0204] It may be necessary to apply the antigen-binding peptides or compositions of this disclosure topically to the site of treatment; this can be achieved (e.g., but not limited to) local infusion during surgery, topical application (e.g., postoperative use in conjunction with wound dressings), injection, via catheter, via suppository, or via implantation, said implantation being a porous, non-porous, or gel-like material, including membranes (such as silicone rubber membranes) or fibers. Preferably, when administering the proteins (including antibodies) of this disclosure, care must be taken to use materials that do not adsorb onto the protein. Composition

[0205] This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0206] In one specific implementation, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeias for animal (especially human) use. Furthermore, "pharmaceutical acceptable carriers" are generally non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of pharmaceutical excipient.

[0207] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils (including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a preferred carrier when the drug composition is administered intravenously. Saline solutions, as well as aqueous solutions of dextran and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. It is also envisioned that the composition include antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and osmolarity regulators such as sodium chloride or dextran. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions would contain a therapeutically effective amount of an antigen-binding polypeptide (preferably in a purified form) and a suitable amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the route of administration. Parenteral formulations can be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0208] In one embodiment, the composition is formulated according to conventional methods to be a pharmaceutical composition suitable for intravenous administration to a human. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic (such as lidocaine) to relieve pain at the injection site. Typically, the components are supplied separately or mixed together in unit dosage forms, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet), with the amount of active substance indicated. In the case of administration by infusion, the composition may be provided in an infusion bottle containing sterile pharmaceutical-grade water or saline. In the case of administration by injection, ampoules of sterile water or saline for injection may be provided to allow the components to be mixed prior to administration.

[0209] The compounds disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions (such as anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.) and those formed with cations (such as cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.). Example Example 1. Generation of bispecific and trispecific antibodies

[0210] The ROR1×4-1BB bispecific antibody was constructed as shown in Figure 1A and Table 3 below. Combining affinity

[0211] FACS binding activity showed that ROR1×4-1BB BsAb bound to A549 tumor cells endogenously expressing ROR1 and HEK293 cells overexpressing 4-1BB in a dose-dependent manner (Figure 2A-B). Epitope grouping

[0212] In the epitope grouping of anti-ROR1 mAb, 3C5 and 8F2 showed that the binding epitopes to the ROR1 antigen were non-competitive or non-overlapping, as detected by Octet (see Figures 4A-C).

[0213] Based on the non-overlapping binding epitopes of 3C5 and 8F2 with ROR1, a double complementary site ROR1×4-1BB antibody was designed (see Figures 1B-C and Table 3). The design is based on the following assumption: a double complementary site antibody targeting the antigen can promote more 4-1BB clusters and induce stronger 4-1BB pathway activation. Table 3. Designed bispecific and trispecific antibody sequences Reporter gene testing

[0214] In this assay, Jurkat cells co-expressing the 4-1BB and NFKB-luciferase reporter genes were used as reporter cell lines, and ROR1-positive or negative cell lines were used as target cells. Reporter and target cells were co-cultured for 6 hours with serially diluted ROR1×4-1BB BsAb or anti-4-1BB urelumab, and then analyzed using One-Globe assay. TM The reagent reads out the 4-1BB NFKB activation signal. In this experiment, 4B-3C5 and 4B-8F2 BsAb showed strong ROR1-dependent 4-1BB activation (Figures 3A-C).

[0215] Similar reporter gene assays were performed using a double-complementary ROR1×4-1BB antibody. The results showed that the double-complementary ROR1×4-1BB TsAb in the form of 4-BiR1 exhibited optimal 4-1BB activation (see Figures 5A-C). Example 2. Humanized Antibody

[0216] Humanized anti-ROR1 antibodies were used to design mono-topic (bispecific) and bi-complementary ROR1×4-1BB TsAbs (see Table 4). Table 4. Humanized bispecific and bicomplementary site antibodies Cell binding affinity and reporter gene assay

[0217] Cell binding of humanized anti-ROR1 mAb was measured using FACS, and ROR1-dependent 4-1BB activation of humanized ROR1×4-1BB BsAb was tested using a similar method as described above. Compared to the chimeric antibody, the humanized mAb showed better or comparable cell binding (Fig. 6A-B), and the humanized BsAb showed better or comparable ROR1-dependent 4-1BB activation (Fig. 6C-D), while the 4-1BB VHH control urerucumab did not activate 4-1BB in ROR1-positive tumor cell lines within the corresponding concentration range.

[0218] Comparisons between humanized antibodies showed that, compared to 4B-3C5 and 4B-8F5, the bicomplementary antibody 4-BiR1 exhibited higher cell binding to ROR1 in ROR1-positive tumor cell lines (Fig. 7A-C) and higher ROR1-dependent 4-1BB activation (Fig. 7D-F), while 4B-3C5 and 4B-8F5 showed similar cell binding affinity and ROR1-dependent 4-1BB activation. In ROR1-negative tumor cell lines, no ROR1-dependent 4-1BB activation was observed with any of the tested antibodies. SPR

[0219] Surface plasmon resonance assays showed that the binding affinity of the humanized antibody to ROR1 was as follows: K4B-h3C5 D The value is 7.93E-08M, K of 4B-h8F5 D The value is 2.37E-07M, K of 4B-hBiR1 D The value is 1.21E-08M (Figure 8A-C). In vivo efficacy

[0220] The in vivo potency of single- and double-complementary ROR1×4-1BB was evaluated using a homologous model (4-1BB humanized mice (Biocytogen)) inoculated with the ROR1-MC38 cell line. All tested humanized antibodies showed significant inhibition of tumor growth (Figure 9A-B). Example 3. Antibody Affinity Maturation

[0221] Considering the low yield of 4-hBiR1, the potential PTM in the anti-ROR1 sequence, and the low affinity of h3C5 and h8F2, the antibody was engineered for affinity maturation (Tables 5A and 5B). Table 5A. Designed bispecific antibody sequences Table 5B. Designed Bicomplementary Antibody Sequences Reporter gene testing

[0222] Similar reporter gene assays were performed using affinity-matured bispecific or dual-complementary ROR1×4-1BB antibodies. Results showed that, compared to the humanized version, the affinity-matured bispecific antibody exhibited stronger ROR1-dependent 4-1BB activation. Figures 14A-14B -E). Compared with the humanized bispecific version and the baseline ROR1×4-1BB BsAb (clone ID: BA6(NA)×1A10M12) from patent WO2021 / 101346 A1, the affinity-matured dual complementary site antibody was observed to exhibit superior ROR1-dependent 4-1BB activation (Figure 11A-C). Cytokine release detection

[0223] 4B-2773, 4B-27, and 4B-73 were selected as representative engineered single- and double-complementary ROR1×4-1BB antibodies and compared with 4B-hBiR1 and the baseline (clone ID: BA6(NA)×1A10 M12) in cytokine release assays. 1 μg / ml anti-CD3 (OKT3) was coated onto culture plates, and MDA-MB-231 cell lines were used as target cells. PBMCs and serially diluted antibodies were co-cultured for 72 hours. IFN-γ and IL-2 induced by 4-1BB were detected. The results showed that all engineered ROR1×4-1BB antibodies tested released higher levels of cytokines than the baseline and ursulcimbrolizumab (Figure 12A-B). In vivo efficacy

[0224] The in vivo potency of engineered single- and double-complementary ROR1×4-1BB was evaluated using a homologous model (4-1BB humanized mice (Biocytogen)) inoculated with the ROR1-MC38 cell line. All tested humanized antibodies showed significant inhibition of tumor growth (Figures 13A-C). Example 4. Tumor growth inhibition of CLDN6-1 x 4-1BB BsAb

[0225] This embodiment reports an early experiment demonstrating that a bispecific antibody with the agonist anti-4-1BB single-domain antibody disclosed herein can achieve tumor suppression by utilizing the effector function of the Fc domain.

[0226] In this embodiment, a bispecific antibody targeting tight junction protein 6 (CLDN6) and 4-1BB was prepared. The single-domain 4-1BB antibody was fused to the C-terminus of the Fc domain (IgG1 Fc wild-type (WT) or IgG1 Fc with N297A (NA) (EU number), while the anti-CLDN6 Fab was placed at the N-terminus.

[0227] CT26 cells endogenously expressing CLDN6 were subcutaneously implanted into BALB / c humanized 4-1BB mice. When the tumor grew to an average size of 100 mm... 3 Mice were treated intraperitoneally with (a) human IgG1, (b) CLDN6-1 x 4-1BB NA (2 mg / kg), (c) CLDN6-1 x 4-1BB WT (2 mg / kg), or (d) a combination of parental CLDN6-1 antibody and 4-1BB sdAb-Fc (1.8 mg / kg and 0.7 mg / kg, respectively). Administered twice weekly for a total of 6 doses. Tumor growth was monitored by volumetric measurements. ​ As shown, both CLDN6-1 x 4-1BB WT and CLDN6-1 x 4-1 BB NA (IgG1 N297A) exhibited antitumor activity, with CLDN6-1 x 4-1 BB WT achieving even stronger activity, with a tumor growth inhibition (TGI) of 75%. Table 6. Exemplary CLDN6 x 4-1BB bispecific antibody

[0228] The scope of this disclosure is not limited to the specific embodiments described, which are merely individual examples of various aspects of this disclosure. Any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

[0229] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated as incorporated by reference.

Claims

1. A multispecific antibody comprising: (1) The first antibody portion specifically binds to the human receptor tyrosine kinase-like orphan receptor 1 (ROR1) protein, and (2) The second antibody portion specifically binds to human 4-1BB protein.

2. The multispecific antibody according to claim 1, wherein the second antibody portion comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence of SNCMG (SEQ ID NO: 47), HCDR2 comprises the amino acid sequence of VICTGGGSPSYADSVKG (SEQ ID NO: 48), and HCDR3 comprises the amino acid sequence of DLLRAGTPLSSYEFNY (SEQ ID NO: 49).

3. The multispecific antibody according to claim 2, wherein the second antibody portion comprises the amino acid sequence of SEQ ID NO: 9, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

9.

4. The multispecific antibody according to any one of claims 1-3, further comprising an Fc domain having retained or enhanced effector function.

5. The multispecific antibody according to any one of claims 1-4, wherein the second antibody portion is sdAb.

6. The multispecific antibody according to any one of the preceding claims, wherein the Fc domain is derived from any one of IgG1, IgG2, IgG3 and IgG4.

7. The multispecific antibody according to claim 6, wherein the Fc domain is derived from IgG1.

8. The multispecific antibody according to claim 7, wherein the Fc domain comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 52 and 140-143.

9. The multispecific antibody according to any one of the preceding claims, wherein... The first antibody portion is Fab', which is fused to the N-terminus of the IgG Fc domain, and The second antibody portion is sdAb, which is fused to the C-terminus of the IgG Fc domain.

10. The multispecific antibody of claim 9, wherein the second antibody portion is fused to the IgG Fc domain via a linker.

11. The multispecific antibody according to any one of the preceding claims, further comprising a third antibody portion, said third antibody portion specifically binding to a second tumor-associated antigen.

12. The multispecific antibody according to claim 11, wherein... The first antibody portion is Fab', which is fused to the N-terminus of the IgG Fc domain. The second antibody portion is sdAb, which is fused to the C-terminus of the IgG Fc domain, and The third antibody portion is scFv, which is fused to the N-terminus of the first antibody portion.

13. The multispecific construct of claim 12, wherein the second antibody portion is fused to the IgG Fc domain via a linker, and the third antibody portion is fused to the first antibody portion via a linker.

14. The multispecific antibody according to claim 11, wherein... The first antibody portion is Fab', which is fused to the N-terminus of the IgG Fc domain. The second antibody portion is sdAb, which is fused to the C-terminus of the IgG Fc domain. The third antibody portion is scFv, which is fused to the N-terminus of the IgG heavy chain constant region (IgG'(CH)). The second antibody portion is sdAb, which is fused to the C-terminus of the IgG'(CH), and The IgG'(CH) contains a paired IgG Fc domain and forms a heterodimer with the IgG Fc domain.

15. The multispecific antibody of claim 14, wherein the second antibody portion is fused to the IgG1 Fc domain via a linker, and the second antibody portion is fused to the IgG'(CH) via a linker.

16. The multispecific antibody according to any one of claims 11-15, wherein the first antibody portion and the third antibody portion specifically bind to human receptor tyrosine kinase-like orphan receptor 1 (ROR1) protein, but bind to different epitopes.

17. The multispecific antibody according to any one of the preceding claims, wherein the first antibody portion or the third antibody portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the following sequences: (a) HCDR1: SYAMS (SEQ ID NO: 10) or RYAMS (SEQ ID NO: 11), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12) or SISSGGNTYYPDTVKGR (SEQ ID NO: 13) or SISSGGTRYYPDTVKGR (SEQ ID NO: 14), HCDR3: DSLYYGSSLYYAMDY (SEQ ID NO: 15), DALYYGGSLYYAMDY (SEQ ID NO: 16) or DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1:RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDG (SEQ ID NO: 18), RENRLVDA (SEQ ID NO: 19) or RANRLVDA (SEQ ID NO: 75), and LCDR3: LQYDEFPYT (SEQ ID NO: 20); (b) HCDR1: TYVMH (SEQ ID NO: 21) or NYVMH (SEQ ID NO: 22), HCDR2: YINPYNGGIRYNEKFKG (SEQ ID NO: 23), YINPYNGVIRYNEKFKG (SEQ ID NO: 24) or YINPYSGGIRYNEKFKG (SEQ ID NO: 24), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), RERGVYYGMSE (SEQ ID NO: 27) or RERGVTAGMDE (SEQ ID NO: 28), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29) or KSSQSLLHSNDKTYLN (SEQ ID NO: 30), LCDR2: LVSKLESG (SEQ ID NO: 31) or LVSKLSSG (SEQ ID NO: 32), and LCDR3: LQATYFPYT (SEQ ID NO: 33) or YQATYFPYT (SEQ ID NO: 34); (c) HCDR1: DYWMH (SEQ ID NO: 35), HCDR2: AIDTSDSSTRNNQKFKG (SEQ ID NO: 36), HCDR3: GARTGTGFGY (SEQ ID NO: 37), LCDR1: KSSQSLLHINGKTYLN (SEQ ID NO: 38), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATHFPYT (SEQ ID NO: 40); or (d) HCDR1: SYGVH (SEQ ID NO: 41), HCDR2: VIWAGGHTNYNSDLMSR (SEQ ID NO: 42), HCDR3: RSIYGDYALDY (SEQ ID NO: 43), LCDR1: VTYMH (SEQ ID NO: 44), LCDR2: DISKLASG (SEQ ID NO: 45), and LCDR3: QQWNYPLMT (SEQ ID NO: 46).

18. The multispecific antibody according to claim 17, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody portion or the third antibody portion respectively comprise the following sequences: (a) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12), HCDR3: DSLYYGSSLYYAMDY (SEQ ID NO: 15), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDG (SEQ ID NO: 18), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), (b) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDA (SEQ ID NO: 75), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), (c) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTTYYPDTVKGR (SEQ ID NO: 12), HCDR3: DALYYGGSLYYAMDY (SEQ ID NO: 16), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RANRLVDA (SEQ ID NO: 75), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), (d) HCDR1: RYAMS (SEQ ID NO: 11), HCDR2: SISSGGNTYYPDTVKGR (SEQ ID NO: 13), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RENRLVDA (SEQ ID NO: 19), and LCDR3: LQYDEFPYT (SEQ ID NO: 20), or (e) HCDR1: SYAMS (SEQ ID NO: 10), HCDR2: SISSGGTRYYPDTVKGR (SEQ ID NO: 14), HCDR3: DALYYGSSLYYAMDY (SEQ ID NO: 74), LCDR1: RASQDIYSYLS (SEQ ID NO: 17), LCDR2: RENRLVDA (SEQ ID NO: 19), and LCDR3:LQYDEFPYT (SEQ ID NO: 20).

19. The multispecific antibody of claim 18, wherein the first antibody portion or the third antibody portion comprises (a) VH, comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and VL, which contains the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b) VH, comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 58, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59; (c) VH, comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 62, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59; (d) VH, comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 63, and VL, comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 64; or (e) VH, comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 65, and VL, which contains the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

64.

20. The multispecific antibody according to claim 17, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first or third antibody portion respectively comprise the following sequences: (a) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGGIRYNEKFKG (SEQ ID NO: 23), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATYFPYT (SEQ ID NO: 33); (b) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGVIRYNEKFKG (SEQ ID NO: 24), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLSSG (SEQ ID NO: 32), and LCDR3: LQATYFPYT (SEQ ID NO: 33); (c) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYNGGIRYNEKFKG (SEQ ID NO: 23), HCDR3: RERGVYYGMSE (SEQ ID NO: 27), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: YQATYFPYT (SEQ ID NO: 34); (d) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO: 25), RERGVYYGMSE (SEQ ID NO: 27), LCDR1: KSSQSLLHSNDKTYLN (SEQ ID NO: 30), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: YQATYFPYT (SEQ ID NO: 34); (e) HCDR1: TYVMH (SEQ ID NO: 21), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO: 25), HCDR3: RERGVTAGMDE (SEQ ID NO: 28), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3: LQATYFPYT (SEQ ID NO: 33); or (f) HCDR1: NYVMH (SEQ ID NO: 22), HCDR2: YINPYSGGIRYNEKFKG (SEQ ID NO: 25), HCDR3: RERGVYYGMDE (SEQ ID NO: 26), LCDR1: KSSQSLLHSNGKTYLN (SEQ ID NO: 29), LCDR2: LVSKLESG (SEQ ID NO: 31), and LCDR3:LQATYFPYT (SEQ ID NO: 33).

21. The multispecific antibody of claim 20, wherein the first antibody portion or the third antibody portion comprises (a) VH, comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3, and VL, which contains the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (b) VH, comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 60, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; (c) VH, comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 66, and VL, which contains the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 67; (d) VH, comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 68, and VL, which contains the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 69; (e) VH, comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70, and VL, which contains the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71; (f) VH, comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; (g) VH, comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 73, and VL, comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; or (h) VH, which contains the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

77.

22. The multispecific antibody of claim 17, wherein the first antibody portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

6.

23. The multispecific antibody of claim 17, wherein the first antibody portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

8.

24. The multispecific antibody according to claim 19 or 21, wherein the first antibody portion comprises (a) VH, comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and VL, which contains the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b) VH, comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 58, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59; (c) VH, comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 62, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59; (d) VH, comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 63, and VL, comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 64; or (e) VH, comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 65, and VL, which contains the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 64; And the third antibody portion contains (a) VH, comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3, and VL, which contains the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (b) VH, comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 60, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; (c) VH, comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 66, and VL, which contains the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 67; (d) VH, comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 68, and VL, which contains the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 69; (e) VH, comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70, and VL, which contains the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71; (f) VH, comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; (g) VH, comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 73, and VL, comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; or (h) VH, which contains the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

77.

25. The multispecific antibody according to claim 19 or 21, wherein the third antibody portion comprises (a) VH, comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and VL, which contains the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b) VH, comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 58, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59; (c) VH, comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 62, and VL, which contains the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59; (d) VH, comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 63, and VL, comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 64; or (e) VH, comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 65, and VL, which contains the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 64; And the first antibody portion contains (a) VH, comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3, and VL, which contains the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 4; (b) VH, comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 60, and VL, which contains the amino acid sequence of SEQ ID NO:61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:61; (c) VH, comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 66, and VL, which contains the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 67; (d) VH, comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 68, and VL, which contains the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 69; (e) VH, comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70, and VL, which contains the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71; (f) VH, comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 72, and VL, which contains the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; (g) VH, comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 73, and VL, comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 61; or (h) VH, which contains the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

77.

26. The multispecific antibody according to claim 14 or 16, wherein the linker comprises the amino acid sequence of SEQ ID NO:

56.

27. The multispecific antibody of claim 15, wherein the first antibody portion and the third antibody portion form a heterodimer by pairing through the Fc region.

28. The multispecific antibody according to any one of the preceding claims, wherein... (a) The first antibody portion comprises an IgG heavy chain constant region (IgG(CH)) containing the amino acid sequence of SEQ ID NO: 51, and the third antibody portion comprises IgG'(CH) containing the amino acid sequence of SEQ ID NO: 53; or (b) The first antibody portion comprises IgG(CH) containing the amino acid sequence of SEQ ID NO: 55, and the third antibody portion comprises IgG'(CH) containing the amino acid sequence of SEQ ID NO:

54.

29. A composition comprising the multispecific antibody as described in any one of the preceding claims and a pharmaceutically acceptable carrier.

30. An isolated cell comprising one or more polynucleotides, said polynucleotides encoding a multispecific antibody according to any one of claims 1-28.

31. A polynucleotide encoding one or more chains of the multispecific antibody according to any one of claims 1-28.

32. A method of treating cancer in a patient in need, comprising administering to the patient the multispecific antibody according to any one of claims 1-28.

33. The method of claim 32, wherein the cancer is selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

34. The method according to any one of claims 32-33, further comprising administering a treatment for the cancer to the patient.

35. The method of claim 34, wherein the therapy is selected from immunotherapy, chemotherapy, and radiotherapy.

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