Binding agents that bind to EPCAM and / or CD137

Abstract

The present invention provides binding agents that bind EpCAM and CD137, useful for treating or preventing tumors or cancer, or for preventing the progression of tumors or cancer.

Description

[Technical Field]

[0001] The present invention relates to binding agents that bind to EpCAM and CD137. Such binding agents are useful for treating or preventing tumors or cancer. [Background technology]

[0002] Epithelial cell adhesion molecule (EpCAM) mediates Ca ion transport in epithelia. 2+ EpCAM is a transmembrane glycoprotein that mediates cell adhesion independent of cytotoxicity. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. Furthermore, EpCAM has oncogenic potential due to its ability to upregulate c-Myc, E-FABP, and cyclins A and E. EpCAM can be used as a diagnostic marker for various cancers. EpCAM also appears to play a role in carcinoma tumorigenesis and metastasis; therefore, EpCAM can also serve as a potential prognostic marker and a potential target for immunotherapy strategies.

[0003] CD137 (4-1BB), a member of the TNFR family, is a costimulatory molecule on CD8+ and CD4+ T cells, regulatory T cells (Tregs), natural killer (T) cells (NK[T] cells), B cells, and neutrophils. On T cells, CD137 is not constitutively expressed but is induced upon T cell receptor (TCR) activation (e.g., on tumor-infiltrating lymphocytes [TILs; Gros et al., J. Clin Invest 2014;124(5):2246-59]). Stimulation with its natural ligand 4-1BBL or agonist antibodies results in signal transduction using TRAF-2 and TRAF-1 as adaptors. Initial signaling by CD137 involves K63 polyubiquitination, which ultimately leads to activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways. Signaling results in T cell costimulation, proliferation, cytokine production, increased maturation, and prolonged CD8+ T cell survival. Agonistic antibodies against CD137 have been shown to promote anti-tumor control by T cells in various preclinical models (Murillo et al., Clin Cancer Res 2008;14(21):6895-906). Antibodies that stimulate CD137 can induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art, including the human IgG4 antibody urelumab (AU 2004279877) and the human IgG2 antibody utomilumab (Fisher et al. 2012 Cancer Immunol. Immunother. 61:1721-1733). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gros et al.,J.Clin Invest 2014;124(5):2246-59 [Non-patent document 2] Murillo et al., Clin Cancer Res 2008;14(21):6895-906 [Non-patent document 3] Fisher et al.2012 Cancer Immunol.Immunother.61:1721-1733 Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for agents and methods for preventing or treating tumors or cancer. [Means for solving the problem]

[0006] In a first aspect, the disclosure provides a binding agent comprising a first antigen-binding region that binds to EpCAM and a second antigen-binding region that binds to CD137.

[0007] In some embodiments, the EpCAM is human EpCAM. In some embodiments, the CD137 is human CD137. In some embodiments, the human EpCAM comprises the sequence set forth in SEQ ID NO: 59. In some embodiments, the human CD137 comprises the sequence set forth in SEQ ID NO: 62.

[0008] In some embodiments, the first antigen-binding region that binds to EpCAM binds to EpCAM expressed on tumor cells.

[0009] In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR3 sequence comprising the sequence set forth in SEQ ID NO: 4. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR2 sequence comprising the sequence set forth in SEQ ID NO: 3. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR1 sequence comprising the sequence set forth in SEQ ID NO: 2. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 3 and 66, respectively.

[0010] In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR3 sequence comprising the sequence set forth in SEQ ID NO: 8. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR2 sequence comprising the sequence set forth in SEQ ID NO: 7. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR1 sequence comprising the sequence set forth in SEQ ID NO: 6. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 67, 68, and 8, respectively.

[0011] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising an HCDR3 sequence and a light chain variable region (VL) comprising an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence set forth in SEQ ID NO: 4 and the LCDR3 sequence comprises the sequence set forth in SEQ ID NO: 8.

[0012] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively.

[0013] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 65 and 66, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 67, 68 and 8, respectively.

[0014] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 3 and 66, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively.

[0015] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1 and / or a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 5.

[0016] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:1.

[0017] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:1.

[0018] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:5.

[0019] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:5.

[0020] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 1, and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 1, and the VL comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 1, and the VL comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 1, and the VL comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 5.

[0021] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 1 and the VL comprises the sequence set forth in SEQ ID NO: 5.

[0022] In some embodiments, the first antigen-binding region that binds to EpCAM comprises heavy and light chain variable regions of an antibody that competes for EpCAM binding, and / or has specificity for EpCAM of an antibody comprising the above-mentioned heavy chain variable region (VH) and / or light chain variable region (VL).

[0023] In some embodiments, the second antigen-binding region that binds CD137 comprises a heavy chain variable region (VH) comprising an HCDR3 sequence comprising the sequence set forth in SEQ ID NO: 14. In some embodiments, the second antigen-binding region that binds CD137 comprises a heavy chain variable region (VH) comprising an HCDR2 sequence comprising the sequence set forth in SEQ ID NO: 13. In some embodiments, the second antigen-binding region that binds CD137 comprises a heavy chain variable region (VH) comprising an HCDR1 sequence comprising the sequence set forth in SEQ ID NO: 12. In some embodiments, the second antigen-binding region that binds CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively. In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 69, 70 and 71, respectively. In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 75, 13 and 71, respectively.

[0024] In some embodiments, the second antigen-binding region that binds CD137 comprises a light chain variable region (VL) comprising an LCDR3 sequence comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the second antigen-binding region that binds CD137 comprises a light chain variable region (VL) comprising an LCDR2 sequence comprising the sequence set forth in SEQ ID NO: 17. In some embodiments, the second antigen-binding region that binds CD137 comprises a light chain variable region (VL) comprising an LCDR1 sequence comprising the sequence set forth in SEQ ID NO: 16. In some embodiments, the second antigen-binding region that binds CD137 comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. In some embodiments, the second antigen-binding region that binds to CD137 comprises a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 72, 73 and 18, respectively.

[0025] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising an HCDR3 sequence and a light chain variable region (VL) comprising an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence set forth in SEQ ID NO: 14 and the LCDR3 sequence comprises the sequence set forth in SEQ ID NO: 18.

[0026] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0027] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 72, 73 and 18, respectively.

[0028] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0029] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11 and / or a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0030] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:11.

[0031] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:11.

[0032] In some embodiments, the second antigen-binding region that binds to CD137 comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 15.

[0033] In some embodiments, the second antigen-binding region that binds to CD137 comprises a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:15.

[0034] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 11, and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 11, and the VL comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 11, and the VL comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 11, and the VL comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 15.

[0035] In some embodiments, the second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 11 and the VL comprises the sequence set forth in SEQ ID NO: 15.

[0036] In some embodiments, the second antigen-binding region that binds to CD137 comprises heavy and light chain variable regions of an antibody that competes for CD137 binding and / or has the specificity for CD137 of an antibody comprising the above-mentioned heavy and / or light chain variable regions.

[0037] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 4; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR3 sequence shown in SEQ ID NO:14.

[0038] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) that includes the LCDR3 sequence set forth in SEQ ID NO: 8; and b) The second antigen-binding region that binds to CD137 comprises a light chain variable region (VL) comprising the LCDR3 sequence shown in SEQ ID NO:18.

[0039] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 4 and a light chain variable region (VL) comprising the LCDR3 sequence set forth in SEQ ID NO: 8; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 14 and a light chain variable region (VL) comprising the LCDR3 sequence set forth in SEQ ID NO: 18.

[0040] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0041] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 67, 68, and 8, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 72, 73 and 18, respectively.

[0042] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0043] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 1, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 5; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0044] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 5; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 11, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0045] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 1 and the VL comprises the sequence set forth in SEQ ID NO: 5; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 11 and the VL comprises the sequence set forth in SEQ ID NO: 15.

[0046] In some embodiments, the first antigen-binding region that binds to EpCAM comprises the heavy and light chain variable regions of an antibody that competes for EpCAM binding with the first antigen-binding region that binds to EpCAM, and / or has the specificity for EpCAM of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the first antigen-binding region that binds to EpCAM, and the second antigen-binding region that binds to CD137 comprises the heavy and light chain variable regions of an antibody that competes for CD137 binding with the second antigen-binding region that binds to CD137, and / or has the specificity for CD137 of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the second antigen-binding region that binds to CD137.

[0047] In some embodiments, the variable region comprises three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).

[0048] In some embodiments, the complementarity determining regions and the framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0049] In some embodiments, the binding agent is in the form of a full-length antibody or antibody fragment.

[0050] In some embodiments, the binding agent is multispecific, such as a bispecific binding agent.

[0051] In some embodiments, the binding agent is multispecific, such as a bispecific antibody.

[0052] In some embodiments, the binder is (i) a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and (ii) a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137. Includes:

[0053] In some embodiments, the binder is i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and ii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137; Includes:

[0054] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein the first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). and The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). Includes:

[0055] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a first heavy chain comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a first light chain comprising a first light chain variable region (VL) and a first light chain constant region (CL); and The second binding arm is i) a second heavy chain comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); ii) a second light chain comprising a second light chain variable region (VL) and a second light chain constant region (CL); Includes:

[0056] In some embodiments, the first binding arm is derived from a full-length antibody. In some embodiments, the first binding arm is derived from a monoclonal antibody. In some embodiments, the first binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the second binding arm is derived from a full-length antibody. In some embodiments, the second binding arm is derived from a monoclonal antibody. In some embodiments, the second binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a full-length antibody, such as a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a monoclonal antibody.

[0057] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises one or more of the constant heavy chain 1 (CH1) region, hinge region, constant heavy chain 2 (CH2) region and constant heavy chain 3 (CH3) region, preferably at least the hinge region, CH2 region and CH3 region.

[0058] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

[0059] In some embodiments, the first heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; the second heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; and the first heavy chain and the second heavy chain do not have substitutions at the same positions.

[0060] In some embodiments, (i) the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said first heavy chain constant region (CH) and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said second heavy chain constant region (CH).

[0061] In some embodiments, the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2, and CH3 regions.

[0062] In some embodiments, the first and second heavy chain constant regions (CHs) are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an identical antibody except for comprising unmodified first and second heavy chain constant regions (CHs).

[0063] In some embodiments, the unmodified first and second heavy chain constant regions (CH) each comprise the amino acid sequence set forth in SEQ ID NO:47.

[0064] In some embodiments, the Fc-mediated effector function is measured by binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors.

[0065] In some embodiments, the Fc-mediated effector function is measured by binding to C1q.

[0066] In some embodiments, the first and second heavy chain constant regions are modified such that C1q binding to the antibody is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is preferably determined by ELISA.

[0067] In some embodiments, in at least one of the first and second heavy chain constant regions, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, P331, and G236 according to EU numbering in a human IgG1 heavy chain are not L, L, D, N, P, and G, respectively.

[0068] In some embodiments, the positions corresponding to positions L234 and L235 according to EU numbering in a human IgG1 heavy chain are F and E in said first and second heavy chains, respectively.

[0069] In some embodiments, the positions corresponding to positions L234, L235 and D265 of a human IgG1 heavy chain according to EU numbering are F, E and A, respectively, in said first and / or second heavy chain constant region (HC), and / or the positions corresponding to positions L234, L235 and G236 of a human IgG1 heavy chain according to EU numbering are F, E and R, respectively, in said first and / or second heavy chain constant region (HC).

[0070] In some embodiments, (i) positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in both the first and second heavy chain constant regions; or (ii) positions corresponding to positions L234, L235, and G236 according to EU numbering in a human IgG1 heavy chain are F, E, and R, respectively, in both the first and second heavy chain constant regions; or (iii) Positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in one of the first heavy chain constant region and the second heavy chain constant region, and positions corresponding to positions L234, L235, and G236 according to EU numbering in the human IgG1 heavy chain are F, E, and R, respectively, in the other of the first heavy chain constant region and the second heavy chain constant region.

[0071] In some embodiments, the positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0072] In some embodiments, positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0073] In some embodiments, the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and the positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R.

[0074] In some embodiments, the constant region of the first and / or second heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:47.

[0075] In some embodiments, a) the constant region of the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 54, and b) the constant region of said second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:52.

[0076] In some embodiments, the binding agent comprises a kappa (κ) light chain constant region.

[0077] In some embodiments, the binding agent comprises a lambda (λ) light chain constant region.

[0078] In some embodiments, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0079] In some embodiments, the second light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0080] In some embodiments, (i) the first light chain constant region and the second light chain constant region are kappa (κ) light chain constant regions; or (ii) the first light chain constant region and the second light chain constant region are lambda (λ) light chain constant regions; or (iii) the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region; or (iv) the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0081] In some embodiments, the kappa (κ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:55.

[0082] In some embodiments, the lambda (λ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:56.

[0083] In some embodiments, the binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment, the isotype is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0084] In some embodiments, the binding agent is a full-length IgG1 antibody.

[0085] In some embodiments, the binding agent is an antibody of the IgG1m(f) allotype.

[0086] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). wherein the first VH comprises a first HCDR1, HCDR2 and HCDR3 sequence, the first VL comprises a first LCDR1, LCDR2 and LCDR3 sequence, the first HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, and the first LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively; The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). wherein the second VH comprises a second HCDR1, HCDR2 and HCDR3 sequence, and the second VL comprises a second LCDR1, LCDR2 and LCDR3 sequence, and the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; wherein positions L234, L235 and G236 according to EU numbering in the human IgG1 heavy chain are F, E and R, respectively, in the first CH; and positions L234, L235 and D265 according to EU numbering in the human IgG1 heavy chain are F, E and A, respectively, in the second CH; and The amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in the first CH, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in the second CH.

[0087] In some embodiments, the binder is i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 9; ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 10; iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20. Includes:

[0088] In some embodiments, the binder is i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9; ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 10; iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20 Includes:

[0089] In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR3 sequence comprising the sequence set forth in SEQ ID NO: 78. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR2 sequence comprising the sequence set forth in SEQ ID NO: 77. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR1 sequence comprising the sequence set forth in SEQ ID NO: 76. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 87, 77 and 84, respectively.

[0090] In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR3 sequence comprising the sequence set forth in SEQ ID NO: 81. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR2 sequence comprising the sequence set forth in SEQ ID NO: 80. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR1 sequence comprising the sequence set forth in SEQ ID NO: 79. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 85, 86, and 81, respectively.

[0091] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising an HCDR3 sequence and a light chain variable region (VL) comprising an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence set forth in SEQ ID NO: 78 and the LCDR3 sequence comprises the sequence set forth in SEQ ID NO: 81.

[0092] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 76, 77 and 78, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 79, 80 and 81, respectively.

[0093] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 82, 83 and 84, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 85, 86 and 81, respectively.

[0094] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 87, 77 and 84, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 79, 80 and 81, respectively.

[0095] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 21, positions 1 to 116, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 22, positions 1 to 112.

[0096] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:21, positions 1 to 116.

[0097] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the sequence shown in positions 1 to 116 of SEQ ID NO:21.

[0098] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 22, positions 1 to 112.

[0099] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising the sequence shown in positions 1 to 112 of SEQ ID NO:22.

[0100] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:21, positions 1-116, and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:22, positions 1-112. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:21, positions 1-116, and the VL comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:22, positions 1-112. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:21, positions 1-116, and the VL comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:22, positions 1-112. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO:21, positions 1-116, and the VL comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO:22, positions 1-112.

[0101] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in SEQ ID NO: 21, positions 1 to 116, and the VL comprises the sequence shown in SEQ ID NO: 22, positions 1 to 112.

[0102] In some embodiments, the first antigen-binding region that binds to EpCAM comprises heavy and light chain variable regions of an antibody that competes for EpCAM binding, and / or has specificity for EpCAM of an antibody comprising the above-mentioned heavy chain variable region (VH) and / or light chain variable region (VL).

[0103] In some embodiments, the second antigen-binding region that binds to CD137 is as described above.

[0104] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 78; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR3 sequence shown in SEQ ID NO:14.

[0105] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) that includes the LCDR3 sequence set forth in SEQ ID NO: 81; and b) The second antigen-binding region that binds to CD137 comprises a light chain variable region (VL) comprising the LCDR3 sequence shown in SEQ ID NO:18.

[0106] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 78 and a light chain variable region (VL) comprising the LCDR3 sequence set forth in SEQ ID NO: 81; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 14 and a light chain variable region (VL) comprising the LCDR3 sequence set forth in SEQ ID NO: 18.

[0107] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0108] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 85, 86, and 81, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 72, 73 and 18, respectively.

[0109] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 87, 77, and 84, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0110] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 21, positions 1 to 116, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 22, positions 1 to 112; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0111] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 21, positions 1 to 116, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 22, positions 1 to 112; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 11, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0112] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in SEQ ID NO: 21, positions 1 to 116, and the VL comprises the sequence shown in SEQ ID NO: 22, positions 1 to 112; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 11 and the VL comprises the sequence set forth in SEQ ID NO: 15.

[0113] In some embodiments, the first antigen-binding region that binds to EpCAM comprises the heavy and light chain variable regions of an antibody that competes for EpCAM binding with the first antigen-binding region that binds to EpCAM, and / or has the specificity for EpCAM of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the first antigen-binding region that binds to EpCAM, and the second antigen-binding region that binds to CD137 comprises the heavy and light chain variable regions of an antibody that competes for CD137 binding with the second antigen-binding region that binds to CD137, and / or has the specificity for CD137 of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the second antigen-binding region that binds to CD137.

[0114] In some embodiments, the variable region comprises three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).

[0115] In some embodiments, the complementarity determining regions and the framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0116] In some embodiments, the binding agent is in the form of a full-length antibody or antibody fragment.

[0117] In some embodiments, the binding agent is multispecific, such as a bispecific binding agent.

[0118] In some embodiments, the binding agent is multispecific, such as a bispecific antibody.

[0119] In some embodiments, the binder is (i) a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and (ii) a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137. Includes.

[0120] In some embodiments, the binder is i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and ii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137; Includes.

[0121] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein the first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). and The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). Includes:

[0122] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a first heavy chain comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a first light chain comprising a first light chain variable region (VL) and a first light chain constant region (CL); and The second binding arm is i) a second heavy chain comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); ii) a second light chain comprising a second light chain variable region (VL) and a second light chain constant region (CL); Includes:

[0123] In some embodiments, the first binding arm is derived from a full-length antibody. In some embodiments, the first binding arm is derived from a monoclonal antibody. In some embodiments, the first binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the second binding arm is derived from a full-length antibody. In some embodiments, the second binding arm is derived from a monoclonal antibody. In some embodiments, the second binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a full-length antibody, such as a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a monoclonal antibody.

[0124] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises one or more of the constant heavy chain 1 (CH1) region, hinge region, constant heavy chain 2 (CH2) region and constant heavy chain 3 (CH3) region, preferably at least the hinge region, CH2 region and CH3 region.

[0125] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

[0126] In some embodiments, the first heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; the second heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; and the first heavy chain and the second heavy chain do not have substitutions at the same positions.

[0127] In some embodiments, (i) the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said first heavy chain constant region (CH) and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said second heavy chain constant region (CH).

[0128] In some embodiments, the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2, and CH3 regions.

[0129] In some embodiments, the first and second heavy chain constant regions (CHs) are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an identical antibody except for comprising unmodified first and second heavy chain constant regions (CHs).

[0130] In some embodiments, the unmodified first and second heavy chain constant regions (CH) each comprise the amino acid sequence set forth in SEQ ID NO:47.

[0131] In some embodiments, the Fc-mediated effector function is measured by binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors.

[0132] In some embodiments, the Fc-mediated effector function is measured by binding to C1q.

[0133] In some embodiments, the first and second heavy chain constant regions are modified such that C1q binding to the antibody is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is preferably determined by ELISA.

[0134] In some embodiments, in at least one of the first and second heavy chain constant regions, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, P331, and G236 according to EU numbering in a human IgG1 heavy chain are not L, L, D, N, P, and G, respectively.

[0135] In some embodiments, the positions corresponding to positions L234 and L235 according to EU numbering in a human IgG1 heavy chain are F and E in said first and second heavy chains, respectively.

[0136] In some embodiments, the positions corresponding to positions L234, L235 and D265 of a human IgG1 heavy chain according to EU numbering are F, E and A, respectively, in said first and / or second heavy chain constant region (HC), and / or the positions corresponding to positions L234, L235 and G236 of a human IgG1 heavy chain according to EU numbering are F, E and R, respectively, in said first and / or second heavy chain constant region (HC).

[0137] In some embodiments, (i) positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in both the first and second heavy chain constant regions; or (ii) positions corresponding to positions L234, L235, and G236 according to EU numbering in a human IgG1 heavy chain are F, E, and R, respectively, in both the first and second heavy chain constant regions; or (iii) Positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in one of the first heavy chain constant region and the second heavy chain constant region, and positions corresponding to positions L234, L235, and G236 according to EU numbering in the human IgG1 heavy chain are F, E, and R, respectively, in the other of the first heavy chain constant region and the second heavy chain constant region.

[0138] In some embodiments, the positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0139] In some embodiments, positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0140] In some embodiments, the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and the positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R.

[0141] In some embodiments, the constant region of the first and / or second heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:47.

[0142] In some embodiments, a) the constant region of the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 54, and b) the constant region of said second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:52.

[0143] In some embodiments, the binding agent comprises a kappa (κ) light chain constant region.

[0144] In some embodiments, the binding agent comprises a lambda (λ) light chain constant region.

[0145] In some embodiments, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0146] In some embodiments, the second light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0147] In some embodiments, (i) the first light chain constant region and the second light chain constant region are kappa (κ) light chain constant regions; or (ii) the first light chain constant region and the second light chain constant region are lambda (λ) light chain constant regions; or (iii) the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region; or (iv) the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0148] In some embodiments, the kappa (κ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:55.

[0149] In some embodiments, the lambda (λ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:56.

[0150] In some embodiments, the binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment, the isotype is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0151] In some embodiments, the binding agent is a full-length IgG1 antibody.

[0152] In some embodiments, the binding agent is an antibody of the IgG1m(f) allotype.

[0153] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). wherein the first VH comprises a first HCDR1, HCDR2 and HCDR3 sequence, the first VL comprises a first LCDR1, LCDR2 and LCDR3 sequence, the first HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 76, 77 and 78, respectively, and the first LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 79, 80 and 81, respectively; The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). wherein the second VH comprises a second HCDR1, HCDR2 and HCDR3 sequence, and the second VL comprises a second LCDR1, LCDR2 and LCDR3 sequence, and the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; wherein positions L234, L235 and G236 according to EU numbering in the human IgG1 heavy chain are F, E and R, respectively, in the first CH; and positions L234, L235 and D265 according to EU numbering in the human IgG1 heavy chain are F, E and A, respectively, in the second CH; and The amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in the first CH, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in the second CH.

[0154] In some embodiments, the binder is i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 21; ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 22; iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20. Includes.

[0155] In some embodiments, the binder is i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21; ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 22; iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20 Includes.

[0156] In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR3 sequence comprising the sequence set forth in SEQ ID NO: 90. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR2 sequence comprising the sequence set forth in SEQ ID NO: 89. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR1 sequence comprising the sequence set forth in SEQ ID NO: 88. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 88, 89, and 90, respectively. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 91, 92, and 90, respectively.

[0157] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising an HCDR3 sequence and a light chain variable region (VL) comprising an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence set forth in SEQ ID NO: 4 and the LCDR3 sequence comprises the sequence set forth in SEQ ID NO: 90.

[0158] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 88, 89 and 90, respectively.

[0159] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 65 and 66, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 91, 92 and 90, respectively.

[0160] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 3 and 66, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 88, 89 and 90, respectively.

[0161] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 25, positions 1 to 115, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 26, positions 1 to 111.

[0162] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 25, positions 1 to 115.

[0163] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the sequence shown in positions 1 to 115 of SEQ ID NO:25.

[0164] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:26, positions 1 to 111.

[0165] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:26, positions 1 to 111.

[0166] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:25, positions 1-115, and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:26, positions 1-111. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:25, positions 1-115, and the VL comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:26, positions 1-111. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:25, positions 1-115, and the VL comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:26, positions 1-111. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO:25, positions 1-115, and the VL comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO:26, positions 1-111.

[0167] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in SEQ ID NO: 25, positions 1 to 115, and the VL comprises the sequence shown in SEQ ID NO: 26, positions 1 to 111.

[0168] In some embodiments, the first antigen-binding region that binds to EpCAM comprises the above-described heavy and light chain variable regions of an antibody that competes for EpCAM binding, and / or has the specificity for EpCAM of an antibody comprising the above-described heavy chain variable region (VH) and / or light chain variable region (VL).

[0169] In some embodiments, the second antigen-binding region that binds to CD137 is as described above.

[0170] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) that includes the LCDR3 sequence set forth in SEQ ID NO: 90; and b) The second antigen-binding region that binds to CD137 comprises a light chain variable region (VL) comprising the LCDR3 sequence shown in SEQ ID NO:18.

[0171] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 4 and a light chain variable region (VL) comprising the LCDR3 sequence set forth in SEQ ID NO: 90; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR3 sequence set forth in SEQ ID NO: 14 and a light chain variable region (VL) comprising the LCDR3 sequence set forth in SEQ ID NO: 18.

[0172] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 88, 89, and 90, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0173] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 91, 92, and 90, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 72, 73 and 18, respectively.

[0174] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 88, 89, and 90, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0175] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 25, positions 1 to 115, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 26, positions 1 to 111; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0176] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, positions 1 to 115, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 26, positions 1 to 111; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 11, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0177] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in SEQ ID NO: 25, positions 1 to 115, and the VL comprises the sequence shown in SEQ ID NO: 26, positions 1 to 111; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 11 and the VL comprises the sequence set forth in SEQ ID NO: 15.

[0178] In some embodiments, the first antigen-binding region that binds to EpCAM comprises the heavy and light chain variable regions of an antibody that competes for EpCAM binding with the first antigen-binding region that binds to EpCAM, and / or has the specificity for EpCAM of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the first antigen-binding region that binds to EpCAM, and the second antigen-binding region that binds to CD137 comprises the heavy and light chain variable regions of an antibody that competes for EpCAM binding with the second antigen-binding region that binds to CD137, and / or has the specificity for CD137 of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the second antigen-binding region that binds to CD137.

[0179] In some embodiments, the variable region comprises three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).

[0180] In some embodiments, the complementarity determining regions and the framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0181] In some embodiments, the binding agent is in the form of a full-length antibody or antibody fragment.

[0182] In some embodiments, the binding agent is multispecific, such as a bispecific binding agent.

[0183] In some embodiments, the binding agent is multispecific, such as a bispecific antibody.

[0184] In some embodiments, the binder is (i) a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and (ii) a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137. Includes:

[0185] In some embodiments, the binder is i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and ii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137; Includes:

[0186] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein the first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). and The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). Includes:

[0187] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a first heavy chain comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a first light chain comprising a first light chain variable region (VL) and a first light chain constant region (CL); and The second binding arm is i) a second heavy chain comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); ii) a second light chain comprising a second light chain variable region (VL) and a second light chain constant region (CL); Includes:

[0188] In some embodiments, the first binding arm is derived from a full-length antibody. In some embodiments, the first binding arm is derived from a monoclonal antibody. In some embodiments, the first binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the second binding arm is derived from a full-length antibody. In some embodiments, the second binding arm is derived from a monoclonal antibody. In some embodiments, the second binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a full-length antibody, such as a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a monoclonal antibody.

[0189] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises one or more of the constant heavy chain 1 (CH1) region, hinge region, constant heavy chain 2 (CH2) region and constant heavy chain 3 (CH3) region, preferably at least the hinge region, CH2 region and CH3 region.

[0190] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

[0191] In some embodiments, the first heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; the second heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; and the first heavy chain and the second heavy chain do not have substitutions at the same positions.

[0192] In some embodiments, (i) the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said first heavy chain constant region (CH) and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said second heavy chain constant region (CH).

[0193] In some embodiments, the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2, and CH3 regions.

[0194] In some embodiments, the first and second heavy chain constant regions (CHs) are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an identical antibody except for comprising unmodified first and second heavy chain constant regions (CHs).

[0195] In some embodiments, the unmodified first and second heavy chain constant regions (CH) each comprise the amino acid sequence set forth in SEQ ID NO:47.

[0196] In some embodiments, the Fc-mediated effector function is measured by binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors.

[0197] In some embodiments, the Fc-mediated effector function is measured by binding to C1q.

[0198] In some embodiments, the first and second heavy chain constant regions are modified such that C1q binding to the antibody is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is preferably determined by ELISA.

[0199] In some embodiments, in at least one of the first and second heavy chain constant regions, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, P331, and G236 according to EU numbering in a human IgG1 heavy chain are not L, L, D, N, P, and G, respectively.

[0200] In some embodiments, the positions corresponding to positions L234 and L235 according to EU numbering in a human IgG1 heavy chain are F and E in said first and second heavy chains, respectively.

[0201] In some embodiments, the positions corresponding to positions L234, L235 and D265 of a human IgG1 heavy chain according to EU numbering are F, E and A, respectively, in said first and / or second heavy chain constant region (HC), and / or the positions corresponding to positions L234, L235 and G236 of a human IgG1 heavy chain according to EU numbering are F, E and R, respectively, in said first and / or second heavy chain constant region (HC).

[0202] In some embodiments, (i) positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in both the first and second heavy chain constant regions; or (ii) positions corresponding to positions L234, L235, and G236 according to EU numbering in a human IgG1 heavy chain are F, E, and R, respectively, in both the first and second heavy chain constant regions; or (iii) Positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in one of the first heavy chain constant region and the second heavy chain constant region, and positions corresponding to positions L234, L235, and G236 according to EU numbering in the human IgG1 heavy chain are F, E, and R, respectively, in the other of the first heavy chain constant region and the second heavy chain constant region.

[0203] In some embodiments, the positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0204] In some embodiments, positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0205] In some embodiments, the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and the positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R.

[0206] In some embodiments, the constant region of the first and / or second heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:47.

[0207] In some embodiments, a) the constant region of the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 54, and b) the constant region of said second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:52.

[0208] In some embodiments, the binding agent comprises a kappa (κ) light chain constant region.

[0209] In some embodiments, the binding agent comprises a lambda (λ) light chain constant region.

[0210] In some embodiments, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0211] In some embodiments, the second light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0212] In some embodiments, (i) the first light chain constant region and the second light chain constant region are kappa (κ) light chain constant regions; or (ii) the first light chain constant region and the second light chain constant region are lambda (λ) light chain constant regions; or (iii) the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region; or (iv) the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0213] In some embodiments, the kappa (κ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:55.

[0214] In some embodiments, the lambda (λ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:56.

[0215] In some embodiments, the binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment, the isotype is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0216] In some embodiments, the binding agent is a full-length IgG1 antibody.

[0217] In some embodiments, the binding agent is an antibody of the IgG1m(f) allotype.

[0218] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). wherein the first VH comprises a first HCDR1, HCDR2 and HCDR3 sequence, the first VL comprises a first LCDR1, LCDR2 and LCDR3 sequence, the first HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, and the first LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 88, 89 and 90, respectively; The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). wherein the second VH comprises a second HCDR1, HCDR2 and HCDR3 sequence, and the second VL comprises a second LCDR1, LCDR2 and LCDR3 sequence, and the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; wherein positions L234, L235 and G236 according to EU numbering in the human IgG1 heavy chain are F, E and R, respectively, in the first CH; and positions L234, L235 and D265 according to EU numbering in the human IgG1 heavy chain are F, E and A, respectively, in the second CH; and The amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in the first CH, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in the second CH.

[0219] In some embodiments, the binder is i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 25; ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 26; iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20. Includes.

[0220] In some embodiments, the binder is i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 25; ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 26; iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20 Includes.

[0221] In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR3 sequence comprising the sequence set forth in SEQ ID NO: 4. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR2 sequence comprising the sequence set forth in SEQ ID NO: 93. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising an HCDR1 sequence comprising the sequence set forth in SEQ ID NO: 2. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 93, and 4, respectively. In some embodiments, the first antigen-binding region that binds EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 96, and 66, respectively. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 93 and 66, respectively.

[0222] In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR3 sequence comprising the sequence set forth in SEQ ID NO: 8. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR2 sequence comprising the sequence set forth in SEQ ID NO: 95. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising an LCDR1 sequence comprising the sequence set forth in SEQ ID NO: 94. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 94, 95, and 8, respectively. In some embodiments, the first antigen-binding region that binds EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 97, 98, and 8, respectively.

[0223] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 93 and 4, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 94, 95 and 8, respectively.

[0224] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 96 and 66, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 97, 98 and 8, respectively.

[0225] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 93 and 66, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 94, 95 and 8, respectively.

[0226] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 27, positions 1 to 115, and / or a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 28, positions 1 to 108.

[0227] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:27, positions 1 to 115.

[0228] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the sequence shown in positions 1 to 115 of SEQ ID NO:27.

[0229] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:28, positions 1 to 108.

[0230] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:28, positions 1 to 108.

[0231] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:27, positions 1-115, and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:28, positions 1-108. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:27, positions 1-115, and the VL comprises a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:28, positions 1-108. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:27, positions 1-115, and the VL comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:28, positions 1-108. In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO:27, positions 1-115, and the VL comprises a sequence having at least 95% identity to the sequence set forth in SEQ ID NO:28, positions 1-108.

[0232] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in SEQ ID NO: 27, positions 1 to 115, and the VL comprises the sequence shown in SEQ ID NO: 28, positions 1 to 108.

[0233] In some embodiments, the first antigen-binding region that binds to EpCAM comprises the above-described heavy and light chain variable regions of an antibody that competes for EpCAM binding, and / or has the specificity for EpCAM of an antibody comprising the above-described heavy chain variable region (VH) and / or light chain variable region (VL).

[0234] In some embodiments, the second antigen-binding region that binds to CD137 is as described above.

[0235] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 93, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 94, 95, and 8, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NOs: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0236] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 64, 96, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 97, 98, and 8, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 72, 73 and 18, respectively.

[0237] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 74, 93, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 94, 95, and 8, respectively; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0238] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 27, positions 1 to 115, and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 28, positions 1 to 108; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and a light chain variable region (VL) comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0239] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 27, positions 1 to 115, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 28, positions 1 to 108; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 11, and the VL comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0240] In some embodiments, a) the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in SEQ ID NO: 27, positions 1 to 115, and the VL comprises the sequence shown in SEQ ID NO: 28, positions 1 to 108; and b) The second antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 11 and the VL comprises the sequence set forth in SEQ ID NO: 15.

[0241] In some embodiments, the first antigen-binding region that binds to EpCAM comprises the heavy and light chain variable regions of an antibody that competes for EpCAM binding with the first antigen-binding region that binds to EpCAM, and / or has the specificity for EpCAM of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the first antigen-binding region that binds to EpCAM, and the second antigen-binding region that binds to CD137 comprises the heavy and light chain variable regions of an antibody that competes for CD137 binding with the second antigen-binding region that binds to CD137, and / or has the specificity for CD137 of an antibody that comprises the heavy chain variable region or light chain variable region or a combination thereof of the second antigen-binding region that binds to CD137.

[0242] In some embodiments, the variable region comprises three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).

[0243] In some embodiments, the complementarity determining regions and the framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0244] In some embodiments, the binding agent is in the form of a full-length antibody or antibody fragment.

[0245] In some embodiments, the binding agent is multispecific, such as a bispecific binding agent.

[0246] In some embodiments, the binding agent is multispecific, such as a bispecific antibody.

[0247] In some embodiments, the binder is (i) a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and (ii) a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137. Includes:

[0248] In some embodiments, the binder is i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first heavy chain variable region (VH) and the first light chain variable region (VL) form the first antigen-binding region that binds to EpCAM; and ii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL), wherein the second heavy chain variable region (VH) and the second light chain variable region (VL) form the second antigen-binding region that binds to CD137; Includes:

[0249] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein the first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). and The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). Includes:

[0250] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a first heavy chain comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a first light chain comprising a first light chain variable region (VL) and a first light chain constant region (CL); and The second binding arm is i) a second heavy chain comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); ii) a second light chain comprising a second light chain variable region (VL) and a second light chain constant region (CL); Includes:

[0251] In some embodiments, the first binding arm is derived from a full-length antibody. In some embodiments, the first binding arm is derived from a monoclonal antibody. In some embodiments, the first binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the second binding arm is derived from a full-length antibody. In some embodiments, the second binding arm is derived from a monoclonal antibody. In some embodiments, the second binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a full-length antibody, such as a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In some embodiments, the first and second binding arms are derived from a monoclonal antibody.

[0252] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises one or more of the constant heavy chain 1 (CH1) region, hinge region, constant heavy chain 2 (CH2) region and constant heavy chain 3 (CH3) region, preferably at least the hinge region, CH2 region and CH3 region.

[0253] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

[0254] In some embodiments, the first heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; the second heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (EU numbering) of a human IgG1 heavy chain; and the first heavy chain and the second heavy chain do not have substitutions at the same positions.

[0255] In some embodiments, (i) the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said first heavy chain constant region (CH) and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said second heavy chain constant region (CH).

[0256] In some embodiments, the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2, and CH3 regions.

[0257] In some embodiments, the first and second heavy chain constant regions (CHs) are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an identical antibody except for comprising unmodified first and second heavy chain constant regions (CHs).

[0258] In some embodiments, the unmodified first and second heavy chain constant regions (CH) each comprise the amino acid sequence set forth in SEQ ID NO:47.

[0259] In some embodiments, the Fc-mediated effector function is measured by binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors.

[0260] In some embodiments, the Fc-mediated effector function is measured by binding to C1q.

[0261] In some embodiments, the first and second heavy chain constant regions are modified such that C1q binding to the antibody is reduced compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is preferably determined by ELISA.

[0262] In some embodiments, in at least one of the first and second heavy chain constant regions, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, P331, and G236 according to EU numbering in a human IgG1 heavy chain are not L, L, D, N, P, and G, respectively.

[0263] In some embodiments, the positions corresponding to positions L234 and L235 according to EU numbering in a human IgG1 heavy chain are F and E in said first and second heavy chains, respectively.

[0264] In some embodiments, the positions corresponding to positions L234, L235 and D265 of a human IgG1 heavy chain according to EU numbering are F, E and A, respectively, in said first and / or second heavy chain constant region (HC), and / or the positions corresponding to positions L234, L235 and G236 of a human IgG1 heavy chain according to EU numbering are F, E and R, respectively, in said first and / or second heavy chain constant region (HC).

[0265] In some embodiments, (i) positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in both the first and second heavy chain constant regions; or (ii) positions corresponding to positions L234, L235, and G236 according to EU numbering in a human IgG1 heavy chain are F, E, and R, respectively, in both the first and second heavy chain constant regions; or (iii) Positions corresponding to positions L234, L235, and D265 according to EU numbering in the human IgG1 heavy chain are F, E, and A, respectively, in one of the first heavy chain constant region and the second heavy chain constant region, and positions corresponding to positions L234, L235, and G236 according to EU numbering in the human IgG1 heavy chain are F, E, and R, respectively, in the other of the first heavy chain constant region and the second heavy chain constant region.

[0266] In some embodiments, the positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0267] In some embodiments, positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0268] In some embodiments, the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and the positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R.

[0269] In some embodiments, the constant region of the first and / or second heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:47.

[0270] In some embodiments, a) the constant region of the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 54, and b) the constant region of said second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:52.

[0271] In some embodiments, the binding agent comprises a kappa (κ) light chain constant region.

[0272] In some embodiments, the binding agent comprises a lambda (λ) light chain constant region.

[0273] In some embodiments, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0274] In some embodiments, the second light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0275] In some embodiments, (i) the first light chain constant region and the second light chain constant region are kappa (κ) light chain constant regions; or (ii) the first light chain constant region and the second light chain constant region are lambda (λ) light chain constant regions; or (iii) the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region; or (iv) the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0276] In some embodiments, the kappa (κ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:55.

[0277] In some embodiments, the lambda (λ) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:56.

[0278] In some embodiments, the binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment, the isotype is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0279] In some embodiments, the binding agent is a full-length IgG1 antibody.

[0280] In some embodiments, the binding agent is an antibody of the IgG1m(f) allotype.

[0281] In some embodiments, the binding agent is an antibody comprising a first binding arm comprising the first antigen-binding region that binds EpCAM and a second binding arm comprising the second antigen-binding region that binds CD137, wherein the first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). wherein the first VH comprises a first HCDR1, HCDR2 and HCDR3 sequence, the first VL comprises a first LCDR1, LCDR2 and LCDR3 sequence, the first HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 93 and 4, respectively, and the first LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 94, 95 and 8, respectively; The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). wherein the second VH comprises a second HCDR1, HCDR2 and HCDR3 sequence, and the second VL comprises a second LCDR1, LCDR2 and LCDR3 sequence, and the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; wherein positions L234, L235 and G236 according to EU numbering in the human IgG1 heavy chain are F, E and R, respectively, in the first CH; and positions L234, L235 and D265 according to EU numbering in the human IgG1 heavy chain are F, E and A, respectively, in the second CH; and The amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in the first CH, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in the second CH.

[0282] In some embodiments, the binder is i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 27; ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 28; iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20. Includes.

[0283] In some embodiments, the binder is i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 27; ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 28; iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20 Includes.

[0284] In a further aspect, the present disclosure provides a binding agent comprising a first binding arm comprising a first antigen binding region and a first heavy chain constant region comprising a constant heavy chain 2 (CH2) region, and a second binding arm comprising a second antigen binding region and a second heavy chain constant region comprising a constant heavy chain 2 (CH2) region, wherein: at least one of the first and second antigen-binding regions binds to EpCAM; and Positions corresponding to positions L234, L235, and G236 according to EU numbering in the human IgG1 heavy chain are F, E, and R, respectively, in at least one of the first and second heavy chain constant regions.

[0285] In some embodiments, the positions corresponding to positions L234, L235 and G236 according to EU numbering in a human IgG1 heavy chain are F, E and R, respectively, in said first and / or second heavy chain constant region.

[0286] In some embodiments, positions corresponding to positions L234, L235 and G236 according to EU numbering in a human IgG1 heavy chain are F, E and R, respectively, in both the first and / or second heavy chain constant regions.

[0287] In some embodiments, the positions corresponding to positions L234, L235, and G236 according to EU numbering in a human IgG1 heavy chain are F, E, and R, respectively, in one of the first or second heavy chain constant regions, and the positions corresponding to positions L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A, respectively, in the other of the first or second heavy chain constant regions.

[0288] In some embodiments, both the first and second antigen-binding regions bind to EpCAM.

[0289] In some embodiments, one of the first and second antigen-binding regions binds to EpCAM, and the other of the first and second antigen-binding regions binds to an antigen other than EpCAM.

[0290] In some embodiments, one of the first and second antigen-binding regions binds to EpCAM and the other of the first and second antigen-binding regions binds to CD137.

[0291] In a further aspect, the present disclosure provides a binding agent comprising a first binding arm comprising a first antigen binding region and a first heavy chain constant region comprising a constant heavy chain 2 (CH2) region, and a second binding arm comprising a second antigen binding region and a second heavy chain constant region comprising a constant heavy chain 2 (CH2) region, wherein: at least one of the first and second antigen-binding regions binds to CD137; and Positions corresponding to positions L234, L235 and G236 according to EU numbering in the human IgG1 heavy chain are F, E and R, respectively, in at least one of the first and second heavy chain constant regions.

[0292] In some embodiments, the positions corresponding to positions L234, L235 and G236 according to EU numbering in a human IgG1 heavy chain are F, E and R, respectively, in said first and / or second heavy chain constant regions.

[0293] In some embodiments, positions corresponding to positions L234, L235 and G236 according to EU numbering in a human IgG1 heavy chain are F, E and R, respectively, in both the first and / or second heavy chain constant regions.

[0294] In some embodiments, the positions corresponding to positions L234, L235, and G236 according to EU numbering in a human IgG1 heavy chain are F, E, and R, respectively, in one of the first or second heavy chain constant regions, and the positions corresponding to positions L234, L235, and D265 according to EU numbering in a human IgG1 heavy chain are F, E, and A, respectively, in the other of the first or second heavy chain constant regions.

[0295] In some embodiments, both the first and second antigen-binding regions bind to CD137.

[0296] In some embodiments, one of the first and second antigen-binding regions binds to CD137, and the other of the first and second antigen-binding regions binds to an antigen other than CD137.

[0297] In some embodiments, one of the first and second antigen-binding regions binds to CD137, and the other of the first and second antigen-binding regions binds to EpCAM.

[0298] In some embodiments, the antigen-binding region that binds EpCAM and / or the antigen-binding region that binds CD137 is as described herein.

[0299] In some embodiments, the EpCAM is human EpCAM.

[0300] In some embodiments, the antigen-binding region that binds to EpCAM binds to EpCAM expressed on tumor cells.

[0301] In some embodiments, the antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively.

[0302] In some embodiments, the antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 1, and the VL comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 5.

[0303] In some embodiments, the antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 1 and the VL comprises the sequence set forth in SEQ ID NO: 5.

[0304] In some embodiments, the CD137 is human CD137.

[0305] In some embodiments, the antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0306] In some embodiments, the antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 11, and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 15.

[0307] In one embodiment, the antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence set forth in SEQ ID NO: 11 and the VL comprises the sequence set forth in SEQ ID NO: 15.

[0308] In some embodiments, the binding agent comprises an antigen-binding region that binds EpCAM and an antigen-binding region that binds CD137, wherein: a) the antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively; and b) The antigen-binding region that binds to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0309] In some embodiments, the binding agent is a bivalent binding agent.

[0310] In some embodiments, the binding agent comprises an Fc region.

[0311] In some embodiments, the binding agent is in the form of an antibody, for example a full-length antibody.

[0312] In some embodiments, the binding agent comprises a first binding arm comprising an antigen-binding region that binds EpCAM and a second binding arm comprising an antigen-binding region that binds CD137, wherein: The first binding arm comprises: i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH); ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL). and The second binding arm is iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL). Includes:

[0313] In some embodiments, the first VH comprises a first HCDR1, HCDR2 and HCDR3 sequence, the first VL comprises a first LCDR1, LCDR2 and LCDR3 sequence, the first HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively, the first LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively; and The second VH comprises second HCDR1, HCDR2 and HCDR3 sequences, the second VL comprises second LCDR1, LCDR2 and LCDR3 sequences, the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

[0314] In some embodiments, The first VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1, and the first VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 5, and The second VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 11, and the second VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0315] In some embodiments, The first VH comprises the amino acid sequence set forth in SEQ ID NO: 1, and the first VL comprises the amino acid sequence set forth in SEQ ID NO: 5, and The second VH comprises the amino acid sequence set forth in SEQ ID NO:11, and the second VL comprises the amino acid sequence set forth in SEQ ID NO:15.

[0316] In some embodiments, each of the first and second heavy chain constant regions (CH) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

[0317] In some embodiments, (i) the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said first heavy chain constant region (CH) and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said second heavy chain constant region (CH).

[0318] In some embodiments, positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

[0319] In some embodiments, the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC), and the positions corresponding to positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), wherein the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L, and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R.

[0320] In a further aspect, the present disclosure provides a polynucleotide or set of polynucleotides that encodes a binding agent described herein.

[0321] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a binding agent described herein or a polynucleotide described herein.

[0322] In a further aspect, the present disclosure provides a method of treating a subject comprising administering to the subject a binding agent described herein, a polynucleotide described herein, or a pharmaceutical composition described herein.

[0323] In some embodiments, the method is a method of treating or preventing cancer in a subject.

[0324] In some embodiments, the subject is a human.

[0325] In a further aspect, the present disclosure provides a binding agent as described herein, a polynucleotide as described herein, or a pharmaceutical composition as described herein for pharmaceutical use, e.g., for use as a medicament.

[0326] In some embodiments, the binding agents described herein, the polynucleotides described herein, or the pharmaceutical compositions described herein are for use in the therapeutic or prophylactic treatment of a disease or disorder.

[0327] In some embodiments, the disease or disorder is cancer.

[0328] In a further aspect, the present disclosure provides a binding agent described herein, a polynucleotide described herein, or a pharmaceutical composition described herein for treating or preventing cancer in a subject.

[0329] In some embodiments, the subject is a human. [Brief explanation of the drawings]

[0330] [Figure 1] Figure 1 shows the relative EpCAM expression on tumor cells as determined by flow cytometry on T84, DiFi, HPAF-II, NCI-N87, Calu-3, NCI-H747, and A549 tumor cell lines. Data shown are the delta geometric mean fluorescence intensity (gMFI) for each tumor cell line, calculated as the geometric mean fluorescence intensity (APC) of the EpCAM antibody minus the geometric mean fluorescence (APC) of a non-binding control antibody. [Figure 2A] Binding of bivalent EpCAM antibodies and their monovalent counterparts to DiFi, HPAF-II, or A549 cells. Monovalent and bivalent binding of EpCAM antibodies to DiFi, HPAF-II, or A549 cell lines was determined by flow cytometry. IgG1-b12-FEAL was included as a negative control in all experiments (open squares). Data shown are the mean fluorescence intensity (MFI; R-phycoerythrin [PE]) ± standard deviation (SD) of duplicate measurements from one representative experiment. A. Binding of bsIgG1-b12-FEALxEpCAM-A37-FEAR (closed circles) and IgG1-EpCAM-A37-FEAR (open triangles) antibodies to DiFi, HPAF-II, or A549 cells. [Figure 2B] B. Binding of bivalent EpCAM antibodies and their monovalent counterparts to DiFi, HPAF-II, or A549 cells. Monovalent and bivalent binding of EpCAM antibodies to DiFi, HPAF-II, or A549 cell lines was determined by flow cytometry. IgG1-b12-FEAL was included as a negative control in all experiments (open squares). Data shown are the mean fluorescence intensity (MFI; R-phycoerythrin [PE]) ± standard deviation (SD) of duplicate measurements from one representative experiment. C. Binding of bsIgG1-b12-FEALxEpCAM-C52-FEAR (closed circles) and IgG1-EpCAM-C52-FEAR (open triangles) antibodies to DiFi, HPAF-II, or A549 cells. [Figure 2C]B. Binding of bivalent EpCAM antibodies and their monovalent counterparts to DiFi, HPAF-II, or A549 cells. Monovalent and bivalent binding of EpCAM antibodies to DiFi, HPAF-II, or A549 cell lines was determined by flow cytometry. IgG1-b12-FEAL was included as a negative control in all experiments (open squares). Data shown are the mean fluorescence intensity (MFI; R-phycoerythrin [PE]) ± standard deviation (SD) of duplicate measurements from one representative experiment. C. Binding of bsIgG1-b12-FEALxEpCAM-UBS-54-FEAR (closed circles) and IgG1-EpCAM-UBS-54-FEAR (open triangles) antibodies to DiFi, HPAF-II, or A549 cells. [Figure 2D] B. Binding of bivalent EpCAM antibodies and their monovalent counterparts to DiFi, HPAF-II, or A549 cells. Monovalent and bivalent binding of EpCAM antibodies to DiFi, HPAF-II, or A549 cell lines was determined by flow cytometry. IgG1-b12-FEAL was included as a negative control in all experiments (open squares). Data shown are the mean fluorescence intensity (MFI; R-phycoerythrin [PE]) ± standard deviation (SD) of duplicate measurements from one representative experiment. C. Binding of IgG1-EpCAM-323-A3-FEAR (open triangle) antibody to DiFi or HPAF-II cells. [Figure 2E]Binding of bivalent EpCAM antibodies and their monovalent counterparts to DiFi, HPAF-II, or A549 cells. Monovalent and bivalent binding of EpCAM antibodies to DiFi, HPAF-II, or A549 cell lines was determined by flow cytometry. IgG1-b12-FEAL was included as a negative control in all experiments (open squares). Data shown are the mean fluorescence intensity (MFI; R-phycoerythrin [PE]) ± standard deviation (SD) of duplicate measurements from one representative experiment. E. Binding of bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-A37-FEAR and bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR antibodies and nonbinding control bispecific antibodies bsIgG1-b12-FERL / CD137-HC7LC2-FEAR and bsIgG1-b12-FERL / b12-FEAR to DiFi cells. [Figure 3A] Binding of EpCAM antibodies to full-length human, cynomolgus monkey, or mouse EpCAM transfected into CHO-S cells. Binding of monovalent and bivalent EpCAM antibodies was analyzed using CHO-S cells transiently transfected with full-length human, cynomolgus monkey, or mouse EpCAM. As a negative control, binding to untransfected CHO-WT cells was assessed. Data are presented as geometric mean fluorescence intensity (gMFI) R-PE values ​​± SD of two technical replicates. A. Binding of IgG1-EpCAM-UBS-54-FEAR, IgG1-EpCAM-A37-FEAR, IgG1-EpCAM-C52-FEAR, and IgG1-EpCAM-343-A3. [Figure 3B]B. Binding of EpCAM antibodies to full-length human, cynomolgus monkey, or mouse EpCAM transfected into CHO-S cells. Binding of monovalent and bivalent EpCAM antibodies was analyzed using CHO-S cells transiently transfected with full-length human, cynomolgus monkey, or mouse EpCAM. As a negative control, binding to untransfected CHO-WT cells was assessed. Data are shown as geometric mean fluorescence intensity (gMFI) R-PE values ​​± SD of two technical replicates. C. Binding of bsIgG1-b12-FEAL / EpCAM-UBS-54-FEAR, bsIgG1-EpCAM-A37-FERL / b12-FEAR, bsIgG1-b12-FEAL / EpCAM-C52-FEAR, and bsIgG1-b12-FEAL / EpCAM-323-A3-FEAR. [Figure 4] Induction of 4-1BB-dependent luciferase activity by the EpCAMx4-1BB bispecific antibody in a cell-based reporter assay. HEK293_NKF_h4-1BB_gfp_luc reporter cells were cocultured with EpCAM-expressing OV-90-SC12 cells overnight in the presence of serial dilutions of BsIgG1-CD137-005-FEAR / EpCAM-323-A3-FEAL or cultured alone (medium). Luciferase activity was quantified by luminometry. Fold induction of luciferase activity compared to cultures without antibody (dotted line) is shown. Error bars indicate SD of duplicate wells. Data from one representative experiment out of three are shown. [Figure 5AB]Enhancement of in vitro PBMC proliferation by EpCAMx4-1BB bispecific antibody in PBMC-DiFi cell cocultures. The EpCAMx4-1BB bispecific antibody was tested in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi cells cocultured with CFSE-labeled PBMCs. Cells were cultured for 96 hours in the presence of anti-CD3 (0.1 μg / mL) with or without the indicated concentrations of EpCAMx4-1BB or EpCAMxb12 bispecific antibody or nonbinding control IgG1-b12-FEAL. The number of CFSE-positive cells was extracted and used to calculate the mitotic index. Data shown are the mean mitotic index ± SD of duplicate measurements from one representative experiment. A. Mitotic index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-UBS54-FEAR (black triangles) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-UBS54-FEAR (black squares) antibodies. B. Mitotic index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-A37-FEAR (black triangles) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-A37-FEAR (black squares) antibodies. [Figure 5CD]Enhancement of in vitro PBMC proliferation by EpCAMx4-1BB bispecific antibody in PBMC-DiFi cell cocultures. The EpCAMx4-1BB bispecific antibody was tested in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi cells cocultured with CFSE-labeled PBMCs. Cells were cultured for 96 hours in the presence of anti-CD3 (0.1 μg / mL) with or without the indicated concentrations of EpCAMx4-1BB or EpCAMxb12 bispecific antibody or nonbinding control IgG1-b12-FEAL. The number of CFSE-positive cells was extracted and used to calculate the mitotic index. Data shown are the mean mitotic index ± SD of duplicate measurements from one representative experiment. C. Mitotic index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-C52-FEAR (black triangles) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-C52-FEAR (black squares) antibodies. D. Mitotic index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-323-A3-FEAR (black triangles) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-323-A3-FEAR (black) antibodies. [Figure 6] Enhancement of in vitro CD4+ and CD8+ T cell proliferation by EpCAMx4-1BB bispecific antibody in PBMC-DiFi cell cocultures. The EpCAMx4-1BB bispecific antibody was tested in an in vitro T cell proliferation assay using EpCAM-expressing DiFi cells cocultured with CFSE-labeled PBMCs. Cells were cultured for 96 hours in the presence of anti-CD3 (0.1 μg / mL) with or without EpCAMx4-1BB (gray bars) or EpCAMxb12 (striped bars) bispecific antibody (10 μg / mL) or the nonbinding control IgG1-b12-FEAL (10 μg / mL; white bars). The number of CFSE-positive cells was assessed as a measure of absolute CD4+ (A) or CD8+ (B) T cell numbers by flow cytometry, and the mitotic index was calculated. Data shown are the mean mitotic index ± SD of duplicate measurements from one representative experiment. [Figure 7ABC]Enhancement of in vitro PBMC proliferation by the EpCAMx4-1BB bispecific antibody in PBMC-tumor cell cocultures using various tumor cell lines. The EpCAMx4-1BB bispecific antibody was tested in an in vitro PBMC proliferation assay using EpCAM-expressing T84, DiFi, HPAF-II, NCI-N87, Calu-3, or NCI-H747 tumor cells cocultured with CFSE-labeled PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL) and EpCAMx4-1BB or EpCAMxb12 bispecific antibody or the nonbinding control IgG1-b12-FEAL. The number of CFSE-positive cells was assessed as a measure of absolute PBMC number by flow cytometry, and a mitotic index was calculated. Data shown are (A) PBMC-T84 coculture, (B) PBMC-DiFi coculture, and (C) PBMC-HPAF-II coculture. [Figure 7DEF] Enhancement of in vitro PBMC proliferation by the EpCAMx4-1BB bispecific antibody in PBMC-tumor cell cocultures using various tumor cell lines. The EpCAMx4-1BB bispecific antibody was tested in an in vitro PBMC proliferation assay using EpCAM-expressing T84, DiFi, HPAF-II, NCI-N87, Calu-3, or NCI-H747 tumor cells cocultured with CFSE-labeled PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL) and EpCAMx4-1BB or EpCAMxb12 bispecific antibody or the nonbinding control IgG1-b12-FEAL. The number of CFSE-positive cells was assessed as a measure of absolute PBMC number by flow cytometry, and a mitotic index was calculated. Data shown are the mean mitotic index ± SD of duplicate measurements from one representative experiment using (D) PBMC-NCI-N87 coculture, (E) PBMC-Calu-3 coculture, and (F) PBMC-NCI-H747 coculture. [Figure 8]Enhancement of in vitro proliferation of human CD4+ and CD8+ T cells in DiFi tumor cell cocultures by EpCAMx4-1BB bispecific antibodies with an Fc domain containing an inactivating mutation. EpCAMx4-1BB bispecific antibodies were tested in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi tumor cells cocultured with CFSE-labeled human PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL) and EpCAM-FERLx4-1BB-FEAR (black diamonds), EpCAM-FEARx4-1BB-FEAL (black squares), b12-FERLx4-1BB-FEAR (black triangles), EpCAM-FERLxb12-FEAR (black triangles), or the nonbinding control b12-FERLxb12-FEAR (open triangles). The number of CFSE-positive (A) CD4+ and (B) CD8+ T cells was assessed as a measure of absolute T cell numbers by flow cytometry, and the mitotic index was calculated. Data shown are nonlinear 4-parameter variable slope fits of the mean mitotic index ± SD of duplicate measurements from one representative experiment using PBMC-DiFi cocultures. [Figure 9] Enhancement of tumor-infiltrating lymphocyte proliferation by EpCAMx4-1BB bispecific antibody in ex vivo patient-derived tumor specimens. Tumor tissue resected from a patient with non-small cell lung cancer was cut into 1-2 mm pieces and cultured for 14 days in the presence of IL-2 (50 U / mL) and 0.2-5 μg / mL of BsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-A37-FEAR or BsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-323-A3-FEAR, or IL-2 alone (no Ab). The IL-2 concentration was gradually reduced to 33 U / mL and 15 U / mL on days 7 and 10, respectively. Absolute cell counts after culture were determined by flow cytometry. Total TIL, NK cell, CD8+, and CD4+ T cell counts are shown for cultures derived from one exemplary patient of the two analyzed patients. Data from 3-4 individual replicates and averages of replicates are shown. Error bars indicate SD. [Figure 10]Antitumor activity of the EpCAMx4-1BB bispecific antibody in hEpCAM mice bearing MC38 tumors overexpressing hEpCAM. Mice transgenic for the human EpCAM protein (hEpCAM mice) were inoculated with 5 × 10 MC38 tumor cells overexpressing human EpCAM protein (MC38_hEpCAM). Treatment began when tumors reached an average size of approximately 30 mm . On days 12, 17, 21, 24, 28, and 31 after tumor inoculation, mice were treated intraperitoneally with 100 μg of BsIgG2amm-EpCAM-323-A3-AALT / m4-1BB-3H3-AAKR or the negative control antibody IgG2amm-b12-AAKR (vertical dotted line). (A) The mean tumor volume per group is shown, and error bars indicate SEM. Growth curves include the last tumor volume measured in sacrificed mice (last observation carried forward). **, P<0.01; two-way repeated measures ANOVA. (B) Percent survival. *, P<0.05; log-rank (Mantel-Cox) test. [Figure 11] Enhancement of human CD4+ and CD8+ T cell proliferation in PBMC-DiFi tumor cell cocultures by the EpCAMx4-1BB bispecific antibody compared with the combination of bivalent EpCAM and 4-1BB-specific monoclonal antibodies. The bispecific antibody bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR was compared with bivalent IgG1-EpCAM-A37-FERL and IgG1-CD137-009-HC7LC2-FEAR (either alone or in combination) in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi tumor cells cocultured with CellTrace Violet (CTV)-labeled human PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL; dotted line) and EpCAMx4-1BB, IgG1-EpCAM-A37-FERL, and / or IgG1-CD137-009-HC7LC2-FEAR. CD4+ and CD8+ T proliferation was assessed by flow cytometry analysis of CTV-labeled dilutions. Data shown are the mean proliferation index ± SD of duplicate measurements from one representative experiment. [Figure 12]Enhancement of human CD4+ and CD8+ T cell proliferation in PBMC-DiFi tumor cell cocultures by the EpCAMx4-1BB bispecific antibody compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR. The bispecific antibody bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR was compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi tumor cells cocultured with CTV-labeled human PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL; dotted line) and bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, or with the combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR. CD4+ and CD8+ T cell proliferation was assessed by flow cytometry analysis of CTV-labeled dilutions. Data shown are the mean dividing cell percentages ± SD of duplicate measurements from one representative experiment. [Figure 13]Enhancement of human CD4+ and CD8+ T cell activation in PBMC-DiFi tumor cell cocultures by EpCAMx4-1BB bispecific antibody compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR. The EpCAMx4-1BB bispecific antibody bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR was compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi tumor cells cocultured with human PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL; dotted line) and bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, or with a combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR. The percentage of CD4+ and CD8+ T cells expressing CD25 (A) or 4-1BB (B), as well as the geometric mean fluorescence intensity (FI) in the CD25+ or 4-1BB+ population, were determined by flow cytometry. Data shown are the mean ± SD of duplicate measurements from one representative experiment. [Figure 14]Enhancement of human CD4+ and CD8+ T cell proliferation in cancer patient-derived PBMC-DiFi tumor cell cocultures by the EpCAMx4-1BB bispecific antibody compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. The EpCAMx4-1BB bispecific antibody bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR was compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR in an in vitro proliferation assay using EpCAM-expressing DiFi tumor cells cocultured with CTV-labeled cancer patient-derived PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL; dotted line) and bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, or with a combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. CD4+ and CD8+ T cell proliferation was assessed by flow cytometry analysis of CTV-labeled dilutions. Data shown are the mean dividing cell percentages ± SD of duplicate measurements from one experiment. [Figure 15A]Enhancement of human CD4+ and CD8+ T cell activation in cancer patient-derived PBMC-DiFi tumor cell cocultures by the EpCAMx4-1BB bispecific antibody compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. The EpCAMx4-1BB bispecific antibody bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR was compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR in an in vitro proliferation assay using EpCAM-expressing DiFi tumor cells cocultured with cancer patient-derived PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL; dotted line) and bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, or with a combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. The percentage of CD4+ and CD8+ T cells expressing CD25 (A), as well as the geometric mean fluorescence intensity (FI) in the CD25+ or 4-1BB+ population, were determined by flow cytometry. Data shown are the mean ± SD of duplicate measurements from one experiment. [Figure 15B]Enhancement of human CD4+ and CD8+ T cell activation in cancer patient-derived PBMC-DiFi tumor cell cocultures by the EpCAMx4-1BB bispecific antibody compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. The EpCAMx4-1BB bispecific antibody bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR was compared to the combinations bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR in an in vitro proliferation assay using EpCAM-expressing DiFi tumor cells cocultured with cancer patient-derived PBMCs. Cells were cultured for 96 hours in the presence of the indicated concentrations of anti-CD3 (0.1 μg / mL; dotted line) and bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, or with a combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. The percentage of CD4+ and CD8+ T cells expressing 4-1BB(B) and the geometric mean fluorescence intensity (FI) in the CD25+ or 4-1BB+ populations were determined by flow cytometry. Data shown are the mean ± SD of duplicate measurements from one experiment. [Figure 16]The EpCAMx4-1BB bispecific antibody enhances CD107a and GzmB expression by CD8+ T cells. PBMC-derived CD8+ T cells expressing a CLDN6-specific TCR were cocultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells for 2 days in the presence of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR or control antibody. CD107a and GzmB expression by CD8+ T cells was analyzed by flow cytometry. (A) Representative flow cytometry plot. (B-C) Dose-response curves for CD107a (C) and GzmB (D) expression levels. Data shown are the mean and SD of duplicate measurements from one of six donors tested. (D) Normalized CD107a and (E) GzmB expression levels (expression levels of bsIgG1-b12-FERL / b12-FEAR were set to 1 for each donor). Pooled data from six donors evaluated in two independent experiments are shown. Error bars indicate SD. ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05; Friedman test with Dunn's multiple comparison test. GMFI = geometric mean fluorescence intensity; GzmB = granzyme B; PBMC = peripheral blood mononuclear cells; SD = standard deviation. [Figure 17]The EpCAMx4-1BB bispecific antibody enhances CD8+ T cell-mediated cytotoxicity against tumor cells. PBMC-derived CD8+ T cells expressing a CLDN6-specific TCR were cocultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells in the presence of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR or control antibody for 5–6 days. Cell index values ​​were derived from impedance measurements performed at 2–3 hour intervals. (A) Cell index curves for cocultures from one representative donor are shown. Symbols represent the average cell index value of duplicate wells. For better visibility, data from each second impedance measurement (i.e., every 4–6 hours) are shown. (B) AUC analysis was performed using the cell index data over the entire coculture period. The AUC for each treatment condition was normalized to that of bsIgG1-ctrl-b12-FERL / b12-FEAR-treated cultures from the same donor. Pooled data from all six donors evaluated in two independent experiments are shown. Error bars indicate SD. ***, P<0.001; **, P<0.01; *, P<0.05; Friedman test with Dunn's multiple comparison test. AUC = area under the curve; PBMC = peripheral blood mononuclear cells; SD = standard deviation. [Figure 18]FcγR binding of EpCAMx4-1BB bispecific antibodies with different Fc inactivating mutations (FER / FEA, FEA / FEA, FER / FER) to immobilized human recombinant FcγR proteins was analyzed by SPR. The binding of EpCAMx4-1BB bispecific antibodies with various combinations of Fc inactivating mutations (FER / FEA, FEA / FEA, FER / FER) and the binding of the monoclonal antibodies IgG1-EpCAM-A37-FERL and IgG1-CD137-009-HC7LC2-FEAR to immobilized human recombinant FcγR proteins was analyzed by SPR. The antibody IgG1-b12 (wild-type Fc) was included as a positive control for FcγR binding. The relative binding response in RU (response units) is measured by setting the sensorgram to 0 RU at the moment of SPR analyte injection. Each sample analyzed on the active surface was also analyzed on a parallel reference surface used for background correction. Data from a single measurement of one experiment are shown. DETAILED DESCRIPTION OF THE INVENTION

[0331] Array Description

[0332] [Table 1] TIFF2025509555000002.tif196158TIFF2025509555000003.tif217158TIFF2025509555000004.t if190158TIFF2025509555000005.tif217158TIFF2025509555000006.tif216158TIFF20255095550 00007.tif216158TIFF2025509555000008.tif185158TIFF2025509555000009.tif204158TIFF202 5509555000010.tif200158TIFF2025509555000011.tif223158TIFF2025509555000012.tif116158

[0333] Detailed Description of the Invention Although the present disclosure will be further described in more detail below, it should be understood that the disclosure is not limited to the specific methods, protocols and reagents described herein, and these may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0334] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0335] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0336] The practice of the present disclosure employs, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques as described in the art (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwiese / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rompp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al., J. Am. Chem. Soc., 1999). al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0337] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not impose limitations on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0338] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually listed herein.

[0339] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0340] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0341] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprising" will be understood to mean the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other member, integer, or step or group of members, integers, or steps. The term "consisting essentially of" means excluding essentially significant other members, integers, or steps. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0342] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0343] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0344] In the context of the present disclosure, the term "about" indicates an interval of precision that a person skilled in the art would understand to still guarantee the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, for example, ±0.01%. As will be understood by a person skilled in the art, such a specific deviation from the numerical value of a given technical effect depends on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger deviation than an artificial or engineered technical effect.

[0345] The term "binding agent" in the context of the present invention refers to any agent capable of binding to a desired antigen. In certain embodiments of the present invention, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also include synthetic, modified, or non-naturally occurring moieties, particularly non-peptide moieties. Such moieties may link the desired antigen-binding functional group or region, such as, for example, an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0346] The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound and soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally called antibodies.

[0347] The structure of immunoglobulins is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2 nd ed. Raven Press, NY (1989). Briefly, immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are primarily V L or VL (variable light chain) domain / region, C L or CL (constant light chain) domain / region, V H or a VH (variable heavy chain) domain / region, and C H or CH (constant heavy chain) domain / region C H 1(CH1), C H 2(CH2), C H 3(CH3) and C HIgG is composed of immunoglobulin domains or regions, such as IgG1, IgG2, and IgG3. The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds within the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically consists of a VL and a CL. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions in which the sequence and / or the configuration of structurally defined loops may be hypervariable), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise specified or contradicted by the context, CDR sequences herein are identified according to the IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc MP Nucleic Acids Res., 38, D301-307 (2010); see also the internet http address www.imgt.org. However, it should be understood that the present disclosure is not limited only to CDR sequences determined according to the IMGT rules. Thus, the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region (VH), e.g., SEQ ID NO: 1 or SEQ ID NO: 11, or the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region (VL), e.g., SEQ ID NO: 5 or SEQ ID NO: 15, encompass CDR sequences determined by any method for determining CDR sequences, for example, according to the IMGT rules or the Kabat rules.Additionally, sequences representing overlapping CDR sequences determined by different methods for determining CDR sequences, for example according to the IMGT rules and the Kabat rules, are included.

[0348] The following table shows the results of determining the CDR sequences according to the IMGT and Kabat rules in relation to SEQ ID NO: 1 and SEQ ID NO: 5 or SEQ ID NO: 11 and SEQ ID NO: 15, and also shows the overlap of the determined CDR sequences.

[0349] [Table 2]

[0350] [Table 3]

[0351] The table below shows the results of determining the CDR sequences according to the IMGT and Kabat rules in relation to SEQ ID NO: 21 and SEQ ID NO: 22, and also shows the overlap of the determined CDR sequences.

[0352] [Table 4]

[0353] The table below shows the results of determining the CDR sequences according to the IMGT and Kabat rules in relation to SEQ ID NO: 25 and SEQ ID NO: 26, and also shows the overlap of the determined CDR sequences.

[0354] [Table 5]

[0355] The table below shows the results of determining the CDR sequences according to the IMGT and Kabat rules in relation to SEQ ID NO: 27 and SEQ ID NO: 28, and also shows the overlap of the determined CDR sequences.

[0356] [Table 6]

[0357] Unless otherwise specified or contradicted by context, references to amino acid positions of constant regions in this disclosure follow EU numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0358] There are five types of mammalian immunoglobulin heavy chains: α, δ, ε, γ, and μ, which constitute the different classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini. In mammals, there are two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within different immunoglobulin isotypes, while the variable region is highly diverse and is responsible for antigen recognition.

[0359] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds containing an amine (-NH2) and a carboxyl (-COOH) functional group along with a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids may be classified based on their structure and chemical properties. Thus, the classes of amino acids may be reflected in one or both of the following tables:

[0360] [Table 7]

[0361] [Table 8]

[0362] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformational variants, isoform variants, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0363] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of an amino acid sequence variant with an insertion, one or more amino acid residues are inserted into a specific site of the amino acid sequence, but random insertion with appropriate screening of the resulting product is also possible.

[0364] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0365] Amino acid deletion mutants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be at any position in the protein. Amino acid deletion mutants containing deletions at the N-terminus and / or C-terminus of the protein are also called N-terminus and / or C-terminus truncation mutants.

[0366] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another in its place. Substitution of one amino acid for another can be classified as conservative or non-conservative. Modifications at positions within the amino acid sequence that are not conserved between homologous proteins or peptides and / or the replacement of an amino acid with another amino acid with similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of a member of a family of amino acids whose side chains are related. In the context of the present disclosure, a "conservative substitution" is the substitution of one amino acid for another amino acid with similar structural and / or chemical properties, such as the substitution of one amino acid residue with another amino acid residue of the same class as defined in either of the two tables above: for example, leucine may be substituted with isoleucine, since both are aliphatic-branched hydrophobic residues. Similarly, aspartic acid may be substituted with glutamic acid, since both are small, negatively charged residues. Naturally occurring amino acids can also generally be divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: -Glycine, alanine; -valine, isoleucine, leucine; -Aspartic acid, glutamic acid; -Asparagine, glutamine; -Serine, threonine; -Lysine, arginine; and -Phenylalanine, tyrosine.

[0367] As used herein, the term "amino acid corresponding to position ..." and similar expressions refer to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence is one that aligns with the other amino acid or segment, typically with default settings, using a standard sequence alignment program such as ALIGN, ClustalW, or the like, and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning sequences or segments in sequences, and thereby determining positions in sequences that correspond to amino acid positions according to the present disclosure, are believed to be well known in the art.

[0368] The term "antibody" (Ab), in the context of the present disclosure, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, that has the ability to specifically bind to an antigen (particularly an epitope on an antigen), typically under physiological conditions, preferably with a half-life of a significant period, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, etc., or any other relevant, functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a period sufficient for the antibody to recruit effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the foregoing. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are referred to as HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), and the CDRs of VL are referred to as LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from the amino terminus to the carboxy terminus in the following order: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0369] The variable regions of the heavy and light chains of immunoglobulin molecules comprise binding domains that interact with antigens. The terms "binding region" and "antigen-binding region" are used interchangeably herein and refer to regions that interact with antigens and include both VH and VL regions. As used herein, antibodies include not only monospecific antibodies but also multispecific antibodies that contain multiple, for example, two or more, for example, three or more different antigen-binding regions.

[0370] As noted above, the term antibody as used herein, unless otherwise specified or clearly contradicted by the context, includes antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed by the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO 2007 / 059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting essentially of a VH domain, also called domain antibodies (Holt et al.; Trends Biotechnol. 2003 Nov;21(11):484-90) (Ward et al., Nature 341,544-546 (1989)); (vi) camelid or nanobody molecules (Revets et al.; Expert Opin Biol Ther. 2005 Jan;5(1):111-24) and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, allowing them to be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv), see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term antibody unless otherwise specified or clearly indicated by the context. Although such fragments are generally included within the meaning of antibody, they collectively and individually are unique features of the present disclosure and exhibit different biological properties and utilities.These and other useful antibody fragments in the context of the present disclosure, as well as bispecific forms of such fragments, are discussed further herein. The term antibody, unless otherwise specified, should also be understood to include antibody-like polypeptides, such as polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0371] The generated antibody can have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG (IgG1, IgG2, IgG3, IgG4, etc.), IgD, IgA (IgA1, IgA2, etc.), IgE, IgM, or IgY) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgG1, than to other isotypes. Thus, for example, the IgG1 antibodies disclosed herein may be sequence variants of naturally occurring IgG1 antibodies, including mutations in the constant region.

[0372] IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some of the embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0373] The term "multispecific antibody" in the context of the present disclosure refers to an antibody having at least two different antigen-binding regions defined by different antibody sequences. In some embodiments, the different antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding regions bind to different target antigens. In one embodiment, the multispecific antibody is a "bispecific antibody" or "bs." Multispecific antibodies, such as bispecific antibodies, can be in any format, including any of the bispecific or multispecific antibody formats described herein below.

[0374] The prefixes BisG1 and bsIgG1 are used interchangeably herein for bispecific antibodies.

[0375] The term "full length" when used in reference to an antibody indicates that the antibody is not a fragment, but includes all of the domains of a particular isotype that are normally found for that isotype in nature, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains of an IgG1 antibody.

[0376] The term "human antibody," as used herein, is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.

[0377] The term "chimeric antibody" as used herein refers to an antibody whose variable region is derived from a non-human species (e.g., from a rodent) and whose constant region is derived from a different species, such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term used to refer to various types of antibody modifications, and the process for antibody engineering is well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques, such as those described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Chimeric antibodies can be genetically engineered or enzymatically engineered recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibodies can be produced by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic applications in humans have been developed to reduce the potential immunogenicity of non-human antibodies, such as rodent antibodies. They typically contain a non-human (e.g., mouse or rabbit) variable region specific for an antigen of interest, as well as human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" as used in the context of a chimeric antibody refers to the region comprising the CDRs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.

[0378] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs) that together form the antigen-binding site into homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0 629 240). Substitution of framework residues (back mutations) from the parent antibody (i.e., non-human antibody) with human framework regions may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help identify amino acid residues within the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions, optionally containing one or more amino acid back mutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily back mutations, can be applied to obtain a humanized antibody with desirable characteristics, such as affinity and biochemical properties.

[0379] As used herein, a protein "derived from" another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical to or similar to one or more amino acid sequences of the other protein or parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, etc. derived from another or parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical to or similar to those of the other or parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, the amino acid sequences of one or more or all of the VH and VL CDRs and / or framework regions, VH, VL, CL, hinge, or CH regions. For example, a humanized antibody can be referred to herein as "derived from" a non-human parent antibody, meaning that at least the VL and VH CDR sequences are identical to or similar to the VH and VL CDR sequences of the non-human parent antibody. A chimeric antibody may be referred to herein as being "derived from" a non-human parent antibody, typically meaning that the VH and VL sequences may be identical or similar to those of the non-human parent antibody. Another example is a binding arm or antigen-binding region, which may be referred to herein as being "derived from" a particular parent antibody, typically meaning that the binding arm or antigen-binding region comprises VH and / or VL CDRs or VH and / or VL sequences that are identical or similar to those of the binding arm or antigen-binding region of the parent antibody. However, as described elsewhere herein, amino acid modifications, such as mutations, can be made in CDRs, constant regions, or elsewhere in the antibody, binding arm, antigen-binding region, etc., to introduce desired properties. When used in reference to one or more sequences derived from a first or parent protein, a "similar" amino acid sequence preferably has at least about 50%, e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, 98%, or 99% sequence identity.

[0380] Non-human antibodies can be produced in many different species, including mice, rabbits, chickens, guinea pigs, llamas, and goats.

[0381] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of antibody genes, and such methods are well known to those skilled in the art.

[0382] Hybridoma production in such non-human species is a very well-established procedure. Immunization protocols and techniques for isolating splenocytes from immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0383] As used herein, unless contradicted by context, the term "Fab arm" or "arm" refers to one heavy chain-light chain pair and is used interchangeably herein with "half molecule."

[0384] The term "binding arm comprising an antigen-binding region" refers to an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm can comprise, for example, six VH and VL CDR sequences, a VH and VL sequence, a Fab or Fab' fragment, or a Fab arm.

[0385] As used herein, unless contradicted by context, the term "Fc region" refers to the region of an antibody consisting of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the term "Fc region" as used herein refers to the region comprising, from the N-terminus to the C-terminus of an antibody, at least the hinge region, the CH2 domain, and the CH3 domain. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0386] In the context of the present disclosure, the term "induce Fc-mediated effector function to a lesser extent", as used in reference to antibodies, including multispecific antibodies, means that the antibody induces an Fc-mediated effector function, in particular selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC or CDC, to a lesser extent compared to a human IgG1 antibody that comprises (i) the same CDR sequences, in particular the same first and second antigen-binding regions as said antibody, and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions.

[0387] Fc-mediated effector function can be measured by binding to FcγR, binding to C1q, or induction of Fc-mediated cross-linking through FcγR.

[0388] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to EU numbering as shown in Kabat (Kabat, E. A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689 (1991)). However, the hinge region may also be of any of the other subtypes described herein.

[0389] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to EU numbering as set forth in Kabat (ibid.). However, the CH1 region may also be of any of the other subtypes described herein.

[0390] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering as set forth in Kabat (ibid.). However, the CH2 region may also be of any of the other subtypes described herein.

[0391] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering as set forth in Kabat (ibid.). However, the CH3 region may also be of any of the other subtypes described herein.

[0392] The term "monovalent antibody" in the context of the present disclosure means that the antibody molecule is capable of binding to a single molecule of antigen and is therefore incapable of antigen cross-linking.

[0393] An "EpCAM antibody" or "anti-EpCAM antibody" is an antibody as described above that specifically binds to the antigen EpCAM.

[0394] A "CD137 antibody" or "anti-CD137 antibody" is an antibody as described above that specifically binds to the antigen CD137.

[0395] An "EpCAMxCD137 antibody" or "anti-EpCAMxCD137 antibody" is a bispecific antibody that contains two different antigen-binding regions, one of which specifically binds to the antigen EpCAM and the other of which specifically binds to the antigen CD137.

[0396] As used herein, the term "biosimilar" (e.g., of an approved reference product / biological drug) refers to a biological product that is similar to the reference product based on: (a) analytical testing showing that the biological product is highly similar to the reference product, despite minor differences in clinically inactive ingredients; (b) animal testing (including toxicity assessment); and / or (c) data from one or more clinical trials (including immunogenicity and pharmacokinetic or pharmacodynamic assessments) sufficient to demonstrate safety, purity, and potency for one or more appropriate conditions of use for which the reference product is approved and intended for use and for which approval is sought (e.g., there are no clinically significant differences in product safety, purity, and potency between the biological product and the reference product). In some embodiments, the biosimilar biological product and the reference product utilize the same mechanism(s) of action for one or more conditions of use prescribed, recommended, or suggested in the proposed labeling, but only to the extent that the mechanism(s) of action are known for the reference product. In some embodiments, one or more conditions of use prescribed, recommended, or suggested in the proposed labeling for the biological product have previously been approved for the reference product. In some embodiments, the route of administration, dosage form, and / or strength of the biological product is the same as that of the reference product. A biosimilar can be, for example, a currently known antibody that has the same primary amino acid sequence as a commercially available antibody, but can be made in a different cell type or by a different production, purification, or formulation method.

[0397] As used herein, the terms "bind" or "capable of binding" in reference to the binding of an antibody to a given antigen or epitope typically refer to a binding activity of about 10, as determined using biolayer interferometry (BLI) or, for example, surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11M or even lower K D An antibody binds with an affinity corresponding to its K for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, D The higher affinity amount binds to a given antigen with an affinity corresponding to the antibody's K D Since it depends on the antibody's K D is very low (i.e., the antibody is highly specific), the degree to which the affinity for the antigen is lower than the affinity for a nonspecific antigen can be at least 10,000-fold.

[0398] As used herein, "k" d ”(seconds -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called value.

[0399] As used herein, "K" D The term "" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0400] Two antibodies have the "same specificity" if they bind to the same antigen and the same epitope. Whether a tested antibody recognizes the same epitope as a particular antigen-binding antibody, i.e., whether the antibody binds to the same epitope, can be tested by various methods well known to those skilled in the art.

[0401] Competition between antibodies can be detected by cross-blocking assay. For example, a competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the well of a microtiter plate, and an antigen-binding antibody and a candidate competing test antibody can be added. The amount of antigen-binding antibody bound to the antigen in the well is indirectly correlated with the binding ability of the candidate competing test antibody that competes for binding to the same epitope. Specifically, the greater the affinity of the candidate competing test antibody for the same epitope, the less antigen-binding antibody will bind to the antigen-coated well. The amount of antigen-binding antibody bound to the well can be measured by labeling the antibody with a detectable or measurable label.

[0402] An antibody that competes with another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, for binding to an antigen, or that has the specificity for the antigen of another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, can be a variant of the heavy and / or light chain variable regions described herein, e.g., an antibody comprising modified and / or a degree of identity of the CDRs described herein.

[0403] As used herein, an "isolated multispecific antibody" is intended to refer to a multispecific antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds EpCAM and CD137 is substantially free of monospecific antibodies that specifically bind EpCAM or CD137).

[0404] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition, which display a single binding specificity and affinity for a particular epitope.

[0405] As used herein, the term "heterodimeric interaction between a first CH3 region and a second CH3 region" refers to the interaction between a first CH3 region and a second CH3 region in a first CH3 / second CH3 heterodimeric antibody.

[0406] As used herein, the term "homodimeric interaction of a first CH3 region and a second CH3 region" refers to the interaction between a first CH3 region and another first CH3 region in a first CH3 / first CH3 homodimeric antibody, and the interaction between a second CH3 region and another second CH3 region in a second CH3 / second CH3 homodimeric antibody.

[0407] As used herein, the term "homodimeric antibody" refers to an antibody comprising two first Fab arms or half molecules, wherein the amino acid sequence of said Fab arms or half molecules is the same.

[0408] As used herein, the term "heterodimeric antibody" refers to an antibody comprising a first and second Fab arm or half molecule, wherein the amino acid sequences of said first and second Fab arms or half molecules are different. In particular, the CH3 regions, or antigen-binding regions, or the CH3 regions and antigen-binding regions of said first and second Fab arms / half molecules are different.

[0409] The term "reducing conditions" or "reducing environment" refers to a state or environment in which a substrate, such as a cysteine ​​residue in the hinge region of an antibody, is more likely to be reduced than oxidized.

[0410] The present disclosure also describes multispecific antibodies, e.g., bispecific antibodies, comprising functional variants of the VL region, VH region, or one or more CDRs of the bispecific antibodies of the examples. The functional variants of the VL, VH, or CDRs used in connection with the bispecific antibodies still allow each antigen-binding region of the bispecific antibody to retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or specificity / selectivity of the parent bispecific antibody, and in some cases, such bispecific antibodies may be associated with greater affinity, selectivity, and / or specificity than the parent bispecific antibody.

[0411] Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988), incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0412] In the context of the present disclosure, unless otherwise indicated, the following notation is used to describe mutations: i) substitution of an amino acid at a given position is described, for example, as K409R, which means substitution of lysine at position 409 of the protein with arginine; and ii) for certain variants, specific three-letter or one-letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, substitution of lysine at position 409 with arginine is designated as K409R, and substitution of lysine at position 409 with any amino acid residue is designated as K409X. In the case of deletion of lysine at position 409, this is indicated by K409*.

[0413] Exemplary variants include those that differ from the VH and / or VL and / or CDRs of a parent sequence by primarily conservative substitutions; for example, 12, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the variant are conservative amino acid residue substitutions.

[0414] In the context of the present disclosure, conservative substitutions may be defined by substitutions within the classes of amino acids defined in Tables 2 and 3.

[0415] Functional variants of the antibody sequences described herein, such as the VL or VH regions, or antibody sequences having a degree of homology or identity to the antibody sequences described herein, such as the VL or VH regions, preferably include modifications or mutations of non-CDR sequences, while the CDR sequences preferably remain unchanged.

[0416] As used herein, the term "EpCAM" refers to epithelial cell adhesion molecule, also known as DIAR5, EGP-2, EGP314, EGP40, ESA, HNPCC8, KS1 / 4, KSA, M4S1, MIC18, MK-1, TACSTD1, TROP1, BerEp4, MOC-31, and Ber-Ep4. EpCAM is believed to have many different roles and appears to play a role in cell adhesion and cancer. In one embodiment, the EpCAM is human EpCAM, having UniProt accession number P16422. The sequence of human EpCAM is also set forth in SEQ ID NO: 59. Amino acids 1-23 of SEQ ID NO: 59 correspond to the signal peptide of human EpCAM, while amino acids 24-265 of SEQ ID NO: 59 correspond to the extracellular domain of human EpCAM; the remainder of the protein, i.e., amino acids 266-288 and 289-314 of SEQ ID NO: 59, are the transmembrane and cytoplasmic domains, respectively.

[0417] As used herein, the term "CD137" refers to CD137(4-1BB), also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the receptor for the ligand TNFSF9 / 4-1BBL. CD137(4-1BB) is thought to be involved in T cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDw137, T cell antigen 4-1BB homolog, and T cell antigen ILA. In one embodiment, CD137(4-1BB) is human CD137(4-1BB) having UniProt accession number Q07011. The sequence of human CD137 is also set forth in SEQ ID NO: 37. Amino acids 1 to 23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; whereas, amino acids 24 to 186 of SEQ ID NO: 37 correspond to the extracellular domain of human CD137; the remainder of the protein, i.e., amino acids 187 to 213 and 214 to 255 of SEQ ID NO: 37, are the transmembrane and cytoplasmic domains, respectively.

[0418] A "treatment cycle" is defined herein as the period within which the effects of adding separate doses of a binding agent result from the pharmacodynamics of the binding agent, or in other words, the period after the subject's body has essentially cleared from the administered binding agent. Multiple small doses within a short time frame, e.g., 2-12 hours or within 2-24 hours, such as on the same day, can be equivalent to a larger single dose.

[0419] In the present context, the terms "treatment," "treating," or "therapeutic intervention" relate to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition from which a subject is afflicted, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, slow the progression of the disease, disorder, or condition, relieve or reduce symptoms and complications, and / or cure or eliminate the disease, disorder, or condition, as well as to prevent the condition, where prevention is to be understood as the management and care of an individual with the aim of combating the disease, condition, or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications. In one embodiment, "treatment" refers to the administration of an effective amount of a therapeutically active binding agent, such as a therapeutically active antibody of the present disclosure, with the aim of alleviating, ameliorating, arresting, or eradicating (curing) the symptoms or disease state.

[0420] Response to treatment with a binding agent of the present disclosure as well as resistance to treatment, non-response to treatment and / or relapse from treatment may be determined according to Response Evaluation Criteria in Solid Tumors; version 1.1 (RECIST criteria v1.1).

[0421] "Best overall response" is the best response recorded from treatment initiation to disease progression / recurrence (using the smallest measurement recorded after treatment initiation as the criterion for PD). Subjects with CR or PR are considered to have an objective response. Subjects with CR, PR, or SD are considered to have disease control. Subjects with NE are considered non-responders. "Best overall response" is the best response recorded from treatment initiation to disease progression / recurrence (using the smallest measurement recorded after treatment initiation as the criterion for PD). Subjects with CR, PR, or SD are considered to have disease control. Subjects with NE are considered non-responders.

[0422] "Duration of response (DOR)" applies only to subjects with a confirmed best overall response of CR or PR and is defined as the time from the first documented objective tumor response (CR or PR) to the date of first PD or death from the underlying cancer.

[0423] "Progression-free survival (PFS)" is defined as the number of days from day 1 of cycle 1 to first documented progression or death from any cause.

[0424] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 to death from any cause. If the subject is not known to have died, OS is censored at the latest date the subject is known to be alive (on or before the cutoff date).

[0425] In the context of this disclosure, the term "therapeutic regimen" refers to a structured treatment plan designed to improve and maintain health.

[0426] The term "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a binding agent, such as an antibody, such as a multispecific antibody or a monoclonal antibody, can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the binding agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the binding agent or fragment thereof are outweighed by therapeutically beneficial effects. If the initial dose is insufficient in the patient, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used. If a dose causes undesirable side effects in the patient, a lower dose (or an effectively lower dose achieved by a different, more localized route of administration) can be used.

[0427] As used herein, the term "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" refer to abnormal cells that grow by rapid and uncontrolled cell proliferation and continue to grow after the stimuli that initiated the new growth have ceased.

[0428] The term "cancer" according to the present disclosure also includes cancer metastasis. "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process, dependent on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and vessels, and then transport by blood before invading the target organ. Finally, the growth of new tumors, i.e., secondary or metastatic tumors, at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present disclosure refers to "distant metastasis," which refers to metastasis away from the primary tumor and regional lymph node system.

[0429] As used herein, terms such as "reduce," "inhibit," "interfere," or "negatively modulate" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or a reduction to essentially zero.

[0430] In one embodiment, the terms "increase" or "enhance" or the like relate to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0431] As used herein, "physiological pH" refers to a pH of 7.5 or about 7.5.

[0432] As used in this disclosure, "wt. %" refers to weight percent, a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total weight of the entire composition in grams (g).

[0433] The term "freezing" refers to the solidification of a liquid, usually accompanied by the removal of heat.

[0434] The term "lyophilize" or "lyophilization" refers to the lyophilization of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0435] The term "recombinant" in the context of the present disclosure means "produced through genetic engineering." In one embodiment, "recombinant" in the context of the present disclosure is not naturally occurring.

[0436] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in living organisms (including viruses), can be isolated from natural sources, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects and objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0437] According to this disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are generally used synonymously herein.

[0438] A "therapeutic protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or pathology. In one embodiment, a therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.

[0439] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous fraction of said structure.

[0440] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a continuous element of said structure. When used in reference to a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0441] With respect to an amino acid sequence (peptide or protein), a "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0442] According to the present disclosure, a portion or fragment of a peptide or protein preferably possesses at least one functional property of the peptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide or protein possesses at least one pharmacological activity of the peptide or protein from which it is derived. A portion or fragment of a peptide or protein preferably comprises a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of up to 8, particularly up to 10, at least 12, at least 15, at least 20, at least 30, or at least 55 consecutive amino acids of the peptide or protein.

[0443] As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence can be a natural or wild-type (WT) amino acid sequence, or can be a modified form of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0444] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0445] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0446] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0447] The terms "% identical" and "% identity" or similar terms are intended to refer to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared.The percentage is purely statistical, and the differences between two sequences may be, but are not necessarily, randomly distributed over the entire length of the sequences being compared.Comparing two sequences is usually carried out by comparing sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, or the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, or the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low-complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0448] The percent identity is obtained by determining the number of identical positions where the compared sequences match, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0449] In some embodiments, the degree of similarity or identity is provided over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is provided over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acid residues, in some embodiments, contiguous amino acid residues. In some embodiments, the degree of similarity or identity is provided over the entire length of the reference sequence.

[0450] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98 or at least 99% of the amino acid residues.

[0451] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0452] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to binding agents, one specific function is one or more binding activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein particularly refers to a variant molecule or sequence that contains an amino acid sequence that has been altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and still performs one or more functions of the parent molecule or sequence, for example, can bind to a target antigen. In one embodiment, alterations to the amino acid sequence of the parent molecule or sequence do not significantly affect or change the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0453] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence, or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence, or a fragment thereof. For example, it will be understood by those skilled in the art that binding agents for use herein can be modified to differ in sequence from the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

[0454] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is. An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell. In one embodiment, the binding agents, e.g., antibodies, described herein are isolated. As used herein, "isolated binding agent" is intended to refer to a binding agent that is substantially free of other binding agents having different antigen specificities. In one embodiment, an isolated bispecific binding agent that specifically binds EpCAM and CD137 is substantially free of monospecific antibodies that specifically bind EpCAM or CD137. In a preferred embodiment, the binding agents used in the present disclosure are in a substantially purified form.

[0455] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, which may be present in a subject, e.g., a patient. Thus, according to this disclosure, cells for transfection with nucleic acids described herein can be present in vitro or in vivo; for example, the cells can form part of a patient's organ, tissue, and / or organism. According to this disclosure, transfection can be transient or stable. In some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, so the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection. Generally, the nucleic acid encoding the antigen is transiently transfected into the cell. RNA can be transfected into the cell to transiently express the encoded protein.

[0456] According to the present disclosure, a peptide or protein analog is a modified form of the peptide or protein from which it is derived, retaining at least one functional property of the peptide or protein. For example, a pharmacologically active analog of a peptide or protein retains at least one pharmacological activity of the peptide or protein from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule associated with the protein or peptide, such as carbohydrates, lipids, and / or proteins or peptides. In one embodiment, a protein or peptide "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the proteins and peptides.

[0457] As used herein, "activation" or "stimulation" refers to the state of immune effector cells, such as T cells, that are stimulated sufficiently to induce detectable cell proliferation. Activation can also involve the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cells" refers, among other things, to immune effector cells undergoing cell division.

[0458] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, resulting in differentiation into an effector cell, such as an effector T cell.

[0459] The term "clonal expansion" or "expansion" refers to the process by which a specific entity multiplies. In the context of the present disclosure, the term is preferably used in reference to an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize said antigen are amplified. Preferably, clonal expansion results in differentiation of immune effector cells.

[0460] According to the present disclosure, an "antigen" encompasses any substance or molecular structure capable of binding to an antibody or a T cell receptor. The presence of an antigen in the body can elicit an immune response. Thus, "antigen" encompasses any substance against which an immune response or immune mechanism is directed. This also includes situations in which an immune response or immune mechanism is directed against one or more antigenic peptides, particularly when the antigen is processed into antigenic peptides and presented in association with MHC molecules. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with an antibody or T lymphocyte (T cell). According to the present disclosure, the term "antigen" includes any molecule containing at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that, optionally after processing, preferably induces an immune response specific to the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0461] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. In one embodiment, "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of surface groupings of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents, but not the latter. Epitopes may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked or covered by the specific antigen-binding peptide (in other words, amino acid residues within the footprint of the specific antigen-binding peptide).

[0462] An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably about 5 to about 100, preferably about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids in length; for example, an epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope in the context of the present disclosure is a T cell epitope.

[0463] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0464] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0465] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition affecting an individual's body. Disease is often interpreted as a medical condition associated with specific symptoms and signs. Disease can be caused by factors from external sources, such as infection, or by internal malfunctions, such as autoimmune disease. In humans, "disease" is often used more broadly to refer to a condition that causes pain, disability, distress, social problems, or death in the affected individual, or similar problems for those who come into contact with the individual. In this broader sense, disease can sometimes include damage, impairment, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Because suffering from and living with many illnesses can alter one's outlook on life and personality, illnesses typically affect individuals not only physically but also emotionally.

[0466] The term "therapeutic treatment" relates to any treatment that improves the health and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, may halt or delay the onset of the disease in an individual, may inhibit or delay the onset of the disease in an individual, may reduce the frequency or severity of symptoms in an individual, and / or may reduce recurrence in an individual who currently has or has previously had the disease.

[0467] The term "prophylactic treatment" or "preventive treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein. Similarly, the term "prophylactic method" in the context of disease progression, such as tumor or cancer progression, relates to any method intended to prevent the disease from progressing in an individual.

[0468] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate), or any other non-mammal, including a bird (chicken), fish, or any other animal species that can suffer from or is susceptible to a disease or disorder (e.g., cancer). Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0469] The term "patient" refers to an individual or subject for treatment, particularly an affected individual or subject.

[0470] As used herein, the term "polynucleotide" or "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0471] The nucleic acid may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retrovirus, adenovirus, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0472] In one embodiment of the present disclosure, nucleic acid is expressed in the cell of the subject that is treated to provide encoded peptide or protein.In one embodiment, nucleic acid is transiently expressed in the cell of the subject.Therefore, in one embodiment, nucleic acid is not integrated into the genome of the cell.In one embodiment, nucleic acid is RNA, preferably in vitro transcribed RNA.

[0473] The nucleic acids described herein can be recombinant and / or isolated molecules.

[0474] In this disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to one or both ends of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0475] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which is related to an RNA transcript encoding a peptide or protein. As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0476] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0477] In one embodiment, the RNA described herein can have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridines.

[0478] In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0479] In some embodiments, the modified nucleoside that replaces one or more uridines in the RNA is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0480] In some embodiments, the RNA of the present disclosure includes a 5' cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified with a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide attached to the mRNA via a 5'-5' triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription, in which the 5' cap is co-transcriptionally expressed on the RNA strand, or can be attached to the RNA post-transcriptionally using a capping enzyme.

[0481] In some embodiments, the RNA comprises cap 0, cap 1, or cap 2. According to the present disclosure, the term "cap 0" refers to the structure "m 7GpppN" where N is any nucleoside having an OH moiety at the 2' position. According to the present disclosure, the term "Cap 1" refers to the structure "m 7 GpppNm, where Nm is any nucleoside having an OCH3 moiety at the 2' position. According to the present disclosure, the term "cap 2" refers to the structure "m 7 GpppNmNm" where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0482] In some embodiments, the RNA is m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3’O G(5')ppp(5')G, and m2 7,3’-O Gppp(m1 2’-O ) ApG.

[0483] In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0484] In some embodiments, an RNA according to the present disclosure comprises a 3'-poly(A) sequence. As used herein, the term "poly(A) sequence" or "poly(A tail)" refers to a continuous or intermittent sequence of adenylic acid residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may follow the 3'UTR of an RNA described herein. The poly(A) sequence may be of any length. In some embodiments, the poly(A) sequence comprises or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides, particularly about 110 nucleotides. In some embodiments, the poly(A) sequence consists exclusively of A nucleotides. In some embodiments, the poly(A) sequence consists essentially of A nucleotides but is interrupted by a random sequence of four nucleotides (A, C, G, and U), as disclosed in International Publication No. WO 2016 / 005324 A1, incorporated herein by reference. Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. Poly(A) cassettes present in the coding strand of DNA that consist essentially of dA nucleotides but are interrupted by a random sequence with an equal distribution of the four nucleotides (dA, dC, dG, dT) and a length of, for example, 5-50 nucleotides, exhibit consistent growth of plasmid DNA in Escherichia coli (E. coli) at the DNA level and are still associated with beneficial properties at the RNA level with respect to supporting RNA stability and translation efficiency. In some embodiments, no nucleotides other than A nucleotides flank the poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked or followed by nucleotides other than A at its 3' end.

[0485] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0486] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Similarly, RNA, such as mRNA, encodes a protein if translation of the RNA produces the protein in a cell or other biological system.

[0487] The RNA may be naked or packaged, for example formulated in particles such as protein and / or lipid particles, eg lipid nanoparticles.

[0488] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0489] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0490] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0491] The Fc region may have a lysine at its C-terminus. The source of this lysine is the naturally occurring sequence found in the human from which these Fc regions are derived. During cell culture production of recombinant antibodies, this terminal lysine can be proteolytically cleaved by one or more endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking the C-terminal lysine. For antibody manufacturing purposes, the DNA encoding this terminal lysine can be omitted from the sequence so that the antibody is produced without the lysine. For example, when using antibodies produced in a CHO-based production system, the degree of processing of the terminal lysine is typically high (Dick, L. Wet et al. Biotechnol. Bioeng. 2008;100:1132-1143), so antibodies produced from nucleic acid sequences that do or do not encode the terminal lysine are substantially identical in sequence and function. Therefore, it is understood that proteins such as antibodies according to the present invention can be produced regardless of whether they encode or have a terminal lysine.

[0492] Aspects and Embodiments of the Disclosure Binding agents that bind to EpCAM and CD137 In a first aspect, the disclosure provides a binding agent comprising a first binding region that binds to EpCAM and a second binding region that binds to CD137.

[0493] The binding agents of the present invention can, in principle, be antibodies of any isotype. The choice of isotype is typically guided by the need for a desired Fc-mediated effector function, such as ADCC induction, or an antibody lacking an Fc-mediated effector function (an "inactive" antibody). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant region kappa or lambda may be used. The effector function of the antibodies described herein can be altered by isotype switching, for example, to IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies for various therapeutic applications. In one embodiment, both heavy chains described herein are of the IgG1 isotype, e.g., IgG1, κ. Optionally, the heavy chains may be modified in the hinge and / or CH3 region, as described elsewhere herein.

[0494] Preferably, each antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. Furthermore, preferably, the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0495] In one embodiment, the binding agent is a full-length antibody, such as a full-length IgG1 antibody. For example, in one embodiment, the binding agent, e.g., a bispecific antibody, comprises two half molecules, each comprising an antigen-binding region.

[0496] Many different formats and uses of bispecific antibodies are known in the art and have been reviewed by Kontermann; Drug Discov Today, 2015 Jul; 20(7): 838-47 and; MAbs, 2012 Mar-Apr; 4(2): 182-97. All of these formats are encompassed herein. Bispecific antibodies according to the present disclosure are not limited to a particular bispecific format or method of making them.

[0497] Examples of bispecific antibody molecules that can be used in the present disclosure include: (i) a single antibody with two arms containing different antigen-binding regions; (ii) a single-chain antibody with specificity for two different epitopes, for example, via two scFvs linked in tandem by an extra peptide linker; and (iii) a dual-variable domain antibody (DVD-Ig) in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin). Heidelberg (2010)); (iv) chemically linked bispecific (Fab') fragments; (v) Tandabs, which are fusions of two single-chain diabodies resulting in tetravalent bispecific antibodies with two binding sites for each of the target antigens; (vi) Flexibodies, which are combinations of scFvs and diabodies resulting in multivalent molecules; (vii) so-called "dock-and-lock" molecules based on the "dimerization and docking domain" of protein kinase A, which when applied to Fabs can result in trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which contain, for example, two scFvs fused to either end of a human Fab arm; and (ix) diabodies.

[0498] In one embodiment, the binding agent described herein is a diabody or crossbody. In one embodiment, the binding agent is a bispecific antibody obtained via controlled Fab arm exchange (e.g., as described in WO2011131746 (Genmab)).

[0499] Examples of different classes of binding agents include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains that force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, where each side of the molecule comprises an Fab fragment or a portion of an Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an extra Fab fragment or a portion of an Fab fragment; (iv) Fc fusion molecules, in which a single-chain Fv molecule or a stabilized diabody is fused to a heavy-chain constant domain, an Fc region, or a portion thereof; (v) Fab fusion molecules, in which different Fab fragments are fused together and fused to a heavy-chain constant domain, an Fc region, or a portion thereof; and (vi) ScFv- and diabody-based antibodies and heavy-chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to a heavy-chain constant domain, an Fc region, or a portion thereof.

[0500] Examples of IgG-like molecules with complementary CH3 domain molecules include Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO 2011069104), so-called knob-into-hole molecules (Genentech, WO 9850431), Cross MAb (Roche, WO 2011117329) and electrostatically compatible molecules (Amgen, EP 1870459 and WO 2009089004; Chugai, U.S. Patent Application No. 201000155133; Oncomed, WO 2010129304), LUZ-Y molecules (Genentech, Wranik et al. al. J. Biol. Chem. 2012, 287(52):43331-9, doi:10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG body and PIG body molecules (Pharmabcine, WO 2010134666, WO 2014081202), strand exchange engineered domain body (SEEDbody) molecules (EMD Serono, WO 2007110205), Biclonics molecules (Merus, WO 2013157953), FcΔAdp molecules (Regeneron, WO 201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO 11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO 2012058768), mAb-Fv molecules (Xencor, WO 2011028952), bivalent bispecific antibodies (WO 2009080254) and DuoBody® molecules (Genmab, WO 2011131746).

[0501] Examples of recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig molecules (WO 2009058383), two-in-one antibodies (Genentech; Bostrom, et al. 2009. Science 323, 1610-1614), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO 2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr; 5(2):208-18), common light chain approaches (Crucell / Merus, U.S. Patent No. 7,262,028), κλ Bodies (NovImmune, WO 2012023053), and CovX-body (CovX / Pfizer; Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17, 501-506).

[0502] Examples of IgG fusion molecules include dual variable domain (DVD)-Ig molecules (Abbott, U.S. Pat. No. 7,612,181), dual-domain double-head antibodies (Unilever; Sanofi Aventis, WO 20100226923), IgG-like bispecific molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb molecules (Zymogenetics, WO 2010111625), HERCULES molecules (Biogen Idec, U.S. Pat. App. No. 007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc., China Patent No. 102250246) and TvAb molecules (Roche, WO 2012025525, WO 2012025530).

[0503] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics / BMS, WO 2010111625), dual affinity retargeting technology (Fc-DART) molecules (MacroGenics, WO 2008157379, WO 2010080538), and dual (ScFv)2-Fab molecules (National Research Center for Antibody Medicine-China).

[0504] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677-86), dual-action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323,1610-1614), dock-and-lock (DNL) molecules (ImmunoMedics, WO 2003074569, WO 2005004809), bivalent bispecific molecules (Biotechnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7), and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[0505] Examples of ScFv-based, diabody-based and domain antibodies include bispecific T cell-triggering (BiTE) molecules (Micromet, WO 2005061547), tandem diabody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66), dual affinity retargeting technology (DART) molecules (MacroGenics, WO 2008157379, WO 2010080538), single chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO 2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. al. Immunol Cell Biol. 2010 Aug;88(6):667-75), dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual targeting heavy chain only domain antibodies.

[0506] In one embodiment, a bispecific antibody of the present disclosure comprises a first Fc sequence comprising a first CH3 region and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and wherein the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. Further details regarding these interactions and how they may be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0507] As further described herein, stable bispecific EpCAMxCD137 antibodies can be obtained in high yield using a particular method based on one homodimeric starting EpCAM antibody and one homodimeric starting CD137 antibody containing only a few conservative asymmetric mutations in the CH3 regions, where asymmetric mutations mean that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions.

[0508] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409 in a human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409 in a human IgG1 heavy chain, wherein the first and second CH3 regions are not substituted at the same positions.

[0509] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises sequences of said first and second CH3 regions comprising asymmetric mutations, i.e. mutations at different positions in the two CH3 regions, for example a mutation at position 405 in one of the CH3 regions and a mutation at position 409 in the other CH3 region. In one embodiment, the mutation at position 405 is F405L. In one embodiment, the mutation at position 409 is K409R.

[0510] In one embodiment, the bispecific antibody comprises first and second heavy chains, each of said first and second heavy chains comprising at least a hinge region, a CH2 and a CH3 region, and wherein (i) the amino acid at the position corresponding to F405 of a human IgG1 heavy chain is L in said first heavy chain and the amino acid at the position corresponding to K409 of a human IgG1 heavy chain is R in said second heavy chain, or (ii) the amino acid at the position corresponding to K409 of a human IgG1 heavy chain is R in said first heavy chain and the amino acid at the position corresponding to F405 of a human IgG1 heavy chain is L in said second heavy chain.

[0511] Conventional methods, such as hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649), can be used to prepare the bispecific antibodies described herein. Co-expression in a host cell of two antibodies consisting of different heavy and light chains results in a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated, for example, by affinity chromatography or similar methods.

[0512] Strategies to promote the formation of functional bispecific products upon coexpression of different antibody constructs, such as those described by Lindhofer et al. (1995 J Immunol 155:219), can also be used. Fusion of rat and mouse hybridomas producing different antibodies results in a limited number of heterodimeric proteins due to selective, species-restricted heavy / light chain pairing. Another strategy to promote heterodimer formation over homodimer formation is the "knob-into-hole" strategy, in which protrusions are introduced into corresponding cavities on the first heavy chain polypeptide and in the second heavy chain polypeptide, so that the protrusions can be positioned within the cavity at the interface of these two heavy chains to promote heterodimer formation and prevent homodimer formation. The "knob" is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains. By replacing large amino acid side chains with smaller ones, a compensatory "cavity" of the same or similar size as the protrusion is created at the interface of the second polypeptide (U.S. Pat. No. 5,731,168). European Patent No. 1870459 (Chugai) and International Publication No. 2009089004 (Amgen) describe other strategies for promoting heterodimer formation during co-expression of different antibody domains in host cells. In these methods, one or more residues constituting the CH3-CH3 interface of both CH3 domains are replaced with charged amino acids so that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. International Publication No. 2007110205 (Merck) describes yet another strategy that exploits the differences between the IgA CH3 domain and the IgG CH3 domain to promote heterodimerization.

[0513] Another in vitro method for producing bispecific antibodies is described in WO2008119353 (Genmab), in which bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (exchange of heavy chains and associated light chains) between two monospecific IgG4 or IgG4-like antibodies upon incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences.

[0514] Preferred methods for preparing the bispecific EpCAMxCD137 antibodies of the present disclosure include those described in WO2011131746 and WO2013060867 (Genmab), comprising the steps of: a) providing a first antibody comprising an Fc region, wherein the Fc region comprises a first CH3 region; b) providing a second antibody comprising a second Fc region, wherein said Fc region comprises a second CH3 region, wherein the first antibody is an EpCAM antibody and the second antibody is a CD137 antibody, or vice versa; the sequences of the first and second CH3 regions are different and a heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions; c) incubating the first antibody with the second antibody under reducing conditions; and d) obtaining said bispecific EpCAMxCD137 antibody The method includes:

[0515] Similarly, there is provided a method for producing an antibody according to the present disclosure, comprising: a) culturing a host cell that produces a first antibody comprising an antigen-binding region capable of binding to human EpCAM as defined herein, and purifying said first antibody from the culture; b) culturing a host cell that produces a second antibody comprising an antigen-binding region capable of binding to human CD137 as defined herein, and purifying said second antibody from the culture; c) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines within the hinge region to undergo disulfide bond isomerization; and d) obtaining the bispecific antibody A method is provided which includes:

[0516] In one embodiment of the present disclosure, the first antibody is incubated with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, wherein the heterodimeric interaction between the first and second antibodies in the resulting heterodimeric antibody is such that Fab arm exchange does not occur in 0.5 mM GSH after 24 hours at 37°C.

[0517] Without being bound by theory, in step c), heavy chain disulfide bonds in the hinge region of the parent antibody are reduced, and the resulting cysteines can then form inter-heavy chain disulfide bonds with cysteine ​​residues of another parent antibody molecule (originally with a different specificity). In one embodiment of this method, the reducing conditions in step c) comprise the addition of a reducing agent, e.g., a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises restoring the conditions to non-reducing or less reducing, e.g., by removing the reducing agent, e.g., by desalting.

[0518] As noted above, in one embodiment, the sequences of the first and second CH3 regions of the homodimeric starting antibody are different, and the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions. Further details regarding these interactions and how they may be achieved are provided in WO2011131746 and WO2013060867 (Genmab), the entire contents of which are incorporated herein by reference.

[0519] In particular, stable bispecific EpCAMxCD137 antibodies can be obtained in high yield using the above methods based on two homodimeric starting antibodies that bind to EpCAM and CD137, respectively, and that contain only minor asymmetric mutations in their CH3 regions, meaning that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions.

[0520] In some embodiments, a binding agent according to the present disclosure comprises, in addition to an antigen-binding region, an Fc region consisting of the Fc sequences of two heavy chains.

[0521] The first and second Fc sequences may each be of any isotype, including, but not limited to, IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, each of the first and second Fc sequences is of or derived from the IgG1 isotype, optionally with one or more mutations or modifications. In one embodiment, each of the first and second Fc sequences is of or derived from the IgG4 isotype, optionally with one or more mutations or modifications. In another embodiment, one of the Fc sequences is of the IgG1 isotype and the other is of the IgG4 isotype, or derived from each such isotype, optionally with one or more mutations or modifications.

[0522] In one embodiment, one or both Fc sequences are effector function-deficient. For example, one or more Fc sequences can be of an IgG1 or non-IgG1 isotype, e.g., IgG2, IgG3, or IgG4, mutated to reduce or eliminate their ability to mediate effector functions such as ADCC.

[0523] As used herein, the term "effector function" includes any function mediated by a component of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, including, for example, the killing of diseased cells, such as tumor cells, or the inhibition of tumor dissemination and metastasis. Preferably, the effector function in the context of the present disclosure is a T cell-mediated effector function. Such functions include ADCC, ADCP, or CDC.

[0524] Antibody-dependent cell-mediated cytotoxicity (ADCC) Antibody-dependent cell-mediated cytotoxicity (ADCC) i...

Claims

1. A binder, The binder is an antibody comprising a first binding arm including a first antigen-binding region that binds to EpCAM, and a second binding arm including a second antigen-binding region that binds to CD137, The first coupling arm described above, i) A polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), ii) Polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL) and The second connecting arm, iii) A polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), iv) Polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL) and The first VH includes the first HCDR1, HCDR2, and HCDR3 sequences, the first VL includes the first LCDR1, HCDR2, and HCDR3 sequences, the first HCDR1, HCDR2, and HCDR3 sequences each include the sequences shown in sequence numbers 2, 3, and 4, and the first LCDR1, HCDR2, and HCDR3 sequences each include the sequences shown in sequence numbers 6, 7, and 8, and A binder wherein the second VH comprises second HCDR1, HCDR2, and HCDR3 sequences, the second VL comprises second LCDR1, LCDR2, and HCDR3 sequences, the second HCDR1, HCDR2, and HCDR3 sequences each comprise the sequences shown in SEQ ID NOs: 12, 13, and 14, and the second LCDR1, LCDR2, and HCDR3 sequences each comprise the sequences shown in SEQ ID NOs: 16, 17, and 18.

2. (i) The EpCAM is a human EpCAM, and / or (ii) The CD137 is human CD137, and / or (iii) The binder according to claim 1, wherein the first antigen-binding domain that binds to EpCAM binds to EpCAM expressed on tumor cells.

3. The aforementioned binder is (i) in the form of a full-length antibody, and / or (ii) A bispecific divalent binder, and / or (iii) The binder according to claim 1, which does not include antigen-binding regions that bind to EpCAM other than the first antigen-binding region that binds to EpCAM, and does not include antigen-binding regions that bind to CD137 other than the second antigen-binding region that binds to CD137.

4. (i) The first VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in Sequence ID No. 1, and the first VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in Sequence ID No. 5, and The second VH includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 11, and the second VL includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 15, (ii) The first VH comprises the amino acid sequence shown in SEQ ID NO: 1, and the first VL comprises the amino acid sequence shown in SEQ ID NO: 5, The second VH comprises the amino acid sequence shown in SEQ ID NO: 11, and the second VL comprises the amino acid sequence shown in SEQ ID NO:

15. The binder according to claim 1.

5. (i) Each of the first and second heavy chain steady regions (CH) includes one or more of the steady heavy chain 1 (CH1) region, the hinge region, the steady heavy chain 2 (CH2) region, and the steady heavy chain 3 (CH3) region, or at least the hinge region, the CH2 region, and the CH3 region, and / or (ii) Each of the first and second heavy chain constant regions (CH) includes a CH3 region, and the two CH3 regions include an asymmetric mutation, and / or (iii) (a) The amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (b) The amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in the first heavy chain constant region (CH), and / or The amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in the second heavy chain constant region (CH), and / or (iv) The binder induces Fc-mediated effector function to a lesser extent than another antibody containing the same first and second antigen-binding regions and two heavy chain constant regions (CH) including the human IgG1 hinge, CH2 and CH3 regions, and / or (v) The binder according to claim 1, wherein the first and second heavy chain constant regions (CH) are modified to induce Fc-mediated effector function to a lesser extent than an antibody which is identical except that it includes an unmodified first and second heavy chain constant region (CH).

6. The binder according to claim 1, wherein in at least one of the first and second heavy chain constant regions (CH), one or more amino acids at positions corresponding to L234, L235, D265, N297, P331, and G236 of the human IgG1 heavy chain according to EU numbering are not L, L, D, N, P, and G, respectively.

7. (i) The positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering are F and E in the first and second heavy chains, and / or (ii) The positions corresponding to the positions L234, L235 and D265 of the human IgG1 heavy chain according to EU numbering are F, E and A in the first and / or second heavy chain steady region (HC), and / or the positions corresponding to the positions L234, L235 and G236 of the human IgG1 heavy chain according to EU numbering are F, E and R in the first and / or second heavy chain steady region (HC), and / or (iii) (a) The positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain according to EU numbering are F, E and A in both the first and second heavy chain steady regions, respectively. (b) The positions corresponding to L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R in both the first and second heavy chain constant regions, or (c) The positions corresponding to human IgG1 heavy chain positions L234, L235 and D265 according to EU numbering are F, E and A in one of the first heavy chain steady region and the second heavy chain steady region, respectively, and the positions corresponding to human IgG1 heavy chain positions L234, L235 and G236 according to EU numbering are F, E and R in the other of the first heavy chain steady region and the second heavy chain steady region, and / or (iv) The positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain steady regions are F and E, respectively, (a) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain steady region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain steady region is R, or (b) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain steady region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain steady region is L, and / or (v) The positions corresponding to L234, L235 and D265 of the human IgG1 heavy chain according to EU numbering are F, E and A in the second heavy chain steady region (HC), and the positions corresponding to L234, L235 and G236 of the human IgG1 heavy chain according to EU numbering are F, E and R in the first heavy chain steady region (HC), (a) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain steady region is L, and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain steady region is R, or (b) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain steady region is R, and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain steady region is L, and / or (vi) The positions corresponding to L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in the second heavy chain steady region (HC); the positions corresponding to L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively, in the first heavy chain steady region (HC); the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain steady region is L; and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain steady region is R. The binder according to claim 6.

8. a) The constant region of the first heavy chain includes the amino acid sequence shown in SEQ ID NO: 54, and b) The constant region of the second heavy chain includes the amino acid sequence shown in SEQ ID NO: 52 The binder according to claim 1.

9. The binder is an antibody comprising a first binding arm including a first antigen-binding region that binds to EpCAM, and a second binding arm including a second antigen-binding region that binds to CD137, The first coupling arm described above, i) A polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), ii) Polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL) The first VH includes the first HCDR1, HCDR2, and HCDR3 sequences, the first VL includes the first LCDR1, HCDR2, and HCDR3 sequences, the first HCDR1, HCDR2, and HCDR3 sequences each include the sequences shown in sequence numbers 2, 3, and 4, and the first LCDR1, HCDR2, and HCDR3 sequences each include the sequences shown in sequence numbers 6, 7, and 8, The second connecting arm, iii) A polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), iv) Polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL) The second VH includes the second HCDR1, HCDR2, and HCDR3 sequences, the second VL includes the second LCDR1, LCDR2, and LCDR3 sequences, the second HCDR1, HCDR2, and HCDR3 sequences each include the sequences shown in sequence numbers 12, 13, and 14, and the second LCDR1, LCDR2, and LCDR3 sequences each include the sequences shown in sequence numbers 16, 17, and 18, Here, the positions L234, L235, and G236 of the human IgG1 heavy chain according to EU numbering are F, E, and R in the first CH, respectively, and the positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A in the second CH, and In the first CH, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L, and in the second CH, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R. The binder according to claim 1.

10. i) A first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in Sequence ID No. 9, ii) A first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in Sequence ID No. 10, iii) A second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in Sequence ID No. 19, and iv) A second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence shown in Sequence ID No.

20. The binder according to claim 1, comprising:

11. i) A first heavy chain containing the amino acid sequence shown in Sequence ID No. 9, ii) A first light chain containing the amino acid sequence shown in Sequence ID No. 10, iii) A second heavy chain containing the amino acid sequence shown in Sequence ID No. 19, and iv) A second light chain containing the amino acid sequence shown in SEQ ID NO: 20 The binder according to claim 1, comprising:

12. A polynucleotide or a set of polynucleotides encoding the binder described in claim 1.

13. A pharmaceutical composition comprising the binder described in claim 1 or the polynucleotide described in claim 12.

14. A pharmaceutical composition according to claim 13, for use in pharmaceutical applications or as a pharmaceutical product.

15. A pharmaceutical composition according to claim 13 for treating or preventing cancer in a subject.

16. The pharmaceutical composition according to claim 15, wherein the subject is a human.

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