Methods of treatment using an agent that binds to epcam and cd137 in combination with a pd-1 axis binding antagonist

Abstract

The present disclosure provides use of a binding agent that binds to EpCAM and CD137 in combination with a PD-1 axis binding antagonist in the treatment or prevention of a tumor or cancer, or in the prevention of progression of a tumor or cancer.

Description

Technical Field

[0001] This disclosure relates to binders that bind to EpCAM and CD137, as well as PD-1 axis binding antagonists. Together, they can be used to treat or prevent tumors or cancer. Background Technology

[0002] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that mediates calcium metabolism in the epithelium. 2+ EpCAM exhibits isotype-independent cell adhesion. It is also involved in cell signaling, migration, proliferation, and differentiation. Furthermore, EpCAM possesses oncogenic potential through its ability to upregulate c-Myc, E-FABP, and cyclin A & E. EpCAM can be used as a diagnostic biomarker for various cancers. In addition, EpCAM appears to play a role in tumorigenesis and metastasis in cancer, thus it may also serve as a potential prognostic biomarker and a potential target for immunotherapy strategies.

[0003] CD137 (4-1BB) is a member of the TNFR family and a co-stimulatory 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 activation of the T-cell receptor (TCR) (e.g., on tumor-infiltrating lymphocytes [TIL; Gros et al., J. Clin Invest 2014;124(5):2246-59]). Stimulation by its natural ligand 4-1BBL or agonist antibodies leads to signaling using TRAF-2 and TRAF-1 as adaptors. Early signaling of CD137 involves K63 polyubiquitination, ultimately leading to activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways. Signaling results in increased T-cell co-stimulation, proliferation, cytokine production, maturation, and prolonged CD8+ T-cell survival. Antibodies targeting CD137 have been shown to promote T-cell antitumor control in various preclinical models (Murillo et al., Clin Cancer Res 2008;14(21):6895-906). Antibodies stimulating CD137 can induce T-cell survival and proliferation, thereby enhancing the antitumor immune response. Antibodies stimulating 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).

[0004] PD-1 and its ligands PD-L1 and PD-L2 are inhibitory checkpoint molecules that regulate the immune system and achieve self-tolerance. Furthermore, inhibitory checkpoint molecules are ideal targets for cancer immunotherapy.

[0005] In tumor draining lymph nodes and the tumor microenvironment, 4-1BB is expressed by a subset of CD4+ and CD8+ T cells, characterized by the co-expression of multiple TCR-inducing molecules, including high levels of programmed cell death 1 (PD-1) (Gros et al., J. Clin Invest 2014;124(5):2246-59; Seifert et al., Cancers (Basel) 12; Simoni et al., Nature 557: 575-579). Upregulation of PD-1 on T cells can promote T-cell exhaustion and reduce T-cell activation upon binding to its ligand, programmed cell death 1 ligand 1 (PD-L1) (Yu et al., Eur JPharmacol 881: 173240). PD-L1 expression is frequently upregulated in tumor cells, particularly in inflammatory tumors (Teng et al., Cancer Res 75: 2139-2145). Therefore, tumor cells provide inhibitory signals to activated T cells, through which they can evade T-cell-mediated cytotoxicity. Antibodies that block the PD-1 / PD-L1 inhibitory axis can restore T-cell function (Boussiotis et al., N Engl J Med 375: 1767-1778; Chen et al., Nature 541: 321-330).

[0006] However, despite these advances in the field, there remains a pressing need to improve therapies to prevent tumor progression or treat cancer. Summary of the Invention

[0007] The inventors have unexpectedly discovered that (i) a combination of a binder comprising a first antigen-binding region that binds to EpCAM and a second antigen-binding region that binds to CD137, and (ii) a PD-1 axis binding antagonist (particularly an inhibitor of the PD-1 / PD-L1 axis), enhances the activated antigen-specificity of human CD8 compared to either component of the combination alone. + T cell cytotoxic activity against tumor cells expressing homologous antigens.

[0008] In a first aspect, this disclosure provides a method for treating or preventing a disease or condition in a subject, the method comprising (i) administering a binder to the subject, the binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, and (ii) administering a PD-1 axis binding antagonist to the subject, wherein the administration of the binder is prior to, concurrent with, or after the administration of the PD-1 axis binding antagonist.

[0009] In some implementations, simultaneous administration means administering the binder and the PD-1 axis binding antagonist simultaneously or within minutes or hours, for example, at 5, 10, 15, 20, 30, 45 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 18 hours.

[0010] In some implementations, prior administration means administering the binder at least one day before administering the PD-1 axis binding antagonist, for example, 1, 2, 3, 4, 5, 6, or 7 days prior, or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks prior, or at least 1, 2, 3, 4, 5, or 6 months prior.

[0011] In some implementations, subsequent administration means administering the binder at least one day after administration of the PD-1 axis binding antagonist, for example, 1, 2, 3, 4, 5, 6 or 7 days later, or at least 1, 2, 3, 4, 5, 6, 7 or 8 weeks later, or at least 1, 2, 3, 4, 5 or 6 months later.

[0012] In some embodiments, the PD-1 axis binding antagonist is administered before or after a treatment cycle.

[0013] In some embodiments, the PD-1 axis binding antagonist comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-PD-L2 antibody, or a nucleic acid, such as RNA, encoding the binding antagonist. In some embodiments, for systemic availability, the encoding RNA is targeted to the liver. Hepatocytes can be efficiently transfected and are able to produce large amounts of protein.

[0014] In some embodiments, EpCAM is human EpCAM. In some embodiments, CD137 is human CD137. In some embodiments, human EpCAM comprises the sequence shown in SEQ ID NO: 59. In some embodiments, human CD137 comprises the sequence shown in SEQ ID NO: 62.

[0015] In some implementations, the first antigen-binding region that binds to EpCAM binds to EpCAM expressed on tumor cells.

[0016] In some embodiments, the PD-1 axis binding antagonist may comprise a PD-1 binding antagonist. In some embodiments, the PD-1 axis binding antagonist may comprise an anti-PD-1 antibody. In some embodiments, the PD-1 antibody may be selected from IgG1-PD1, nivolumab, cemiplimab, dostarlimab, or pembrolizumab. In some embodiments, the PD-1 antibody may be IgG1-PD1.

[0017] In some embodiments, the PD-1 axis binding antagonist may comprise a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist may comprise an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody may be selected from atezolizumab, avelumab, or durvalumab.

[0018] In some embodiments, the PD-1 axis binding antagonist may comprise a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist may comprise an anti-PD-L2 antibody. In some embodiments, the anti-PD-L2 antibody may be selected from OT17B10 and PDL2 / 1850.

[0019] In some implementations, the disease or condition involves disordered cell growth, such as cancer, tumor, or solid tumor.

[0020] In some embodiments, the cancer, tumor, or solid tumor may be selected from cholangiocarcinoma, gastric / gastroesophageal junction (GEJ) cancer, melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, urothelial carcinoma, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Merkel cell carcinoma, and mesothelioma. In some embodiments, the disease or condition may be cholangiocarcinoma or gastric / gastroesophageal junction (GEJ) cancer.

[0021] In some implementations, the subjects can be mammal subjects, such as human subjects.

[0022] In some embodiments, the subject has not received prior treatment for the said disease or condition. In some embodiments, the subject has received prior treatment for the said disease or condition. In some embodiments, the subject has not received prior treatment with a checkpoint inhibitor, such as CTLA-4 treatment.

[0023] In some embodiments, the method may further include administering one or more additional therapeutic agents to the subject. Such additional therapeutic agents may comprise one or more chemotherapeutic agents, such as platinum-based compounds (e.g., cisplatin, oxaliplatin, and carboplatin), taxane-based compounds (e.g., paclitaxel and nab-paclitaxel), nucleoside analogs (e.g., 5-fluorouracil and gemcitabine), and combinations thereof (e.g., cisplatin / carboplatin + 5-fluorouracil or nab-paclitaxel + gemcitabine).

[0024] In some embodiments, administering the binder to the subject comprises administering a nucleic acid encoding the binder. In some embodiments, administering the PD(L)-1 axis binding antagonist to the subject comprises administering a nucleic acid encoding the PD(L)-1 axis binding antagonist. In some embodiments, administering both the binder and the PD(L)-1 binding antagonist to the subject comprises administering a nucleic acid encoding both the binder and the PD(L)-1 binding antagonist. In some embodiments, the nucleic acid is RNA.

[0025] In one aspect, this disclosure provides a polynucleotide or a group of polynucleotides encoding a binding agent and / or a PD-1 axis binding antagonist as defined above.

[0026] In one aspect, this disclosure provides a kit comprising, in one or more vials: (i) a binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, or a nucleic acid encoding said binder; (ii) a PD-1 axis binding antagonist, or a nucleic acid encoding said PD-1 axis binding antagonist; and optionally, (iii) one or more therapeutic agents. In some embodiments, the kit also includes instructions for using the kit to treat or prevent a disease or condition in a subject. In some embodiments of the kit, the binder or encoding nucleic acid and the PD-1 binding antagonist or encoding nucleic acid are in different vials.

[0027] In one aspect, this disclosure provides a kit for treating or preventing a disease or condition in a subject, preferably wherein the disease or condition is a tumor, solid tumor, or cancer, wherein the method comprises (i) administering a binding agent to the subject, and (ii) administering a PD-1 binding antagonist to the subject, wherein the administration of the binding agent is prior to, concurrent with, or after the administration of the PD-1 binding antagonist.

[0028] In one aspect, this disclosure provides a kit for reducing or preventing the progression of a tumor, solid tumor, or cancer in a subject, wherein the method includes (i) administering a binding agent to the subject and (ii) administering a PD-1 binding antagonist to the subject, wherein the administration of the binding agent is prior to, concurrent with, or after the administration of the PD-1 binding antagonist.

[0029] Various embodiments relating to the treatment methods of this disclosure are equally applicable to aspects relating to the kits of this disclosure and their uses.

[0030] One aspect of this disclosure provides a binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, for treating or preventing a disease or condition in a subject, the method comprising co-administering the binder to the subject with a PD-1 axis binding antagonist, wherein the administration of the binder is prior to, concurrent with, or after the administration of the PD-1 axis binding antagonist.

[0031] One aspect of this disclosure provides a PD-1 axis binding antagonist for treating or preventing a disease or condition in a subject, the method comprising co-administering the PD-1 axis binding antagonist to the subject with a binder, the binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, wherein the administration of the PD-1 axis binding antagonist is prior to, concurrent with, or after the administration of the binder.

[0032] One aspect of this disclosure provides a binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, and a PD-1 axis binding antagonist, for treating or preventing a disease or condition in a subject, the method comprising administering the binder and the PD-1 axis binding antagonist to the subject, wherein the administration of the binder is prior to, concurrent with, or after the administration of the PD-1 axis binding antagonist.

[0033] In some implementations, the methods used to treat or prevent a subject's disease or condition are methods used to reduce or prevent the progression of a subject's tumor or to treat the subject's cancer.

[0034] One aspect of this disclosure provides the use of a binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137 in the preparation of a medicament, in combination with a PD-1 axis binding antagonist, for the treatment or prevention of a disease or condition in a subject.

[0035] One aspect of this disclosure provides the use of a PD-1 axis binding antagonist in the preparation, in combination with a binding agent, in a medicament for treating or preventing a disease or condition in a subject, said binding agent comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137.

[0036] One aspect of this disclosure provides (i) a binder comprising a first antigen-binding region that binds to EpCAM and a second antigen-binding region that binds to CD137, and (ii) the use of a PD-1 axis binding antagonist in the preparation of a medicament for treating or preventing a disease or condition in a subject.

[0037] In some implementations, the disease or condition is a tumor, a solid tumor, or cancer.

[0038] The implementation plan related to treatment methods also applies to aspects related to the use of binders and PD-1 axis binding antagonists.

[0039] One aspect of this disclosure provides a medical preparation comprising a binder and a PD-1 axis binding antagonist as defined herein. In one embodiment, the medical preparation is used for any treatment method disclosed herein. Attached Figure Description

[0040] Figure 1 : Relative EpCAM expression on tumor cells.

[0041] Figure 1 The relative EpCAM expression on T84, DiFi, HPAF-II, NCI-N87, Calu-3, NCI-H747, and A549 tumor cells, as determined by flow cytometry, is shown. The data presented are the Δ geometric mean fluorescence intensity (gMFI) for each tumor cell line, calculated as: geometric mean fluorescence intensity (APC) of the EpCAM antibody – geometric mean fluorescence intensity (APC) of the unbound control antibody.

[0042] Figure 2 The binding of bivalent EpCAM antibodies and their monovalent counterparts to DiFi, HPAF-II, or A549 cells.

[0043] The 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 (hollow square) in all experiments. The data shown are mean fluorescence intensity (MFI; R-phycoerythrin [PE]) ± standard deviation (SD) for repeated measurements of a representative experiment. A. Binding of bsIgG1-b12-FEALxEpCAM-A37-FEAR (solid circle) and IgG1-EpCAM-A37-FEAR (hollow triangle) antibodies to DiFi, HPAF-II, or A549 cells. B. Binding of bsIgG1-b12-FEALxEpCAM-C52-FEAR (solid circle) and IgG1-EpCAM-C52-FEAR (hollow triangle) antibodies to DiFi, HPAF-II, or A549 cells. C. Binding of bsIgG1-b12-FEALxEpCAM-UBS-54-FEAR (solid circle) and IgG1-EpCAM-UBS-54-FEAR (hollow triangle) antibodies to DiFi, HPAF-II, or A549 cells. D. Binding of IgG1-EpCAM-323-A3-FEAR (hollow triangle) antibody to DiFi or HPAF-II cells. E. Binding of bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-A37-FEAR and bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, as well as the non-binding control bispecific antibodies bsIgG1-b12-FERL / CD137-HC7LC2-FEAR and bsIgG1-b12-FERL / b12-FEAR to DiFi cells.

[0044] Figure 3 The binding of EpCAM antibody to full-length human, cynomolgus monkey, or mouse EpCAM transfected into CHO-S cells.

[0045] Binding to monovalent and bivalent EpCAM antibodies was analyzed using CHO-S cells transiently transfected with full-length human, cynomolgus monkey, or mouse EpCAM. Binding to untransfected CHO-WT cells was assessed as a negative control. Data are presented as geometric mean fluorescence intensity (gMFI) R-PE values ​​± SD for two technical replicates. A. Binding of IgG1-EpCAM-UBS-54-FEAR, IgG1-EpCAM-A37-FEAR, IgG1-EpCAM-C52-FEAR, and IgG1-EpCAM-343-A3. B. 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.

[0046] Figure 4 In a cell-based reporter assay, the EpCAMx4-1BB bispecific antibody induced 4-1BB-dependent luciferase activity.

[0047] HEK293_NKF_h4-1BB_gfp_luc reporter cells were co-cultured with EpCAM-expressing OV-90-SC12 cells, or cultured alone (medium), overnight in the presence of serial dilutions of BsIgG1-CD137-005-FEAR / EpCAM-323-A3-FEAL. Luciferase activity was quantified by luminescence assay. The fold induction of luciferase activity relative to antibody-free culture (dashed line) is shown. Error bars indicate the SD of replicate wells. Data from a representative experiment of three experiments are shown.

[0048] Figure 5 : Enhanced in vitro proliferation of PBMCs in PBMC-DiFi cell co-culture using EpCAMx4-1BB bispecific antibody.

[0049] DiFi cells expressing EpCAM were co-cultured with CFSE-labeled PBMCs, and the EpCAMx4-1BB bispecific antibody was tested in an in vitro PBMC proliferation assay. Cells were cultured for 96 h with or without the indicated concentrations of EpCAMx4-1BB or EpCAMxb12 bispecific antibody or the unbound control IgG1-b12-FEAL in the presence of anti-CD3 (0.1 µg / mL). The number of CFSE-positive cells was extracted and used to calculate the mitotic index. The data shown are mean mitotic index ± SD from repeated measurements obtained from a representative experiment. A. Mitotic index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-UBS54-FEAR (solid triangle) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-UBS54-FEAR (solid square) antibodies. B. Migration index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-A37-FEAR (solid triangle) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-A37-FEAR (solid square) antibodies. C. Migration index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-C52-FEAR (solid triangle) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-C52-FEAR (solid square) antibodies. D. Migration index of PBMC proliferation induced by bsIgG1-b12-FEAL / EpCAM-323-A3-FEAR (solid triangle) and bsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-323-A3-FEAR (solid square) antibodies.

[0050] Figure 6 : Enhanced in vitro proliferation of CD4+ and CD8+ T- cells in PBMC-DiFi cell co-culture using EpCAMx4-1BB bispecific antibody.

[0051] DiFi cells expressing EpCAM were co-cultured with CFSE-labeled PBMCs, and the EpCAMx4-1BB bispecific antibody was tested in an in vitro T-cell proliferation assay. Cells were cultured for 96 h with or without EpCAMx4-1BB (gray bar) or EpCAMxb12 (striped bar) bispecific antibody (10 μg / mL) or the unbound control IgG1-b12-FEAL (10 μg / mL; empty bar) in the presence of anti-CD3 (0.1 µg / mL). The number of CFSE-positive cells was assessed by flow cytometry as a measure of absolute CD4+ (A) or CD8+ (B) T-cell counts, and the mitotic index was calculated. Data shown are mean mitotic index ± SD of repeated measurements obtained from a representative experiment.

[0052] Figure 7 : Enhanced in vitro proliferation of PBMCs in PBMC-tumor cell cocultures using various tumor cell lines via EpCAMx4-1BB bispecific antibody.

[0053] Tumor cells expressing EpCAM (T84, DiFi, HPAF-II, NCI-N87, Calu-3, or NCI-H747) were co-cultured with CFSE-labeled PBMCs, and the EpCAMx4-1BB bispecific antibody was tested in an in vitro PBMC proliferation assay. Cells were cultured for 96 h in the presence of anti-CD3 (0.1 µg / mL) and indicated concentrations of EpCAMx4-1BB or EpCAMxb12 bispecific antibody or the unbound control IgG1-b12-FEAL. The number of CFSE-positive cells was assessed by flow cytometry as a measure of absolute PBMC count, and the mitotic index was calculated. The data shown are the mean splitting index ± SD of repeated measurements obtained from a representative experiment using (A) PBMC-T84 coculture, (B) PBMC-DiFi coculture, (C) PBMC-HPAF-II coculture, (D) PBMC-NCI-N87 coculture, (E) PBMC-Calu-3 coculture, or (F) PBMC-NCI-H747 coculture.

[0054] Figure 8 The in vitro proliferation of human CD4+ and CD8+ T-cells was enhanced in DiFi tumor cell co-culture using an EpCAMx4-1BB bispecific antibody containing an inert Fc-domain.

[0055] EpCAMx4-1BB bispecific antibody was tested in an in vitro PBMC proliferation assay using DiFi tumor cells expressing EpCAM co-cultured with CFSE-labeled human PBMCs. Cells were cultured for 96 h in the presence of anti-CD3 (0.1 µg / mL) and, as indicated, EpCAM-FERLx4-1BB-FEAR (solid rhombus), EpCAM-FEARx4-1BB-FEAL (solid square), b12-FERLx4-1BB-FEAR (left half solid triangle), EpCAM-FERLxb12-FEAR (right half solid triangle) bispecific antibody or the unbound control b12-FERLxb12-FEAR (hollow triangle). The number of CFSE-positive (A) CD4+ and (B) CD8+ T cells was assessed by flow cytometry as a measure of absolute T-cell count, and the mitotic index was calculated. The data shown are a nonlinear 4-parameter variable slope fit of mean mitotic index ± SD obtained from a representative experiment using PBMC-DiFi co-cultures.

[0056] Figure 9 In ex vivo patient-derived tumor specimens, the EpCAMx4-1BB bispecific antibody enhanced the expansion of tumor-infiltrating lymphocytes.

[0057] The tumor tissue removed from a non-small cell lung cancer patient was cut into 1-2 mm pieces. 3 Fragments were cultured for 14 days in the presence of IL-2 (50 U / mL) and 0.2–5 µg / mL BsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-A37-FEAR or BsIgG1-CD137-009-HC7LC2-FEAL / EpCAM-323-A3-FEAR, or for 14 days with IL-2 alone (w / o Ab). IL-2 concentrations were gradually decreased on days 7 and 10, to 33 U / mL and 15 U / mL, respectively. Absolute cell numbers after culture were determined by flow cytometry. Total TILs, NK cells, CD8+, and CD4+ T cell numbers from cultures derived from one exemplary patient are shown in the analysis of two patients. Data from 3–4 individual replicates and the mean of the replicates are shown. Error bars indicate SD.

[0058] Figure 10 Antitumor activity of EpCAMx4-1BB bispecific antibody in hEpCAM mice carrying hEpCAM-overexpressing MC38 tumors.

[0059] Use 5×10 5One MC38 tumor cell line overexpressing human EpCAM protein (MC38_hEpCAM) was inoculated into a human EpCAM protein transgenic mouse (hEpCAM mouse). When the average tumor size reached approximately 30 mm... 3 Treatment began at that time. 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 on days 12, 17, 21, 24, 28, and 31 post-tumor inoculation (vertical dashed line). (A) shows the mean tumor volume for each group, with error bars representing SEM. Growth curves include the tumor volume of the last euthanized mice (last observation transfer method). P<0.01; two-way repeated measures ANOVA. (B) Survival percentage. P<0.05; Log-rank (Mantel-Cox) test.

[0060] Figure 11 Compared with the combination of bivalent EpCAM- and 4-1BB-specific monoclonal antibodies, the EpCAMx4-1BB bispecific antibody enhanced the proliferation of human CD4+ and CD8+ T- cells in PBMC-DiFi tumor cell co-culture.

[0061] DiFi tumor cells expressing EpCAM were co-cultured with CellTrace Violet (CTV)-labeled human PBMCs. In in vitro PBMC proliferation assays, bispecific antibodies bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR were compared with bivalent IgG1-EpCAM-A37-FERL and IgG1-CD137-009-HC7LC2-FEAR (alone or in combination). Cells were cultured for 96 h in the presence of anti-CD3 (0.1 µg / mL; dashed line) and the indicated concentrations of EpCAMx4-1BB, IgG1-EpCAM-A37-FERL, and / or IgG1-CD137-009-HC7LC2-FEAR. CD4+ and CD8+ T cell proliferation was assessed by flow cytometry analysis of CTV-labeled dilutions. Data presented are mean amplification index ± SD of repeated measurements obtained from a representative experiment.

[0062] Figure 12Compared with the combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR, the EpCAMx4-1BB bispecific antibody enhanced the proliferation of human CD4+ and CD8+ T- cells in PBMC-DiFi tumor cell co-culture.

[0063] DiFi tumor cells expressing EpCAM were co-cultured with CTV-labeled human PBMCs. In in vitro PBMC proliferation assays, the bispecific antibodies bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR were compared with combinations of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR. Cells were cultured for 96 h in the presence of anti-CD3 (0.1 µg / mL; dashed line) and either bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR or combinations of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR at the indicated concentrations. CD4+ and CD8+ T cell proliferation was assessed by flow cytometry analysis of CTV-labeled dilutions. The data shown are the mean percentage of dividing cells ± SD obtained from repeated measurements of a representative experiment.

[0064] Figure 13 Compared with the combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR, the EpCAMx4-1BB bispecific antibody enhanced human CD4+ and CD8+ T-cell activation in PBMC-DiFi tumor cell co-culture.

[0065] DiFi tumor cells expressing EpCAM were co-cultured with human PBMCs. In in vitro PBMC proliferation assays, the bispecific antibodies bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR were compared with combinations of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR. Cells were cultured for 96 h in the presence of anti-CD3 (0.1 µg / mL; dashed line) and the indicated concentrations of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR or combinations of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-HC7LC2-FEAR. The percentages of CD4+ and CD8+ T cells expressing CD25 (A) or 4-1BB (B) and the geometric mean fluorescence intensity (FI) in the CD25+ or 4-1BB+ population were determined by flow cytometry. The data shown are the mean ± SD of repeated measurements obtained from a representative experiment.

[0066] Figure 14 Compared with the combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR, the EpCAMx4-1BB bispecific antibody enhanced the proliferation of human CD4+ and CD8+ T- cells in PBMC-DiFi tumor cell co-cultures derived from cancer patients.

[0067] DiFi tumor cells expressing EpCAM were co-cultured with CTV-labeled cancer patient-derived PBMCs. In in vitro proliferation assays, the bispecific antibodies bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR were compared with combinations of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. Cells were cultured for 96 h in the presence of anti-CD3 (0.1 µg / mL; dashed line) and the indicated concentrations of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR or combinations 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 using CTV-labeled dilutions. Data shown are mean percentage of dividing cells ± SD from repeated measurements obtained from one experiment.

[0068] Figure 15 Compared with the combination of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR, the EpCAMx4-1BB bispecific antibody enhanced human CD4+ and CD8+ T-cell activation in PBMC-DiFi tumor cell co-cultures derived from cancer patients.

[0069] DiFi tumor cells expressing EpCAM were co-cultured with PBMCs derived from cancer patients. In in vitro proliferation assays, the bispecific antibodies bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR were compared with combinations of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. Cells were cultured for 96 h in the presence of anti-CD3 (0.1 µg / mL; dashed line) and the indicated concentrations of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR or combinations of bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR. The percentages of CD4+ and CD8+ T cells expressing CD25 (A) and 4-1BB (B) were determined by flow cytometry, along with the geometric mean fluorescence intensity (FI) in either the CD25+ or 4-1BB+ population. Data shown are the mean ± SD of repeated measurements obtained from one experiment.

[0070] Figure 16 EpCAMx4-1BB bispecific antibody enhances CD8 + CD107a and GzmB expression on T cells.

[0071] In the presence of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR and control antibody, PBMC-derived CD8 expressing CLDN6-specific TCRs were... + T cells were co-cultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells for 2 days. CD8+ was analyzed by flow cytometry. + CD107a and GzmB expression on T cells. (A) Representative flow cytometry plot. (BC) Dose-response curves of CD107a (C) and GzmB (D) expression levels. Data shown are the mean and SD of repeated measurements from one of the six donors tested. (D) Normalized CD107a and (E) GzmB expression levels (bsIgG1-b12-FERL / b12-FEAR expression levels for each donor were set to 1). Pooled data from the six donors are shown, evaluated in two independent experiments. Error bars represent SD. P < 0.0001; P < 0.001; P<0.01; P < 0.05; the Friedman test of Dunn's multiple comparison test was used. GMFI = geometric mean fluorescence intensity; GzmB = granzyme B; PBMC = peripheral blood mononuclear cells; SD = standard deviation.

[0072] Figure 17 EpCAMx4-1BB bispecific antibody enhances CD8 + T-cell-mediated cytotoxicity against tumor cells.

[0073] In the presence of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR and control antibody, PBMC-derived CD8 expressing CLDN6-specific TCRs were... + T cells were co-cultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells for 5–6 days. Cell index values ​​were derived from impedance measurements performed at 2–3 h intervals. (A) Cell index curves from a representative donor are shown. The symbols represent the mean cell index values ​​of replicate wells. For better visibility, data are shown at every other impedance measurement (i.e., every 4–6 h). (B) AUC analysis was performed using cell index data throughout the co-culture process. AUC for each treatment condition was normalized to cultures treated with bsIgG1-ctrl-b12-FERL / b12-FEAR from the same donor. Summary data from all six donors are shown, evaluated in two independent experiments. Error bars represent SD. P < 0.001; P<0.01; P < 0.05; the Friedman test of Dunn's multiple comparison test was used. AUC = area under the curve; PBMC = peripheral blood mononuclear cells; SD = standard deviation.

[0074] Figure 18 The binding of EpCAMx4-1BB bispecific antibodies with different Fc-inert mutations (FER / FEA, FEA / FEA, FER / FER) to FcγR.

[0075] The binding of EpCAMx4-1BB bispecific antibodies with various combinations of Fc-inert mutations (FER / FEA, FEA / FEA, FER / FER) to immobilized human recombinant FcγR protein was analyzed by SPR, as well as the binding of monoclonal antibodies IgG1-EpCAM-A37-FERL and IgG1-CD137-009-HC7LC2-FEAR to immobilized human recombinant FcγR protein: FcγRIa, FcγRIIa-H131, FcγRIIa-R131, FcγRIIb, FcγRIIIa-F158, and FcγRIIIa-V158. Antibody IgG1-b12 (wild-type Fc) was included as a positive control for FcγR binding. Relative binding responses in RU (units of response) were measured by setting the sensor map to 0 RU at the time of SPR analyte injection. Each sample analyzed on the active surface was also analyzed on a parallel reference surface used for background correction. The data shown is from a single measurement in one experiment.

[0076] Figure 19 In PBMC-EpCAM+ tumor cell co-culture, the combination of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR and pembrolizumab induced IFNγ secretion.

[0077] Healthy donor PBMCs were co-cultured with EpCAM (EpCAM+)-expressing DiFi or CAL27 tumor cells (16:1 ratio) in the presence of anti-CD3 antibody (0.1 μg / mL) and treated for 96 h with bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, 1 μg / mL pembrolizumab (alone or in combination), or control antibodies (bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR [3 μg / mL each] or IgG4 [1 μg / mL]) to determine IFNγ secretion. IFNγ concentration in the supernatant was analyzed by ELISA. The data shown are single IFNγ measurements from representative donors (n=5 [DiFi] or n=4 [CAL27]). The black dashed line represents a co-culture that is anti-CD3 stimulated without antibody treatment.

[0078] Figure 20 In PBMC-EpCAM+ tumor cell co-culture, the combination of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR and IgG1-PD1 induced IFNγ secretion.

[0079] Healthy donor PBMCs were co-cultured with EpCAM (EpCAM+)-expressing DiFi or CAL27 tumor cells (16:1 ratio) in the presence of anti-CD3 antibody (0.1 μg / mL) and treated for 96 h with bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, 1 μg / mL IgG1-PD1 (alone or in combination), or control antibodies (bsIgG1-EpCAM-A37-FERL / b12-FEAR and bsIgG1-b12-FERL / CD137-009-HC7LC2-FEAR [3 μg / mL each] or IgG1-b12-FERR [1 μg / mL]) to determine IFNγ secretion. IFNγ concentration in the supernatant was analyzed by ELISA. The data shown are single IFNγ measurements from representative donors (n=5 [DiFi] or n=4 [CAL27]). The black dashed line represents a co-culture that is anti-CD3 stimulated without antibody treatment.

[0080] Figure 21 Combining EpCAMx4-1BB bispecific antibody with anti-PD-1 antibody enhances the activity of a single agent, leading to increased CD8 activity in isolated human CRC tissue cultures. + Enhanced T cell proliferation.

[0081] Tumor tissue fragments excised from CRC patients were cultured for 2 weeks in the presence of IL-2 (≤50 U / mL) and treated on days 0 and 3 with bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR (0.2 µg / mL), pembrolizumab (0.8 µg / mL), IgG1-PD1 (0.8 µg / mL), or a combination thereof as indicated. CD8+ T-cell counts were determined by flow cytometry at the end of the culture. Data shown are from 3–4 replicates from one of the three patients tested and the median of these replicates. The dashed line represents the median CD8+ T-cell count in the untreated culture.

[0082] Figure 22 Binding the EpCAMx4-1BB bispecific antibody to the anti-PD-(L)1 antibody enhances the activity of a single drug, leading to CD8... + Enhanced expression of CD107a and GzmB on T cells.

[0083] In the presence of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, anti-PD-(L)1 antibody, a combination of both, or a control antibody, PBMC-derived CD8 expressing CLDN6-specific TCRs will be... + T cells were co-cultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells for 2 days. Flow cytometry analysis was used to determine the expression of CD107a and GzmB on CD8 cells. + The percentage of T cells. The data shown are the mean and standard deviation from replicate wells from representative donors (4–11 donors tested). Using PBMCs from different donors, combinations of (A) with nivolumab, pembrolizumab, IgG1-PD1, and atezolizumab were tested in independent experiments, as well as (B) with cimipril and dotalipramab. As previously stated, the concentration of anti-PD-(L)1 antibody used was sufficient to target human CD8+. + The maximum single-drug activity was achieved in T-cell antigen-specific proliferation assays.

[0084] Figure 23 Combining the EpCAMx4-1BB bispecific antibody with the anti-PD-(L)1 antibody enhances the activity of a single drug, leading to CD8... + Enhanced T-cell-mediated tumor cell killing.

[0085] In the presence of bsIgG1-EpCAM-A37-FERL / CD137-009-HC7LC2-FEAR, anti-PD-(L)1 antibody, a combination of both, or a control antibody, PBMC-derived CD8 expressing CLDN6-specific TCRs will be... +T cells were co-cultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells for 5–6 days. Tumor cell killing was assessed by impedance measurements of adherent tumor cell layers at 2– to 3–h intervals. (A) Cell index curves normalized to the co-culture start point (set to 1) are shown in co-cultures from a representative donor and pembrolizumab as an exemplary anti-PD-(L)1 antibody. The symbols represent the mean cell index values ​​of the replicate wells. For better visibility, data are shown every other impedance measurement (i.e., every 4–6 h). (B, C) AUC analysis was performed using normalized cell index data throughout the co-culture process. AUC for each treatment condition was normalized to cultures treated with bsIgG1-b12-FERL / b12-FEAR from the same donor. Using PBMCs from different donors, combinations with nivolumab, pembrolizumab, IgG1-PD1, and atezolizumab were tested in independent experiments (B) and combinations with cimipril and dotalimab (C). The data shown are AUCs for representative donors (4–11 donors tested). As previously mentioned, the concentrations of anti-PD-(L)1 antibodies used were sufficient for human CD8... + The maximum single-drug activity was achieved in T-cell antigen-specific proliferation assays.

[0086] Figure 24 Antitumor activity of EpCAMx4-1BB bispecific antibody in human EpCAM transgenic mice carrying MC38 tumors expressing hEpCAM.

[0087] Human EpCAM transgenic mice (n=11 per group) were fed 5 × 10⁻⁶ mice. 5 MC38_hEpCAM tumor cells were seeded. When the average tumor size reached approximately 75 mm... 3 Treatment began at that time. Mice were treated intraperitoneally with BisIgG2amm-EpCAM-A37-AALT / m4-1BB-3H3-AAKR (1, 5, or 15 mg / kg) or the negative control antibody IgG2amm-b12-AAKR (15 mg / kg) at days 17, 20, 24, 27, 31, and 34 post-tumor inoculation (vertical dashed lines). (A) Tumor growth curves for individual mice are shown. The number of mice with complete tumor regression (CR) is shown. (B) Kaplan-Meier survival analysis. P<0.01; P<0.05; Log-rank (Mantel-Cox) test.

[0088] Figure 25Peripheral immune regulation of EpCAMx4-1BB bispecific antibody in human EpCAM transgenic mice carrying MC38 tumors expressing human EpCAM.

[0089] Human EpCAM transgenic mice were inoculated with MC38_hEpCAM tumor cells, and as follows Figure 24 The following treatments were performed. Peripheral blood was analyzed by flow cytometry 3 days after the third treatment. (A) CD8 cells expressing 4-1BB or PD-1 + The frequency of T cells. (B) Has naive (CD62L) + CD44 - ), central memory (CD62L) + CD44 + ) or effector / memory (CD62L) - CD44 + ) phenotype CD8 + T cell frequency. Mean frequency versus SD is shown. P < 0.001; P<0.01; P < 0.05; the Kruskal-Wallis test of Dunn's multiple comparison test was used.

[0090] Figure 26 The combination of EpCAMx4-1BB bispecific antibody and anti-PD-1 antibody demonstrated antitumor activity in human EpCAM transgenic mice carrying MC38_hEpCAM tumors.

[0091] Human EpCAM transgenic mice were used with 5 × 10 5 MC38_hEpCAM tumor cells were inoculated. Mice were randomly divided into treatment groups (n=11 per group), and the tumors were divided into treatment groups when the average size reached 30 mm. 3 Treatment began at [time]. Mice were treated intraperitoneally on days 14, 17, 21, 24, 28, and 31 post-tumor inoculation with BisIgG2amm-EpCAM-A37-AALT / m4-1BB-3H3-AAKR (1 mg / kg), IgG2amm-mPD1-RMP114-AALT (10 mg / kg), a combination of both, or the negative control antibody BisIgG2amm-b12-AALT / b12-AAKR (11 mg / kg) (vertical dashed line). (A) Tumor growth curves for individual mice are shown. The number of mice with complete tumor regression (CR) is shown. (B) Kaplan-Meier survival analysis. P < 0.0001; Log-rank (Mantel-Cox) test.

[0092] Figure 27 Tumor immunomodulation following treatment of human EpCAM transgenic mice carrying MC38 tumors expressing hEpCAM with a combination of EpCAMx4-1BB bispecific anti- and anti-PD-1 antibodies.

[0093] Human EpCAM transgenic mice (n=5 per group) were inoculated with MC38_hEpCAM tumor cells, and as follows Figure 26 The following treatments were performed. Two days after the second treatment, the tumor was removed and analyzed by flow cytometry. (A) Relative number of immune cell populations (i.e., the ratio of immune cells to tumor cells). Mean frequency and SD are shown. (B) Frequency of T cells. P<0.05; Kruskal-Wallis test with Dunn's multiple comparison test was used. (C) Simple linear regression of T cell frequency and tumor volume.

[0094] Figure 28 The combination of EpCAMx4-1BB bispecific antibody and anti-PD-1 antibody demonstrated antitumor activity in human EpCAM transgenic mice carrying B16F10_hEpCAM tumors.

[0095] Human EpCAM transgenic mice were used with 3 × 10 5 1 B16F10_hEpCAM tumor cell line was seeded. When the average tumor size reached approximately 12 mm... 3 Treatment began at that time. Mice were treated intraperitoneally on days 12, 15, 18, 22, 25, and 29 post-tumor inoculation with BisIgG2amm-EpCAM-A37-AALT / m4-1BB-3H3-AAKR (0.4, 1.8, or 9 mg / kg) in combination with IgG2amm-mPD1-RMP114-AALT (10 mg / kg) or the negative control antibody IgG2amm-b12-AAKR (15 mg / kg) (vertical dashed line). (A) Tumor growth curves for individual mice are shown. The number of mice with complete tumor regression (CR) is shown. (B) Group mean tumor volume over time. (LOCF applied until three mice in a group were still alive). P < 0.001, a two-way ANOVA (mixed effects) analysis using Dunnett's multiple comparison test was performed. (C) Kaplan-Meier survival analysis. P < 0.001; P<0.05; Log-rank (Mantel-Cox) test.

[0096] Figure 29 Pharmacodynamic activity of the combination of EpCAMx4-1BB bispecific antibody and anti-PD-1 antibody in human EpCAM transgenic mice carrying B16F10_hEpCAM tumors.

[0097] Human EpCAM transgenic mice were inoculated with B16F10_hEpCAM tumor cells, and as follows Figure 28 The following treatment was performed. Two days after the second treatment, peripheral blood was collected and the tumor was excised for flow cytometry analysis. (A) Peripheral blood CD8 expressing PD-1 or Ki-67 + and CD4 + (B) T cell frequency. + The frequency of T cells and CD8 cells expressing PD-1 or granzyme B (GzmB) + The frequency of T cells. P<0.01; P < 0.05, so the Kruskal-Wallis test of Dunn's multiple comparison test was used.

[0098] Figure 30 In human EpCAM transgenic mice carrying B16F10_hEpCAM tumors, the combination of EpCAMx4-1BB bispecific antibody and anti-PD-1 antibody demonstrated antitumor activity compared to single treatment.

[0099] Human EpCAM transgenic mice were used with 3 × 10 5 One B16F10_hEpCAM tumor cell was seeded. When the average tumor size reached approximately 15 mm... 3 Treatment began at that time. Mice were treated intraperitoneally on days 10, 13, 17, 20, 24, and 27 post-tumor inoculation with BisIgG2amm-EpCAM-A37-AALT / m4-1BB-3H3-AAKR (9 mg / kg), IgG2amm-mPD1-RMP114-AALT (10 mg / kg), a combination of both, or the negative control antibody BisIgG2amm-b12-AALT / b12-AAKR (19 mg / kg) (vertical dashed line). (A) Tumor growth curves for individual mice are shown. The number of mice with complete tumor regression (CR) is shown. (B) Group mean tumor volume over time. (LOCF applied until three mice in a group were still alive). P<0.01; P < 0.05, a two-way ANOVA (mixed effects) analysis using Dunnett's multiple comparison test was performed. (C) Kaplan-Meier survival analysis. P<0.001; P<0.01; Log-rank (Mantel-Cox) test.

[0100] Figure 31 The pharmacodynamic activity of the combination of EpCAMx4-1BB bispecific antibody and anti-PD-1 antibody compared with monotherapy in human EpCAM transgenic mice carrying B16F10_hEpCAM tumors.

[0101] Human EpCAM transgenic mice were inoculated with B16F10_hEpCAM tumor cells, and as follows Figure 30 The following treatments were performed. Peripheral blood was collected one day after the second treatment and one day after the fourth treatment for flow cytometry analysis. (A) CD8 cells expressing Lag-3 or Ki67 + The frequency of T cells, or (B) co-expression of two markers. (C) Having naive (CD62L) + CD44 - ), central memory (CD62L) + CD44 + ) or effector / memory (CD62L) - CD44 + ) phenotype CD8 + Frequency of T cells. (D) CD4 cells expressing Lag-3 or Ki67. + Frequency of T cells. Group mean versus SD is shown. P < 0.0001; P < 0.001; P<0.01; P < 0.05, so the Kruskal-Wallis test of Dunn's multiple comparison test was used.

[0102] Sequence Description

[0103] Table 1 – Sequence Detailed Implementation

[0104] Although this disclosure is described in more detail below, it should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0105] The elements of this disclosure will be described in more detail below. These elements are listed with specific embodiments; however, it should be understood that they can be combined in any way and in any number to produce other embodiments. The examples and preferred embodiments described differently should not be construed as limiting this disclosure to the explicitly described embodiments. This specification should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as disclosed by the specification of this application.

[0106] Preferably, the terms used herein are as defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0107] Unless otherwise stated, the practice of this disclosure will employ conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques as explained in the literature in the field (see, for example, Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / 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; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J.). Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0108] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise claimed, the use of any and all instances or exemplary language (e.g., “such”) provided herein is for the purpose of better illustrating this disclosure and does not constitute a limitation on the scope of this disclosure. No language in this specification should be construed as illustrating any unclaimed element essential to the practice of this disclosure.

[0109] The list of ranges of values ​​in this document is provided solely as a shorthand method for individually referring to each distinct value falling within the range. Unless otherwise stated herein, each individual value is incorporated into this specification as it is individually stated herein.

[0110] Several documents are referenced throughout the text of this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.), whether mentioned above or below, is incorporated herein in its entirety. Nothing herein should be construed as an admission that the invention did not precede these disclosures by virtue of prior invention.

[0111] definition

[0112] Definitions applicable to all aspects of this disclosure are provided below. Unless otherwise stated, the following terms have the following meanings. Any undefined term has its generally accepted meaning.

[0113] Throughout the specification and appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to mean including the stated members, integers, or steps, or groups of members, integers, or steps, but not excluding any other members, integers, or steps, or groups of members, integers, or steps. The term “substantially constitutes…” excludes any other members, integers, or steps of any substantial significance. The term “comprise” encompasses the term “substantially constitutes…”, which in turn encompasses the term “consistent with…”. Therefore, whenever the term “comprises” appears in this application, it may be replaced by the term “substantially constitutes…” or “consistent with…”. Similarly, whenever the term “substantially constitutes…” appears in this application, it may be replaced by the term “consistent with…”.

[0114] The terms “a,” “an,” and “this,” as well as similar designations, used in the context of describing this disclosure (particularly in the context of the claims) should be understood to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context.

[0115] When used herein, “and / or” is considered as a specific disclosure of each of the two specified features or components, including or excluding the other. For example, “X and / or Y” is considered as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were listed separately herein.

[0116] In the context of this disclosure, the term "about" refers to a range of accuracy, which, as will be understood by those skilled in the art, still ensures 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%, and, for example, ±0.01%. As will be understood by those skilled in the art, such specific deviation from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects can generally have a larger such deviation than man-made or engineered effects.

[0117] In the context of this disclosure, the term "binding agent" refers to any agent capable of binding to a desired antigen. In some embodiments of this disclosure, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also comprise synthetic, modified, or non-naturally occurring portions, particularly non-peptide portions. For example, such portions may be linked to a desired antigen-binding function or region such as an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antibody-binding CDR or variable region.

[0118] The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably antigen receptors such as antibodies or B cell receptors (BCRs). Immunoglobulins are characterized by structural domains that form the characteristic immunoglobulin (Ig) fold, known as immunoglobulin domains. The term encompasses both membrane-bound immunoglobulins and soluble immunoglobulins. Membrane-bound immunoglobulins, also called surface immunoglobulins or membrane immunoglobulins, are typically part of the BCR. Soluble immunoglobulins are generally referred to as antibodies.

[0119] The structures of immunoglobulins have been well characterized. See, for example, Fundamental Immunology Ch.7 (Paul, W., ed., 2). nd ed. Raven Press, NY (1989). Simply put, immunoglobulins generally consist of several chains, typically two identical heavy chains and two identical light chains linked by disulfide bonds. These chains primarily contain immunoglobulin domains or regions, such as V... L Or VL (variable light chain) structural domain / region, C L Or CL (constant light chain) structural domain / region, V H Or VH (variable heavy chain) structural domains / regions and C H Or CH (constant heavy chain) domain / region C H 1 (CH1), C H 2(CH2), C H3 (CH3) and C H 4 (CH4). The heavy chain constant region typically contains three domains: CH1, CH2, and CH3. The hinge region is the area between the CH1 and CH2 domains of the heavy chain and is highly flexible. The disulfide bonds in the hinge region are part of the interaction between the two heavy chains in the IgG molecule. Each light chain typically contains a VH and a CL. The light chain constant region typically contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions, which may be hypervariable in the sequence and / or form of the structure-defined ring), also called complementarity-determining regions (CDRs), which are scattered among more conserved regions called framework regions (FRs). Each VH and VL typically contains 3 CDRs and 4 FRs, arranged from the amino-terminus to the carboxyl-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 stated or contradicted by the context, the CDR sequences described herein are identified using DomainGapAlign according to the IMGT rules (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also the Internet http address "www.imgt.org". However, it should be understood that this disclosure is not limited to CDR sequences identified solely according to the IMGT rules. Therefore, HCDR1, HCDR2, and HCDR3 sequences of the heavy chain variable region (VH), e.g., SEQ ID NO:1 or SEQ ID NO:11, or LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region (VL), e.g., SEQ ID NO:5 or SEQ ID NO:11, are also included. 15. This should cover those CDR sequences determined by any method used to determine CDR sequences (e.g., according to the IMGT rule or the Kabat rule). Furthermore, it should also include overlapping sequences representing CDR sequences determined by different methods used to determine CDR sequences (e.g., according to the IMGT rule and the Kabat rule).

[0120] The following table shows the results of determining the CDR sequences associated with SEQ ID NO: 1 and SEQ ID NO: 5 or SEQ ID NO: 11 and SEQ ID NO: 15 according to the IMGT rule and the Kabat rule, and also shows the overlap of the determined CDR sequences.

[0121] The following table shows the results of determining the CDR sequences associated with SEQ ID NO: 21 and SEQ ID NO: 22 according to the IMGT rule and the Kabat rule, and also shows the overlap of the determined CDR sequences.

[0122] The following shows the results of determining the CDR sequences associated with SEQ ID NO: 25 and SEQ ID NO: 26 according to the IMGT rule and the Kabat rule, and also shows the overlap of the determined CDR sequences.

[0123] The following table shows the results of determining the CDR sequences associated with SEQ ID NO: 27 and SEQ ID NO: 28 according to the IMGT rule and the Kabat rule, and also shows the overlap of the determined CDR sequences.

[0124] Unless otherwise stated or contradicted by the context, the positions of amino acids in the constant region in this disclosure are based on EU numbers (Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(1):78-85; Kabatet al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0125] There are five types of mammalian immunoglobulin heavy chains: α, δ, ε, γ, and µ, which constitute 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 transmembrane domains and short cytoplasmic domains at their C-termini. There are two types of light chains in mammals: λ and κ. Immunoglobulin chains contain variable and constant regions. The constant region is largely conserved across different isotypes of immunoglobulins, while the variable portion is highly differentiated and responsible for antigen recognition.

[0126] The terms "amino acid" and "amino acid residue" are used interchangeably herein and should not be construed as limiting. An amino acid is an organic compound containing amine (-NH₂) and carboxyl (-COOH) functional groups, as well as a specific side chain (R group) for each amino acid. In the context of this disclosure, amino acids can be classified based on structural and chemical characteristics. Therefore, the categories of amino acids may be reflected in one or both of the following tables:

[0127] Table 2: Major classifications based on the structure and general chemical characteristics of the R group

[0128] Table 3: Optional physical and functional classifications of amino acid residues

[0129] For the 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, splicing variants, post-translational modification variants, conformations, isotypes, allele variants, species variants, and species homologs, particularly those that are naturally occurring. In particular, the term “variant” includes fragments of amino acid sequences.

[0130] Amino acid insertion variants involve the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertions are also possible with appropriate screening of the resulting product.

[0131] Amino acid addition variants contain amino- and / or carboxyl-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

[0132] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can occur at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of a protein are also called N-terminal and / or C-terminal truncated variants.

[0133] Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue at its position. The substitution of one amino acid for another can be classified as conserved or non-conserved substitution. Modification at non-conserved amino acid sequence positions between homologous proteins or peptides and / or substitution of amino acids with other amino acids having similar properties are preferred. Preferably, amino acid changes in peptide and protein variants are conserved amino acid changes, i.e., substitutions of amino acids with similar or no charge. Conservative amino acid changes involve the substitution of one of the amino acid families associated with its side chain. In the context of this disclosure, "conservative substitution" means replacing one amino acid with another amino acid having similar structure and / or chemical characteristics, thus replacing another amino acid residue of the same category as defined in either of the two tables above with one amino acid residue: for example, isoleucine can be used to replace leucine because they are both aliphatic, branched, hydrophobic compounds. Similarly, glutamic acid can be used to replace aspartic acid because they are both small, negatively charged residues. Naturally occurring amino acids can 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 collectively classified as aromatic amino acids. In one embodiment, conserved amino acid substitutions include substitutions within the following group: - Glycine, alanine; - Valine, Isoleucine, Leucine; - Aspartic acid, glutamic acid; - Asparagine, glutamine; - Serine, threonine; - Lysine, arginine; and - Phenylalanine, tyrosine.

[0134] As used herein, the term "amino acid corresponding to position..." and similar expressions refer to amino acid position numbers in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by comparison with human IgG1. Therefore, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence is an amino acid or segment that, using standard sequence alignment programs such as ALIGN, ClustalW, or similar programs, is typically aligned with another amino acid or segment by default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. How to align sequences or segments within sequences to determine positions in the sequence corresponding to amino acid positions according to this disclosure is well known in the art.

[0135] In the context of this disclosure, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind to an antigen (particularly an epitope on the antigen), typically under physiological conditions, preferably having a significant half-life, 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, etc., or any other relevant functionally defined time (such as the time sufficient to induce, promote, enhance, and / or regulate an antibody-related physiological response to an antigen and / or the time sufficient to recruit the antibody to effector activity). Specifically, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected 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 comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The variable region and constant region are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains 3 CDRs and 4 FRs, arranged in the following order from the amino-terminus to the carboxyl-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 binding domains that interact with the antigen. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino-terminus to the carboxyl-terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins 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. Antibodies can be complete immunoglobulins derived from natural or recombinant sources and can be the immunologically active portion of a complete immunoglobulin. Antibodies are typically tetramers 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.

[0136] The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The terms "binding region" and "antigen-binding region" are used interchangeably herein, referring to the region that interacts with the antigen and contains both VH and VL regions. Antibodies, as used herein, include not only monospecific antibodies but also multispecific antibodies, which contain multiple, such as two or more, for example, three or more distinct antigen-binding regions.

[0137] As stated above, unless otherwise specified or clearly contradicted by the context, the term "antibody" as used herein includes fragments of antibodies that are antigen-binding fragments, i.e., retain the ability to bind specifically to an antigen. The antigen-binding function of antibodies has been shown to be achieved by fragments of full-length antibodies. Examples of antigen-binding fragments covered by the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of 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 disulfide bonds in the hinge region; (iii) Fd fragments, consisting essentially of VH and CH1 domains; (iv) Fv fragments, consisting essentially of the VL and VH domains of a single arm of the antibody; and (v) dAb fragments (Ward et al.). . Nature 341 , 544-546 (1989)), which is basically composed of VH domains, and is also called a domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90); (vi) Camel or nanobody molecules (Revets et al; Expert Opinion Biol Ther. 2005 Jan; 5 (1):111-24); and (vii) the separated complementarity-determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via synthetic linkers using recombination methods, allowing them to form a single protein chain where the VL and VH regions pair to form a monovalent molecule (called 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)). Unless otherwise stated or the context clearly indicates, such single-chain antibodies are encompassed within the term antibody. While such fragments are generally included in the meaning of antibody, they collectively and independently constitute a unique feature of this disclosure, exhibiting distinct biological properties and utilities. In the context of this disclosure, these and other useful antibody fragments, as well as bispecific forms of such fragments, are further discussed herein. It should also be understood that, unless otherwise stated, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like peptides such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to bind specifically to antigens, provided by any known technique such as enzymatic digestion, peptide synthesis, and recombinant techniques.

[0138] The resulting antibodies can be of any isotype. As used herein, the term "isotype" refers to a class of immunoglobulins (e.g., IgG (such as IgG1, IgG2, IgG3, IgG4), IgD, IgA (such as IgA1, IgA2), IgE, IgM, or IgY) encoded by the heavy chain constant region gene. When a specific isotype such as IgG1 is mentioned herein, the term is not limited to a specific isotype sequence, such as a specific IgG1 sequence, but is used to indicate that the antibody is sequence-closer to that isotype, such as IgG1, compared to other isotypes. Thus, for example, the IgG1 antibody of this disclosure can be a sequence variant of a naturally occurring IgG1 antibody, including variations in the constant region.

[0139] IgG1 antibodies can exist in a variety of polymorphic variants known as allotypes (reviewed in Jefferis and Lefranc 2009). mAbs Vol 1 Issue 4 1-7), any of which are applicable to some embodiments described herein. Common allotypes in the population are those named with the letters a, f, n, z, or combinations thereof. In any embodiment described herein, the antibody may comprise a heavy chain Fc region containing a human IgG Fc region. In other embodiments, the human IgG Fc region comprises human IgG1.

[0140] In the context of this disclosure, the term "multispecific antibody" refers to an antibody having at least two distinct antigen-binding regions defined by different antibody sequences. In some embodiments, the distinct antigen-binding regions bind to different epitopes on the same antigen. However, in a preferred embodiment, the distinct antigen-binding regions bind to different target antigens. In one embodiment, a multispecific antibody is a "bispecific antibody" or "bs". Multispecific antibodies, such as bispecific antibodies, can be in any form, including any bispecific or multispecific antibody forms described below.

[0141] For bispecific antibodies, the prefixes BisG1 and bsIgG1 can be used interchangeably in this article.

[0142] When used in the context of antibodies, the term "full-length" means that the antibody is not a fragment, but contains all the domains of a particular isotype that are commonly found in nature, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0143] As used herein, the term "human antibody" is intended to include antibodies having variable and frame regions derived from human germline immunoglobulin sequences, as well as constant domains of human immunoglobulins. 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 mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another non-human species, such as a mouse, has been grafted onto a human frame sequence.

[0144] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., rodents) and the constant region is derived from a different species, such as humans. Chimeric antibodies can be produced through antibody engineering. "Antibody engineering" is a general term used for the modification of various types of antibodies, and the methods of antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies can be genetically or enzymatically engineered recombinant antibodies. The production of 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 have been developed for therapeutic applications in humans to reduce the intended antibody immunogenicity of non-human antibodies (e.g., rodent antibodies). They typically contain a non-human (e.g., mouse or rabbit) variable region specific to the antigen of interest, as well as human constant antibody heavy and light chain domains. In the context of chimeric antibodies, the term "variable region" or "variable domain" refers to the region containing the CDR and framework regions of the immunoglobulin heavy and light chains, as described below.

[0145] As used herein, the term "humanized antibody" refers to a genetically engineered nonhuman antibody containing a human antibody constant domain and a nonhuman variable domain, wherein the nonhuman variable domain is modified to have a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six nonhuman antibody complementarity-determining regions (CDRs) that co-form the antibody binding site onto a homologous human receptor frame region (FR) (see WO 92 / 22653 and EP 0 629 240). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the frame residues from the parent antibody (i.e., the nonhuman antibody) with human frame regions (reversion mutations). Structural homology modeling may help identify amino acid residues in the frame regions that are important for the antibody's binding properties. Therefore, humanized antibodies may contain nonhuman CDR sequences, primarily human frame regions optionally containing one or more amino acid reversion mutations to nonhuman amino acid sequences, and fully human constant regions. Optionally, additional amino acid modifications (not necessarily reversion mutations) can be used to obtain humanized antibodies with preferred characteristics, such as affinity and biochemical properties.

[0146] As used herein, a protein “derived from” another protein (e.g., a parental protein) means that one or more amino acid sequences of that protein are identical or similar to one or more amino acid sequences of the other or parental protein. For example, in an antibody, binding arm, antigen-binding region, or constant region derived from another or parental antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to the amino acid sequences of the other or parental 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 the VH and VL CDRs and / or one or more or all of the frame regions, VH, VL, CL, hinge, or CH regions. For example, a humanized antibody may be described herein as “derived from” a non-human parental antibody, meaning that at least the VL and VH CDR sequences are identical or similar to the VH and VL CDR sequences of the non-human parental antibody. A chimeric antibody may be described herein as “derived from” a non-human parental antibody, meaning that typically the VH and VL sequences may be identical or similar to the VH and VL sequences of the non-human parental antibody. Another example is a binding arm or antigen-binding region, which may be described herein as “derived” from a specific parent antibody. This means that the binding arm or antigen-binding region typically contains the same or similar VH and / or VL CDR, or VH and / or VL sequences as 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 to the CDR, constant region, or other parts of the antibody, binding arm, antigen-binding region, etc., to introduce desired characteristics. When used in the context of one or more sequences derived from a first or parent protein, the “similar” amino acid sequence preferably has at least about 50%, such as 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.

[0147] Non-human antibodies can be produced in many different species, such as mice, rabbits, chickens, guinea pigs, llama, and goats.

[0148] Monoclonal antibodies can be prepared using a variety of techniques, including conventional monoclonal antibody methods such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies can be employed, such as viral or oncogenic transformation of B-lymphocytes or phage display using antibody gene libraries, and such methods are well known to those skilled in the art.

[0149] Hybridoma generation in such non-human species is a well-established procedure. Immunization protocols and techniques for isolating spleen cells from immunized animals / non-human species for fusion are known in the art. Fusion pairs (e.g., mouse myeloma cells) and fusion methods are also known.

[0150] When used herein, unless contradicted by the context, the term “Fab-arm” or “arm” refers to a heavy-light chain pair and is used interchangeably with “half-molecule”.

[0151] The term "binding arm containing an antigen-binding region" refers to an antibody molecule or fragment containing an antigen-binding region. Therefore, a binding arm can contain, for example, six VH and VL CDR sequences, VH and VL sequences, Fab or Fab' fragments, or a Fab-arm.

[0152] As used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of two Fc sequences of the immunoglobulin heavy chain, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, as used herein, the term "Fc region" refers to a region comprising at least a hinge region, a CH2 region, and a CH3 region in a direction from the N-terminus to the C-terminus of the antibody. The Fc region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0153] In the context of this disclosure, the term “lower degree of induction of Fc-mediated effector function” used in relation to antibodies (including multispecific antibodies) means that an antibody induces Fc-mediated effector function to a lower degree than a human IgG1 antibody comprising (i) the same CDR sequence as the antibody, particularly comprising the same first antigen-binding region and second antigen-binding region, and (ii) two heavy chains comprising the hinge region, CH2 region and CH3 region of human IgG1, such function being particularly selected from the list of IgG Fc receptor (FcgammaR, FcγR) binding, C1q binding, ADCC or CDC.

[0154] Fc-mediated effector functions can be measured by binding to FcγR, binding to C1q, or by FcγR-induced Fc-mediated crosslinking.

[0155] As used herein, the term "hinge region" refers to the hinge region of the immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to EU numbers as shown in Kabat (Kabat, EA 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 can also be any other subtype as described herein.

[0156] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of the immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to EU numbers as shown in Kabat (ibid.). However, the CH1 region can also be any other subtype as described herein.

[0157] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of the immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbers as shown in Kabat (ibid.). However, the CH2 region can also be any other subtype as described herein.

[0158] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of the immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbers as shown in Kabat (ibid.). However, the CH3 region can also be any other subtype as described herein.

[0159] In the context of this disclosure, the term "monovalent antibody" means that an antibody molecule is able to bind to a single antigen molecule and therefore cannot undergo antigen cross-linking.

[0160] "EpCAM antibody" or "anti-EpCAM antibody" is an antibody as described above that specifically binds to the antigen EpCAM.

[0161] "CD137 antibody" or "anti-CD137 antibody" is an antibody as described above that specifically binds to the antigen CD137.

[0162] "EpCAMxCD137 antibody" or "anti-EpCAMxCD137 antibody" is a bispecific antibody containing two distinct antigen-binding regions, one of which specifically binds to the antigen EpCAM and the other specifically binds to the antigen CD137.

[0163] As used herein, the term "biosimilar" (e.g., a biosimilar of an approved reference product / biologic) refers to a biologic product that is similar to a reference product based on data from: (a) analytical studies demonstrating that the biologic product is highly similar to the reference product, although there are minor differences in clinically inactive components; (b) animal studies (including toxicity assessments); and / or (c) one or more clinical studies (including assessments of immunogenicity and pharmacokinetic or pharmacodynamics) sufficient to demonstrate safety, purity, and potency under one or more appropriate conditions of use where the reference product has been approved and is intended for use and is seeking approval (e.g., there are no clinically significant differences between the biologic product and the reference product in terms of safety, purity, and potency). In some embodiments, the biosimilar biologic product and the reference product employ the same one or more mechanisms of action for one or more conditions of use specified, recommended, or suggested in the proposed label, but only to the extent known to the reference product. In some embodiments, one or more conditions of use specified, recommended, or suggested in the proposed label for the biologic product have previously been approved for use with the reference product. In some implementations, the route of administration, dosage form, and / or potency of the biosimilar are the same as those of the reference product. A biosimilar may be, for example, an antibody known to have the same primary amino acid sequence as a commercially available antibody, but may be prepared in different cell types or by different production, purification, or formulation methods.

[0164] As used herein, in the context of antibody binding to a predetermined antigen or epitope, the terms "binding" or "capable of binding" are generally used in the context of approximately 10 -7 M or smaller K D The combination of affinity, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller, approximately 10 -10 M or smaller, or about 10 -11 Or even smaller, when determined using biolayer interferometry (BLI), or, for example, when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument with the antigen as ligand and the antibody as the analyte. The affinity of an antibody for a predetermined antigen corresponds to a higher Kb than its affinity for nonspecific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. D At least 10 times lower K DFor example, at least 100 times lower, at least 1,000 times lower, at least 10,000 times lower, at least 100,000 times lower. The amount of high affinity depends on the antibody's K. D Therefore, when the antibody's K D When the affinities are very low (i.e., the antibodies are highly specific), the degree to which the affinity for the antigen is lower than that for the affinity for the nonspecific antigen can be at least 10,000 times.

[0165] As used in this article, the term "k" d (sec) -1 This refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also called k. off value.

[0166] As used in this article, the term "K" D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction."

[0167] If two antibodies bind to the same antigen and the same epitope, they have "the same specificity." Whether an antibody to be tested binds to a certain antigen or recognizes the same epitope, i.e., whether the antibody binds to the same epitope, can be tested using various methods known to those skilled in the art.

[0168] Competition between antibodies can be detected through cross-blocking assays. For example, competitive ELISA assays can be used as cross-blocking assays. For instance, target antigens can be coated onto the wells of a microtiter plate, and antigen-binding antibodies and candidate competitive test antibodies can be added. The amount of antigen-binding antibody binding to the antigen in the well is indirectly related to the binding ability of the candidate competitive test antibody that competes for the same epitope. Specifically, the greater the affinity of the candidate competitive test antibody for the same epitope, the less antigen-binding antibody binds to the antigen-coated well. The amount of antigen-binding antibody binding to the well can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0169] An antibody that competes with another antibody (e.g., an antibody containing heavy and light chain variable regions as described herein) for binding to an antigen, or an antibody that is specific to an antigen of another antibody (e.g., an antibody containing heavy and light chain variable regions as described herein), may be an antibody containing variants of the heavy chain and / or light chain variable regions as described herein, such as modifications and / or a degree of similarity in the CDR as described herein.

[0170] As used herein, “isolated multispecific antibodies” refers to multispecific antibodies that are substantially free of other antibodies with different antigen specificities (e.g., isolated bispecific antibodies that specifically bind to EpCAM and CD137 are substantially free of monospecific antibodies that specifically bind to EpCAM or CD137).

[0171] As used herein, the term "monoclonal antibody" refers to an antibody molecule consisting of a single molecule. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0172] When used herein, the term "heterodimeric interaction between the first CH3 region and the second CH3 region" refers to the interaction between the first CH3 region and the second CH3 region in the first CH3 / second-CH3 heterodimeric antibody.

[0173] When used herein, the term “homodimal interaction between the first CH3 region and the second CH3 region” refers to the interaction between the first CH3 region and another first CH3 region in a first-CH3 / first-CH3 homodimeric antibody, and the interaction between the second CH3 region and another second CH3 region in a second-CH3 / second-CH3 homodimeric antibody.

[0174] When used herein, the term "homodimolecular antibody" refers to an antibody comprising two first Fab-arms or half-molecules, wherein the Fab-arms or half-molecules have identical amino acid sequences.

[0175] When used herein, the term "heterodimeric antibody" refers to an antibody comprising first and second Fab-arms or hemimolecules, wherein the amino acid sequences of the first and second Fab-arms or hemimolecules are different. In particular, the CH3 region or antigen-binding region, or the CH3 region and antigen-binding region of the first and second Fab-arms / hemimolecules, are different.

[0176] The term “reduction conditions” or “reduction environment” refers to conditions or environments in which substrates (such as cysteine ​​residues in the hinge region of an antibody) are more likely to be reduced rather than oxidized.

[0177] This disclosure also describes multispecific antibodies, such as bispecific antibodies, comprising functional variants of the VL region, VH region, or one or more CDRs of an example bispecific antibody. The functional variants of the VL, VH, or CDR used in the case of bispecific antibodies still allow each antigen-binding region of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) of the affinity and / or specificity / selectivity of the parent bispecific antibody. In some cases, such bispecific antibodies may have greater affinity, selectivity, and / or specificity than the parent bispecific antibody.

[0178] These functional variants typically maintain significant sequence identity with the parental bispecific antibody. The percentage identity between two sequences is a function of the number of common positions (i.e., % homology = # of common positions / total # of positions × 100). Given the number and length of nicks, nicks need to be introduced to achieve optimal alignment of the two sequences. The percentage 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 nick length penalty of 12, and a nick penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0179] In the context of this disclosure, unless otherwise stated, the following symbols are used to describe mutations: i) an amino acid substitution at a given position is written as, for example, K409R, which indicates that a lysine residue at position 409 of a protein is replaced by an arginine residue; and ii) for a particular variant, a specific three-letter code or a single-letter code is used, including codes Xaa and X, to represent any amino acid residue. Thus, a lysine substitution at position 409 with arginine is named K409R, and a lysine substitution at position 409 with any amino acid residue is named K409X. The case of a lysine deletion at position 409 is represented by K409. express.

[0180] Exemplary variants include those that differ primarily from the VH and / or VL and / or CDR regions of the parental sequence through conserved substitutions; for example, 12 substitutions, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in a variant are conserved amino acid residue substitutions.

[0181] In the context of this disclosure, conservative substitutions can be defined by substitutions within the amino acid categories defined in Tables 2 and 3.

[0182] The antibody sequences described herein, such as functional variants of the VL or VH regions, or antibody sequences that have a certain degree of homology or similarity to the antibody sequences described herein, such as the VL or VH regions, preferably contain modifications or variations in non-CDR sequences, while the CDR sequences preferably remain unchanged.

[0183] As used herein, the term "EpCAM" refers to the 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 functions and appears to play a role in cell adhesion and cancer. In one embodiment, EpCAM is human EpCAM, which has UniProt accession number P16422. The sequence of human EpCAM is also shown 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; and the remainder of the protein, namely amino acids 266-288 and 289-314 from SEQ ID NO: 59, are the transmembrane domain and cytoplasmic domain, respectively.

[0184] 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 believed 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), which has UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO: 37. Amino acids 1-23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; amino acids 24-186 of SEQ ID NO: 37 correspond to the extracellular domain of human CD137; and the remaining parts of the protein, namely amino acids 187-213 and 214-255 from SEQ ID NO: 37, are the transmembrane domain and the cytoplasmic domain, respectively.

[0185] "Treatment period" is defined in this document as the time period within the influence of a single dose of the binder due to the pharmacodynamics of the binder, or in other words, the time period after the administered binder has been substantially cleared from the subject's body. Multiple small doses within a small time window (e.g., within 2–24 hours, such as 2–12 hours or on the same day) may be equivalent to a larger single dose.

[0186] In this document, the terms "treatment," "treating," or "therapeutic intervention" refer to the management and care of a subject for the purpose of combating a disease condition such as a disease or symptom. The term is intended to encompass the full spectrum of treatment for a given disease condition suffered by a subject, such as administering a therapeutically effective compound to alleviate symptoms or complications, delay the progression of the disease, symptom, or disease condition, reduce or alleviate symptoms and complications, and / or cure or eliminate the disease, symptom, or disease condition, as well as prevention of the disease condition, wherein prevention is understood as the management and care of an individual for the purpose of combating a disease, disease condition, or symptom, and includes administering an active compound to prevent the occurrence of symptoms or complications. In one embodiment, "treatment" means administering an effective amount of a therapeutically active conjugate of this disclosure, such as a therapeutically active antibody, for the purpose of reducing, improving, preventing, or eradicating (curing) symptoms or disease conditions.

[0187] The response to treatment, as well as resistance, non-response, and / or recurrence to treatment with the binders of this disclosure, can be determined according to the Solid Tumor Response Evaluation Criteria; version 1.1 (RECIST Criteria v1.1).

[0188] "Optimal overall response" is the best response recorded from the start of treatment until disease progression / relapse (the minimum measurement recorded since the start of treatment is used as a reference 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 to be non-responders.

[0189] "Duration of Response (DOR)" applies only to subjects whose best overall response is confirmed as CR or PR, and is defined as the time from the first recorded objective tumor response (CR or PR) to the first PD or to the date of death from underlying cancer.

[0190] "Progression-free survival (PFS)" is defined as the number of days from day 1 of cycle 1 to the first recorded progression or death from any cause.

[0191] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 until death from any cause. If it is unknown that a subject has died, the OS will be revised on the latest known date of the subject's survival (on or before the cutoff date).

[0192] In the context of this disclosure, the term "treatment plan" refers to a structured treatment program designed to improve and maintain health.

[0193] The term "effective dose" or "therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dose and time period. The therapeutically effective dose of a conjugate (e.g., an antibody, such as a multispecific antibody or a monoclonal antibody) can vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the conjugate's ability to elicit the desired response in the individual. Therapeutic effective dose is also the amount in which the beneficial therapeutic effect significantly outweighs any toxic or harmful effects of the conjugate or its fragments. Higher doses (or higher effective doses achieved through different, more localized routes of administration) may be used when a patient's response to an initial dose is insufficient. Lower doses (or lower effective doses achieved through different, more localized routes of administration) may be used when a patient experiences undesirable side effects at a given dose.

[0194] As used herein, the term "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cell" refers to abnormal cells that grow through rapid, uncontrolled cell proliferation and continue to grow even after the stimuli that initiated new growth have ceased.

[0195] According to the terminology of this disclosure, "cancer" 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 and depends 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 blood vessels, and then, after being transported via the bloodstream, infiltration of target organs. Finally, the growth of new tumors at the target site, i.e., secondary or metastatic tumors, depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, as tumor cells or components can be retained and develop metastatic potential. In one embodiment, the term "metastasis" according to this disclosure refers to "distant metastasis," which involves metastasis distant from the primary tumor and the regional lymph node system.

[0196] As used herein, terms such as “reduce,” “inhibit,” “interference,” and “negative regulation” indicate the ability to cause an overall reduction in levels, for example, a reduction of 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 include complete or substantially complete inhibition, i.e., a reduction to 0 or substantially to 0.

[0197] In one implementation, terms such as “increase” or “enhance” refer 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%.

[0198] As used in this article, "physiological pH" refers to a pH of 7.5 or approximately 7.5.

[0199] As used in this disclosure, “weight%” means weight percentage, which is a unit of concentration that measures the amount of substance in grams (g) and is expressed as a percentage of the total weight of the total composition in grams (g).

[0200] The term "freezing" refers to the solidification of a liquid, usually accompanied by the removal of heat.

[0201] The terms “lyophilizing” or “lyophilization” refer to the freeze-drying of a substance by freezing it and then reducing the ambient pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or lower) to allow the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Therefore, the terms “lyophilizing” and “freeze-drying” are used interchangeably herein.

[0202] In the context of this disclosure, the term "recombinant" means "prepared by genetic engineering." In one embodiment, the "recombinant object" in the context of this disclosure is not naturally occurring.

[0203] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory are naturally occurring. The term "found in nature" means "existing in nature," including known objects as well as 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.

[0204] 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 a large peptide, particularly a peptide having at least about 151 amino acids; however, the terms "peptide" and "protein" are generally used synonymously herein.

[0205] When administered to a subject in a therapeutically effective amount, a "therapeutic protein" has a positive or beneficial effect on the subject's condition or disease state. In one embodiment, the therapeutic protein has therapeutic or palliative properties and can be administered to improve, reduce, alleviate, reverse, delay the onset of one or more symptoms of a disease or condition, or reduce the severity of one or more symptoms of a disease or condition. Therapeutic proteins may 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 may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens used for vaccination and immunostimulants such as cytokines.

[0206] The term "portion" refers to a small part. Regarding a specific structure such as an amino acid sequence or a protein, the term "portion" can refer to a continuous or discontinuous part of that structure.

[0207] The terms “part” and “fragment” are used interchangeably herein and refer to a continuous element. For example, a structure such as an amino acid sequence or a portion of a protein refers to a continuous element of that structure. When used in the context of a composition, the term “part” means a portion of the composition. For example, a part of the composition can be any portion ranging from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0208] Regarding amino acid sequences (peptides or proteins), a "fragment" refers to a portion of the amino acid sequence, specifically a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end, provided the truncated open reading frame contains a start codon for initiating translation. The amino acid sequence fragment 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. The amino acid sequence fragment 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.

[0209] According to this disclosure, a portion or fragment of a peptide or protein preferably has 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 to nucleic acids. For example, a pharmacologically active fragment of a peptide or protein has at least one pharmacological activity of the peptide or protein from which that fragment 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, up to 12, up to 15, up to 20, up to 30, or up to 55 consecutive amino acids of the peptide or protein.

[0210] In this document, "variant" refers to an amino acid sequence that differs from the parent amino acid sequence due to at least one amino acid modification. The parent amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence, or it 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, for example, 1 to about 20 amino acid modifications compared to the parent, and more preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0211] In this article, "wild-type," "WT," or "natural" refers to an amino acid sequence found in nature, including allelic variations. Wild-type amino acid sequences, peptides, or proteins have amino acid sequences that have not been intentionally modified.

[0212] Preferably, the similarity, preferably the degree of identity, between a given amino acid sequence and an amino acid sequence variant thereof is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Preferably, the degree of similarity or identity is given for 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 amino acid region of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, it is preferable to give a degree of similarity or identity 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, which in some embodiments are consecutive amino acids. In some embodiments, a degree of similarity or identity is given for the entire length of the reference amino acid sequence. Sequence similarity can be determined using tools known in the art, preferably sequence identity alignment, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0213] "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between those sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between those sequences.

[0214] Specifically, the terms "% identical" and "% similarity," or similar terms, refer to the percentage of identical nucleotides or amino acids in the best alignment between the sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may, but do not necessarily, be randomly distributed across the entire length of the sequences to be compared. Typically, the two sequences are compared by comparing the sequences after the best alignment of the fragments or "comparison window" to identify local regions of the respective sequences. The best alignment for comparison can be performed manually, or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2,482; the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some implementations, the percentage similarity between two sequences is determined using the BLASTN or BLASTP algorithm, which is available on the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi). In some implementations, the algorithm parameters for the BLASTN algorithm used on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 28; (iii) a maximum match in the query range set to 0; (iv) match / no-match scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for the low-complexity region being used. In some implementations, the algorithm parameters for the BLASTP algorithm used on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 3; (iii) a maximum match in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to exist: 11, extend: 1; and (vi) conditional composition score matrix adjustment.

[0215] The percentage similarity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0216] In some embodiments, a degree of similarity or identity is given for regions of 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, a degree of similarity is given for 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, which in some embodiments are consecutive amino acid residues. In some embodiments, a degree of similarity or identity is given for the entire length of the reference sequence.

[0217] According to this disclosure, the homologous amino acid sequences exhibit at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98%, or at least 99% similarity of amino acid residues.

[0218] Technicians can readily prepare the amino acid sequence variants described herein, for example, through recombinant DNA manipulation. For instance, Sambrook et al. (1989) described in detail the procedures for preparing DNA sequences of peptides or proteins with substitutions, additions, insertions, or deletions. Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, through solid-phase synthesis and similar methods.

[0219] In one embodiment, the fragment or variant of the 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 derived from the amino acid sequence; that is, it is functionally equivalent. Regarding the binding agent, a specific function is one or more binding activities exhibited by the amino acid sequence of the derived fragment or variant. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence that comprises an amino acid sequence altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, and is still capable of performing one or more functions of the parent molecule or sequence, such as binding to a target antigen. In one embodiment, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of said 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 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.

[0220] The phrase "derived from" a specific amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence derived from the specific amino acid sequence has the same, substantially the same, or homologous amino acid sequence as that specific sequence or a fragment thereof. The amino acid sequence derived from the specific amino acid sequence can be a variant of that specific sequence or a fragment thereof. For example, those skilled in the art will understand that the binding agents used herein can be modified so that their sequences differ from those of the naturally occurring sequences from which they are derived, while retaining the desired activity of the natural sequence.

[0221] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living organisms are not "separated," but the same nucleic acid or peptide partially or completely separated from its native coexisting substance is "separated." Separated nucleic acids or proteins may exist in a substantially purified form or may exist in a non-natural environment, such as a host cell. In one embodiment, the conjugates (e.g., antibodies) described herein are separated. As used herein, "separated conjugates" means conjugates that are substantially free of other conjugates with different antigen specificities. In one embodiment, a separated bispecific conjugate that specifically binds to EpCAM and CD137 is substantially free of monospecific antibodies that specifically bind to EpCAM or CD137. In a preferred embodiment, the conjugates used in this disclosure are in a substantially purified form.

[0222] The term “genetic modification” or simply “modification” includes transfecting cells with nucleic acids. The term “transfection” involves introducing nucleic acids, particularly RNA, into cells. For the 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, such as a patient. Thus, according to this disclosure, cells used for transfecting the nucleic acids described herein may be present in vitro or in vivo, for example, the cells may constitute part of an organ, tissue, and / or organism of a patient. According to this disclosure, transfection can be transient or stable. For some applications of transfection, transient expression of the transfected genetic material is sufficient. RNA can be transfected into cells to transiently express its encoded protein. Because the nucleic acids introduced during transfection typically do not integrate into the nuclear genome, the exogenous nucleic acids are diluted or degraded by mitosis. Cells that allow free amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acids actually remain in the genome of the cell and its daughter cells, stable transfection must be performed. Such stable transfection can be achieved by using a virus-based system or a transposon-based system. Typically, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells to transiently express the protein it encodes.

[0223] According to this disclosure, a peptide or protein analog is a modified form of the peptide or protein from which it is derived and has at least one functional property of the peptide or protein. For example, a pharmacologically active peptide or protein analog has at least one pharmacological activity of the peptide or protein from which the analog is derived. Such modifications include any chemical modifications and include 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 those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristylation, isopreneation, esterification, alkylation, derivatization, introduction of a protecting / blocking group, protease cleavage, or binding to an antibody or another cellular ligand. The term “analog” also extends to all functional chemical equivalents of the protein and peptide.

[0224] As used herein, “activation” or “stimulation” refers to the state of immune effector cells, such as T cells, that have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signaling pathways, induced cytokine production, and detectable effector function. The term “activated immune effector cells” refers to immune effector cells undergoing cell division.

[0225] The term "initiation" refers to the process in which immune effector cells, such as T cells, come into contact with their specific antigens for the first time, leading to differentiation into effector cells, such as effector T cells.

[0226] The term "clonal expansion" or "expansion" refers to a process in which a specific entity multiplies. In the context of this disclosure, the term is preferably used in the context of an immune response, in which immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize said antigen expand. Preferably, clonal expansion leads to the differentiation of immune effector cells.

[0227] According to this disclosure, "antigen" encompasses any substance or molecular structure that can bind to an antibody or T-cell receptor. The presence of an antigen in the body can trigger an immune response. Therefore, "antigen" encompasses any substance targeted by an immune response or immune mechanism. This also includes cases where the antigen is processed into an antigenic peptide and the immune response or immune mechanism targets one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, "antigen" refers to any substance that specifically reacts with an antibody or T-lymphocyte (T-cell) receptor, preferably a peptide or protein. According to this disclosure, the term "antigen" includes any molecule containing at least one epitope, such as a T-cell epitope. Preferably, in the context of this disclosure, an antigen is a molecule, optionally after processing, that induces an immune response, preferably antigen- (including cells expressing the antigen) specific. In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, or an epitope derived from such an antigen.

[0228] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, that is, a portion or fragment of a molecule recognized by the immune system, for example, a portion or fragment of a molecule recognized by antibody T cells or B cells, particularly when presented in the context of MHC molecules. In one embodiment, "epitope" refers to a protein determinant capable of specifically binding to an antibody. Epitopes typically consist of surface groups of a molecule, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features and specific charge features. The difference between conformational and non-conformational epitopes is that the former, but not the latter, loses binding in the presence of denaturing solvents. Epitopes may contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked or covered by the specific antigen-binding peptide (in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide).

[0229] The epitope of the protein preferably comprises a continuous or discontinuous portion of the protein, and preferably has a length of about 5 to about 100, more preferably about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids. For example, the epitope may preferably have a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In one embodiment, the epitope in the context of this disclosure is a T-cell epitope.

[0230] As used herein, the terms “optional” or “optionally” mean that an event, situation, or condition described below may or may not occur, and the description includes both the occurrence and non-occurrence of the event, situation, or condition.

[0231] As used herein, the terms “connected,” “integrated,” or “integrated” are used interchangeably. These terms refer to two or more elements or components or domains connected together.

[0232] The term "disease" (also referred to as "symptom" in this text) refers to an abnormal condition affecting an individual's body. Disease is generally interpreted as a medical condition associated with specific symptoms or signs. Diseases may be caused by factors originating from external sources, such as infectious diseases, or they may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death in an affected individual or similar problems in those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, symptoms, syndromes, infections, isolation symptoms, deviant behaviors, and atypical changes in structure and function, but in other contexts and for other purposes, these can be considered distinct categories. Diseases often affect individuals not only physically but also emotionally, as infection and suffering from many diseases can alter a person's outlook on life and their personality.

[0233] The term "therapeutic treatment" refers to any treatment that improves an individual's health and / or prolongs (increases) their lifespan. Such treatment may eliminate an individual's disease, stop or slow the progression of the disease, inhibit or slow the progression of the disease, reduce the frequency or severity of symptoms, and / or reduce recurrence in individuals currently suffering from or previously suffering from the disease.

[0234] The term "preventive treatment" or "preventive therapy" refers to any treatment designed to prevent the occurrence of a disease in an individual. The terms "preventive treatment" or "preventive therapy" are used interchangeably herein. Similarly, in the context of disease progression (e.g., the progression of a tumor or cancer), the term "method for prevention" refers to any method designed to prevent the progression of a disease in an individual.

[0235] The terms “individual” and “subject” are used interchangeably herein. They refer to a person or other mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) who may have a disease or condition (e.g., cancer) or is susceptible to a disease or condition (e.g., cancer), or any other non-mammal, including birds (chickens), fish, or any other animal species. Unless otherwise stated, the terms “individual” and “subject” do not indicate a specific age and therefore cover adults, older adults, children, and newborns. In embodiments of this disclosure, “individual” or “subject” is a “patient.”

[0236] The term "patient" refers to an individual or subject of treatment, particularly an individual or subject who is ill.

[0237] As used herein, the terms "polynucleotide" or "nucleic acid" are intended to include DNA and RNA such as genomic DNA, cDNA, and mRNA, as well as molecules produced through recombination and chemical synthesis. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0238] Nucleic acids can be contained in vectors. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, bacteriophage vectors such as λ phage, viral vectors such as retroviruses, adenoviruses, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Vectors include expression and cloning vectors. Expression vectors contain plasmids and viral vectors and generally contain the desired coding sequence as well as the appropriate DNA sequence necessary for the operatively linked coding sequence to be expressed 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 engineer and amplify a desired DNA fragment and may lack the functional sequence required for expressing the desired DNA fragment.

[0239] In one embodiment of this disclosure, the nucleic acid is expressed in the cells of the treated subject to provide the encoded peptide or protein. In one embodiment, the nucleic acid is transiently expressed in the subject's cells. Therefore, in one embodiment, the nucleic acid is not integrated into the cell's genome. In one embodiment, the nucleic acid is RNA, preferably in vitro transcribed RNA.

[0240] The nucleic acids described in this article can be recombinant and / or isolated molecules.

[0241] In this disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the 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 the β-D-furanose group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of a non-nucleotide substance to an internal RNA nucleotide or the terminus 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. For the purposes of this disclosure, these altered RNAs are considered analogs of naturally occurring RNA.

[0242] In some embodiments of this disclosure, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As established in the art, mRNA generally contains 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, wherein the DNA refers to a nucleic acid containing deoxyribonucleotides.

[0243] In one implementation, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of 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. cDNA can be obtained by reverse transcription of RNA.

[0244] In one embodiment, the RNA described herein may have modified nucleosides. In some embodiments, the RNA contains modified nucleosides in place of at least one (e.g., each) uridine.

[0245] In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0246] In some implementations, the nucleoside replacing one or more uridines in the RNA can be any one or more of the following: 3-methyluridine (m 3 U), 5-methoxyuridine (mo) 5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thiouridine (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-oxyacetic acid methyl ester (mcmo) 5 U), 5-carboxymethyluridine (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-thio-uridine (mcm) 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm) 5 s 2 U), 5-methylaminomethyluridine (mnm) 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm) 5 se 2 U), 5-carbamoylmethyluridine (ncm) 5 U), 5-Carboxymethylaminomethyluridine (cmnm) 5 U), 5-Carboxymethylaminomethyl-2-thio-uridine (cmnm) 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauric acid methyl-uridine (τm) 5 U), 1-Taurate methyl-pseudouridine, 5-Taurate methyl-2-thio-uridine (τm5s2U), 1-Taurate methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseuuridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine (D), dihydrouridine, 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-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m) 5 Um), 2′-O-methyl-pseuuridine (ψm), 2-thio-2′-O-methyl-uridine (s) 2 Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm) 5 Um), 5-carbamoylmethyl-2′-O-methyluridine (ncm) 5 Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm) 5 Um), 3,2′-O-dimethyluridine (m) 3 Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm) 5 Um), 1-thio-uridine, deoxythymidine, 2′-F-arglucuridine, 2′-F-uridine, 2′-OH-arglucuridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other modified uridine known in the art.

[0247] In some embodiments, the RNA according to this disclosure comprises a 5'-cap. In one embodiment, the RNA of this disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified with a 5'-cap analogue. The term "5'-cap" refers to a structure found at the 5'-terminus of an mRNA molecule and generally consists of a guanosine nucleotide linked to the mRNA via a 5'-to-5'-triphosphate bond. In one embodiment, the guanosine is methylated at position 7. RNA providing a 5'-cap or a 5'-cap analogue can be provided by in vitro transcription, wherein the 5'-cap is co-transcribed into the RNA strand, or it can be ligated to the RNA post-transcriptionally using a capping enzyme.

[0248] In some embodiments, the RNA comprises cap0, cap1, or cap2. According to this disclosure, the term "cap0" indicates the structure "m 7 "GpppN", where N is any nucleoside with an OH moiety at the 2' position. According to this disclosure, the term "cap1" indicates the structure "m 7 "GpppNm", where Nm is any nucleotide with an OCH3 moiety at the 2' position. According to this disclosure, the term "cap2" represents the structure "m". 7 "GpppNmNm", where each Nm is independently any nucleoside with an OCH3 motif at the 2' position.

[0249] In some implementations, the RNA includes a 5'-cap structure selected from 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.

[0250] In some embodiments, the RNA according to this disclosure comprises a 5'-UTR and / or a 3'-UTR. The terms "untranslated region" or "UTR" refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule such as mRNA. An untranslated region (UTR) may be present at the 5' (upstream) (5'-UTR) and / or the 3' (downstream) (3'-UTR) of an open reading frame. If present, the 5'-UTR is located at the 5' end, upstream of the start codon of the protein-coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, 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, but the term "3'-UTR" preferably does not include the poly(A) sequence. Thus, the 3'-UTR is located upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0251] In some embodiments, the RNA according to this disclosure comprises a 3'-poly(A) sequence. As used herein, the term "poly(A) sequence" or "poly-A tail" refers to a continuous or discontinuous sequence of adenosine residues, typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may be located after the 3' UTR of the RNA described herein. Poly(A) sequences can have 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 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 only of A nucleotides. In some embodiments, the poly(A) sequence consists essentially of A nucleotides but is interrupted by random sequences of four nucleotides (A, C, G, and U), as disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. The poly(A) box present in the DNA coding strand is essentially composed of dA nucleotides, but is interrupted by random sequences of, for example, 5-50 nucleotides in length, with equal distributions of four nucleotides (dA, dC, dG, dT). This results in plasmid DNA appearing in *E. coli* at the DNA level. E. coli It proliferates consistently in the RNA and remains associated with beneficial properties supporting RNA stability and translation efficiency at the RNA level. In some embodiments, there are no nucleotides other than A nucleotides flanking the 3' end of the poly(A) sequence; that is, the poly(A) sequence is not masked or followed by nucleotides other than A at its 3' end.

[0252] In the context of this disclosure, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into peptides or proteins.

[0253] "Encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, so that it can be used as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological characteristics. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes a protein. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, as well as the non-coding strand used as a template for gene or cDNA transcription, can both be referred to as encoding the protein or other product of that gene or cDNA. Similarly, if the translation of RNA produces a protein in a cell or other biological system, then that RNA, such as mRNA, encodes a protein.

[0254] RNA can be naked or packaged, such as formulated in particles, like protein and / or lipid particles, such as lipid nanoparticles.

[0255] As used in this article, “endogenous” means any substance that originates from or is produced within an organism, cell, tissue, or system.

[0256] As used herein, the term “exogenous” means any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0257] As used in this article, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence.

[0258] The Fc region may have a lysine residue at its C-terminus. This lysine residue originates from naturally occurring sequences found in humans, from which these Fc regions are derived. During the production of recombinant antibodies in cell culture, this terminal lysine residue can be proteasically cleaved away by an endogenous carboxypeptidase, producing a constant region with the same sequence but lacking the C-terminal lysine residue. For antibody preparation purposes, the DNA encoding this terminal lysine residue can be omitted from the sequence, resulting in an antibody without the lysine residue. Antibodies produced from nucleic acid sequences encoding or not encoding the terminal lysine residue are substantially identical in sequence and function because the degree of processing of the terminal lysine residue is typically high when, for example, antibodies produced in a CHO-based generation system are used (Dick, LWet al. Biotechnol. Bioeng. 2008;100: 1132–1143). Therefore, it should be understood that proteins such as antibodies according to this disclosure can be produced with or without encoding a terminal lysine residue.

[0259] Aspects and implementation methods disclosed herein

[0260] In a first aspect, this disclosure provides a method for treating or preventing a disease or condition in a subject, the method comprising (i) administering a binder to the subject, the binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, and (ii) administering a PD(L)-1 axis binding antagonist to the subject, wherein the administration of the binder occurs before, simultaneously with, or after the administration of the PD(L)-1 axis binding antagonist.

[0261] Binders that bind to EpCAM and CD137

[0262] In some embodiments, EpCAM is human EpCAM. In some embodiments, CD137 is human CD137. In some embodiments, human EpCAM comprises the sequence shown in SEQ ID NO: 59. In some embodiments, human CD137 comprises the sequence shown in SEQ ID NO: 62.

[0263] In some implementations, the first antigen-binding region that binds to EpCAM binds to EpCAM expressed on tumor cells.

[0264] In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR3 sequence, wherein the HCDR3 sequence contains the sequence shown in SEQ ID NO: 4. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR2 sequence, wherein the HCDR2 sequence contains the sequence shown in SEQ ID NO: 3. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR1 sequence, wherein the HCDR1 sequence contains the sequence shown in SEQ ID NO: 2. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences contain the sequences shown in SEQ ID NO: 2, 3, and 4, respectively. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 64, 65, and 66, respectively. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 74, 3, and 66, respectively.

[0265] In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR3 sequence, the LCDR3 sequence containing the sequence shown in SEQ ID NO: 8. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR2 sequence, the LCDR2 sequence containing the sequence shown in SEQ ID NO: 7. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR1 sequence, the LCDR1 sequence containing the sequence shown in SEQ ID NO: 6. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences contain the sequences shown in SEQ ID NO: 6, 7, and 8, respectively. In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 67, 68, and 8, respectively.

[0266] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) containing an HCDR3 sequence and a light chain variable region (VL) containing an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence shown in SEQ ID NO: 4 and the LCDR3 sequence comprises the sequence shown in SEQ ID NO: 8.

[0267] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 6, 7, and 8, respectively.

[0268] In some embodiments, the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 67, 68, and 8, respectively.

[0269] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 6, 7, and 8, respectively.

[0270] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region (VH) includes the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1, and the light chain variable region (VL) includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 5.

[0271] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 1.

[0272] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 1.

[0273] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 5.

[0274] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing a sequence as shown in SEQ ID NO: 5.

[0275] In some embodiments, the first antigen-binding region binding 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 5. In some embodiments, the first antigen-binding region binding 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 with the sequence shown in SEQ ID NO: 1, and the VL comprises a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 5. In some embodiments, the first antigen-binding region binding 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 with the sequence shown in SEQ ID NO: 1, and the VL comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 5. In some embodiments, the first antigen-binding region binding 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 with the sequence shown in SEQ ID NO: 1, and the VL comprises a sequence having at least 95% identity with the sequence shown in SEQ ID NO: 5.

[0276] 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 as shown in SEQ ID NO: 1, and the VL comprises a sequence as shown in SEQ ID NO: 5.

[0277] In some embodiments, the first antigen-binding region that binds to EpCAM comprises heavy chain and light chain variable regions of an antibody, which competes with antibodies comprising the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding and / or for the specificity of antibodies containing the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding.

[0278] In some embodiments, the second antigen-binding region binding to CD137 includes a heavy chain variable region (VH) containing an HCDR3 sequence, the HCDR3 sequence containing the sequence shown in SEQ ID NO: 14. In some embodiments, the second antigen-binding region binding to CD137 includes a heavy chain variable region (VH) containing an HCDR2 sequence, the HCDR2 sequence containing the sequence shown in SEQ ID NO: 13. In some embodiments, the second antigen-binding region binding to CD137 includes a heavy chain variable region (VH) containing an HCDR1 sequence, the HCDR1 sequence containing the sequence shown in SEQ ID NO: 12. In some embodiments, the second antigen-binding region binding to CD137 includes a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences contain the sequences shown in SEQ ID NO: 12, 13, and 14, respectively. In some embodiments, the second antigen-binding region binding to CD137 includes a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 69, 70, and 71, respectively. In some embodiments, the second antigen-binding region binding to CD137 includes a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 75, 13, and 71, respectively.

[0279] In some embodiments, the second antigen-binding region binding to CD137 includes a light chain variable region (VL) containing an LCDR3 sequence, the LCDR3 sequence containing the sequence shown in SEQ ID NO: 18. In some embodiments, the second antigen-binding region binding to CD137 includes a light chain variable region (VL) containing an LCDR2 sequence, the LCDR2 sequence containing the sequence shown in SEQ ID NO: 17. In some embodiments, the second antigen-binding region binding to CD137 includes a light chain variable region (VL) containing an LCDR1 sequence, the LCDR1 sequence containing the sequence shown in SEQ ID NO: 16. In some embodiments, the second antigen-binding region binding to CD137 includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences contain the sequences shown in SEQ ID NO: 16, 17, and 18, respectively. In some embodiments, the second antigen-binding region that binds to CD137 includes a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 72, 73, and 18, respectively.

[0280] In some embodiments, the second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) containing an HCDR3 sequence and a light chain variable region (VL) containing an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence shown in SEQ ID NO: 14 and the LCDR3 sequence comprises the sequence shown in SEQ ID NO: 18.

[0281] In some embodiments, the second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 12, 13, and 14, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17, and 18, respectively.

[0282] In some embodiments, the second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 69, 70, and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 72, 73, and 18, respectively.

[0283] In some embodiments, the second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 75, 13, and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17, and 18, respectively.

[0284] In some embodiments, the second antigen-binding region binding to CD137 includes a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region (VH) includes the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and the light chain variable region (VL) includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0285] In some embodiments, the second antigen-binding region that binds to CD137 includes a heavy chain variable region (VH) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 11.

[0286] In some embodiments, the second antigen-binding region that binds to CD137 includes a heavy chain variable region (VH) containing a sequence as shown in SEQ ID NO: 11.

[0287] In some embodiments, the second antigen-binding region binding to CD137 includes a light chain variable region (VL) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO: 15.

[0288] In some embodiments, the second antigen-binding region that binds to CD137 includes a light chain variable region (VL) containing a sequence as shown in SEQ ID NO: 15.

[0289] In some embodiments, the second antigen-binding region binding 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 15. In some embodiments, the second antigen-binding region binding 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 with the sequence shown in SEQ ID NO: 11, and the VL comprises a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 15. In some embodiments, the second antigen-binding region binding 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 with the sequence shown in SEQ ID NO: 11, and the VL comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 15. In some embodiments, the second antigen-binding region binding 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 with the sequence shown in SEQ ID NO: 11, and the VL comprises a sequence having at least 95% identity with the sequence shown in SEQ ID NO: 15.

[0290] In some embodiments, the second antigen-binding region binding to CD137 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: 11, and the VL comprises the sequence shown in SEQ ID NO: 15.

[0291] In some embodiments, the first antigen-binding region that binds to CD137 includes a heavy chain and a light chain variable region of the antibody, the antibody competing with antibodies containing the heavy chain variable region and / or the light chain variable region as shown above for CD137 binding and / or specificity against antibodies that have CD137 containing the heavy chain variable region and / or the light chain variable region as shown above.

[0292] In some implementation schemes, a) The first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing the HCDR3 sequence as shown in SEQ ID NO: 4, and b) The second antigen-binding region that binds to CD137 includes a heavy chain variable region (VH) containing the HCDR3 sequence as shown in SEQ ID NO: 14.

[0293] In some implementation schemes, a) The first antigen-binding region binding to EpCAM includes a light chain variable region (VL) comprising the LCDR3 sequence as shown in SEQ ID NO: 8, and b) The second antigen-binding region that binds to CD137 includes a light chain variable region (VL) containing the LCDR3 sequence as shown in SEQ ID NO: 18.

[0294] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR3 sequence as shown in SEQ ID NO: 4, and the light chain variable region (VL) comprises the LCDR3 sequence as shown in SEQ ID NO: 8, 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 heavy chain variable region (VH) comprises the HCDR3 sequence as shown in SEQ ID NO: 14, and the light chain variable region (VL) comprises the LCDR3 sequence as shown in SEQ ID NO: 18.

[0295] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 2, 3, and 4, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 6, 7, and 8; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0296] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 64, 65, and 66, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 67, 68, and 8; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 72, 73 and 18, respectively.

[0297] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 74, 3, and 66, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 6, 7, and 8; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0298] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 1, and the light chain variable region (VL) comprises 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) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and the light chain variable region (VL) comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0299] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 SEQ ID NO: 1, and the 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 SEQ ID NO: 5; and b) The second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 SEQ ID NO: 11, and the 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 SEQ ID NO: 15.

[0300] In some implementation schemes, a) The first antigen-binding region binding 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: 1, and the VL comprises the sequence shown 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 shown in SEQ ID NO: 11, and the VL comprises the sequence shown in SEQ ID NO: 15.

[0301] In some embodiments, the binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region that binds to EpCAM, and the second binding arm comprising a second antigen-binding region that binds to CD137, wherein... The first connecting arm includes i) Peptides containing a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) A polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), The first VH comprises the first HCDR1, HCDR2 and HCDR3 sequences, and the first VL comprises the first LCDR1, LCDR2 and LCDR3 sequences, wherein the first HCDR1, HCDR2 and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 2, 3 and 4, and the first LCDR1, LCDR2 and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 6, 7 and 8; And the second connecting arm includes iii) Peptides containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) Peptides containing a second light chain variable region (VL) and a second light chain constant region (CL), The second VH comprises the second HCDR1, HCDR2 and HCDR3 sequences, and the second VL comprises the second LCDR1, LCDR2 and LCDR3 sequences, wherein the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NO:12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NO:16, 17 and 18, respectively. In the first CH, positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively; in the second CH, positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively; and In the first CH, the amino acid corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second CH, the amino acid corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R.

[0302] In some implementations, the binder comprises 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 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 with the amino acid sequence shown in SEQ ID NO: 10. iii) A second 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 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 with the amino acid sequence shown in SEQ ID NO: 20.

[0303] In some implementations, the binder comprises i) The first heavy chain, which contains the amino acid sequence shown in SEQ ID NO: 9, ii) A first light chain comprising the amino acid sequence shown in SEQ ID NO: 10. iii) A second chain comprising the amino acid sequence shown in SEQ ID NO: 19; and iv) The second light chain contains the amino acid sequence shown in SEQ ID NO: 20.

[0304] In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR3 sequence, wherein the HCDR3 sequence contains the sequence shown in SEQ ID NO: 78. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR2 sequence, wherein the HCDR2 sequence contains the sequence shown in SEQ ID NO: 77. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR1 sequence, wherein the HCDR1 sequence contains the sequence shown in SEQ ID NO: 76. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences contain the sequences shown in SEQ ID NO: 76, 77, and 78, respectively. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 82, 83, and 84, respectively. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 87, 77, and 84, respectively.

[0305] In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR3 sequence, the LCDR3 sequence containing the sequence shown in SEQ ID NO: 81. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR2 sequence, the LCDR2 sequence containing the sequence shown in SEQ ID NO: 80. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR1 sequence, the LCDR1 sequence containing the sequence shown in SEQ ID NO: 79. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences contain the sequences shown in SEQ ID NO: 79, 80, and 81, respectively. In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences respectively contain sequences as shown in SEQ ID NO: 85, 86, and 81.

[0306] In some embodiments, the first antigen-binding region that binds to EpCAM comprises a heavy chain variable region (VH) containing an HCDR3 sequence and a light chain variable region (VL) containing an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence shown in SEQ ID NO: 78 and the LCDR3 sequence comprises the sequence shown in SEQ ID NO: 81.

[0307] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 76, 77, and 78, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 79, 80, and 81, respectively.

[0308] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 82, 83, and 84, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 85, 86, and 81, respectively.

[0309] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 87, 77, and 84, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 79, 80, and 81, respectively.

[0310] In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region (VH) includes HCDR1, HCDR2, and HCDR3 sequences at positions 1-116 of SEQ ID NO: 21, and the light chain variable region (VL) includes LCDR1, LCDR2, and LCDR3 sequences at positions 1-112 of SEQ ID NO: 22.

[0311] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown in positions 1-116 of SEQ ID NO: 21.

[0312] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing the sequence shown in positions 1-116 of SEQ ID NO: 21.

[0313] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown at positions 1-112 of SEQ ID NO: 22.

[0314] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing the sequence shown in positions 1-112 of SEQ ID NO: 22.

[0315] In some embodiments, the first antigen-binding region binding 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 with the sequences shown 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 with the sequences shown in SEQ ID NO: 22 positions 1-112. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in SEQ ID NO: 21 positions 1-116, and the VL comprises a sequence having at least 80% identity with the sequences shown in SEQ ID NO: 22 positions 1-112. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in positions 1-116 of SEQ ID NO: 21, and the VL comprises a sequence having at least 90% identity with the sequences shown in positions 1-112 of SEQ ID NO: 22. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in positions 1-116 of SEQ ID NO: 21, and the VL comprises a sequence having at least 95% identity with the sequences shown in positions 1-112 of SEQ ID NO: 22.

[0316] 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 as shown in positions 1-116 of SEQ ID NO: 21, and the VL comprises a sequence as shown in positions 1-112 of SEQ ID NO: 22.

[0317] In some embodiments, the first antigen-binding region that binds to EpCAM comprises heavy chain and light chain variable regions of an antibody, which competes with antibodies comprising the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding and / or for the specificity of antibodies containing the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding.

[0318] In some implementations, the second antigen-binding region that binds to CD137 is shown above.

[0319] In some implementation schemes, a) The first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing the HCDR3 sequence as shown in SEQ ID NO: 78, and b) The second antigen-binding region that binds to CD137 includes a heavy chain variable region (VH) containing the HCDR3 sequence as shown in SEQ ID NO: 14.

[0320] In some implementation schemes, a) The first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing the LCDR3 sequence as shown in SEQ ID NO: 81, and b) The second antigen-binding region that binds to CD137 includes a light chain variable region (VL) containing the LCDR3 sequence as shown in SEQ ID NO: 18.

[0321] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR3 sequence as shown in SEQ ID NO: 78, and the light chain variable region (VL) comprises the LCDR3 sequence as shown in SEQ ID NO: 81, 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 heavy chain variable region (VH) comprises the HCDR3 sequence as shown in SEQ ID NO: 14, and the light chain variable region (VL) comprises the LCDR3 sequence as shown in SEQ ID NO: 18.

[0322] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 76, 77, and 78, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 79, 80, and 81; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0323] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 82, 83, and 84, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 85, 86, and 81; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 72, 73 and 18, respectively.

[0324] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 87, 77, and 84, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 79, 80, and 81; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0325] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR1, HCDR2, and HCDR3 sequences at positions 1-116 of SEQ ID NO: 21, and the light chain variable region (VL) comprises the LCDR1, LCDR2, and LCDR3 sequences at positions 1-112 of SEQ ID NO: 22; 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 heavy chain variable region (VH) comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and the light chain variable region (VL) comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0326] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 at positions 1-116 of SEQ ID NO: 21, and the 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 at positions 1-112 of SEQ ID NO: 22; and b) The second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 SEQ ID NO: 11, and the 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 SEQ ID NO: 15.

[0327] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in positions 1-116 of SEQ ID NO: 21, and the VL comprises the sequence shown in positions 1-112 of SEQ ID NO: 22; 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 shown in SEQ ID NO: 11, and the VL comprises the sequence shown in SEQ ID NO: 15.

[0328] In some embodiments, the binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region that binds to EpCAM, and the second binding arm comprising a second antigen-binding region that binds to CD137, wherein... The first connecting arm includes: i) Peptides containing a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) A polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), The first VH comprises the first HCDR1, HCDR2 and HCDR3 sequences, and the first VL comprises the first LCDR1, LCDR2 and LCDR3 sequences, wherein the first HCDR1, HCDR2 and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO:76, 77 and 78, and the first LCDR1, LCDR2 and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO:79, 80 and 81; And the second connecting arm includes: iii) Peptides containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) Peptides containing a second light chain variable region (VL) and a second light chain constant region (CL), The second VH comprises the second HCDR1, HCDR2 and HCDR3 sequences, and the second VL comprises the second LCDR1, LCDR2 and LCDR3 sequences, wherein the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NO:12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NO:16, 17 and 18, respectively. In the first CH, positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively; in the second CH, positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively; and In the first CH, the amino acid corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second CH, the amino acid corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R.

[0329] In some implementations, the binder comprises 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 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 with the amino acid sequence shown in SEQ ID NO: 22. iii) A second 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 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 with the amino acid sequence shown in SEQ ID NO: 20.

[0330] In some implementations, the binder comprises i) The first heavy chain, which contains the amino acid sequence shown in SEQ ID NO: 21, ii) A first light chain comprising the amino acid sequence shown in SEQ ID NO: 22, iii) A second chain comprising the amino acid sequence shown in SEQ ID NO: 19; and iv) The second light chain contains the amino acid sequence shown in SEQ ID NO: 20.

[0331] In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR3 sequence, the LCDR3 sequence containing the sequence shown in SEQ ID NO: 90. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR2 sequence, the LCDR2 sequence containing the sequence shown in SEQ ID NO: 89. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR1 sequence, the LCDR1 sequence containing the sequence shown in SEQ ID NO: 88. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences contain the sequences shown in SEQ ID NO: 88, 89, and 90, respectively. In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences respectively contain sequences as shown in SEQ ID NO: 91, 92, and 90.

[0332] In some embodiments, the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) containing an HCDR3 sequence and a light chain variable region (VL) containing an LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence shown in SEQ ID NO: 4, and the LCDR3 sequence comprises the sequence shown in SEQ ID NO: 90.

[0333] In some embodiments, the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 88, 89, and 90, respectively.

[0334] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 91, 92, and 90, respectively.

[0335] In some embodiments, the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 88, 89, and 90, respectively.

[0336] In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region (VH) includes HCDR1, HCDR2, and HCDR3 sequences at positions 1-115 of SEQ ID NO: 25, and the light chain variable region (VL) includes LCDR1, LCDR2, and LCDR3 sequences at positions 1-111 of SEQ ID NO: 26.

[0337] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown in positions 1-115 of SEQ ID NO: 25.

[0338] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing the sequence shown in positions 1-115 of SEQ ID NO: 25.

[0339] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown at positions 1-111 of SEQ ID NO: 26.

[0340] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing the sequence shown in positions 1-111 of SEQ ID NO: 26.

[0341] In some embodiments, the first antigen-binding region binding 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 with the sequences shown 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 with the sequences shown in SEQ ID NO: 26 positions 1-111. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in SEQ ID NO: 25 positions 1-115, and the VL comprises a sequence having at least 80% identity with the sequences shown in SEQ ID NO: 26 positions 1-111. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in positions 1-115 of SEQ ID NO: 25, and the VL comprises a sequence having at least 90% identity with the sequences shown in positions 1-111 of SEQ ID NO: 26. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in positions 1-115 of SEQ ID NO: 25, and the VL comprises a sequence having at least 95% identity with the sequences shown in positions 1-111 of SEQ ID NO: 26.

[0342] 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 as shown in positions 1-115 of SEQ ID NO: 25, and the VL comprises a sequence as shown in positions 1-111 of SEQ ID NO: 26.

[0343] In some embodiments, the first antigen-binding region that binds to EpCAM comprises heavy chain and light chain variable regions of an antibody, which competes with antibodies comprising the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding and / or for the specificity of antibodies containing the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding.

[0344] In some implementations, the second antigen-binding region that binds to CD137 is shown above.

[0345] In some implementation schemes, a) The first antigen-binding region binding to EpCAM includes a light chain variable region (VL) comprising the LCDR3 sequence as shown in SEQ ID NO: 90, and b) The second antigen-binding region that binds to CD137 includes a light chain variable region (VL) containing the LCDR3 sequence as shown in SEQ ID NO: 18.

[0346] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR3 sequence as shown in SEQ ID NO: 4, and the light chain variable region (VL) comprises the LCDR3 sequence as shown in SEQ ID NO: 90, 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 heavy chain variable region (VH) comprises the HCDR3 sequence as shown in SEQ ID NO: 14, and the light chain variable region (VL) comprises the LCDR3 sequence as shown in SEQ ID NO: 18.

[0347] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 2, 3, and 4, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 88, 89, and 90; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0348] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 64, 65, and 66, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 91, 92, and 90; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 72, 73 and 18, respectively.

[0349] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 74, 3, and 66, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 88, 89, and 90; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0350] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR1, HCDR2, and HCDR3 sequences at positions 1-115 of SEQ ID NO: 25, and the light chain variable region (VL) comprises the LCDR1, LCDR2, and LCDR3 sequences at positions 1-111 of SEQ ID NO: 26; 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 heavy chain variable region (VH) comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and the light chain variable region (VL) comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0351] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 at positions 1-115 of SEQ ID NO: 25, and the 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 at positions 1-111 of SEQ ID NO: 26; and b) The second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 SEQ ID NO: 11, and the 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 SEQ ID NO: 15.

[0352] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in positions 1-115 of SEQ ID NO: 25, and the VL comprises the sequence shown in positions 1-111 of SEQ ID NO: 26; 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 shown in SEQ ID NO: 11, and the VL comprises the sequence shown in SEQ ID NO: 15.

[0353] In some embodiments, the binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region that binds to EpCAM, and the second binding arm comprising a second antigen-binding region that binds to CD137, wherein... The first connecting arm includes: i) Peptides containing a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) A polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), The first VH comprises the first HCDR1, HCDR2 and HCDR3 sequences, and the first VL comprises the first LCDR1, LCDR2 and LCDR3 sequences, wherein the first HCDR1, HCDR2 and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 2, 3 and 4, and the first LCDR1, LCDR2 and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 88, 89 and 90; And the second connecting arm includes iii) Peptides containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) Peptides containing a second light chain variable region (VL) and a second light chain constant region (CL), The second VH comprises the second HCDR1, HCDR2 and HCDR3 sequences, and the second VL comprises the second LCDR1, LCDR2 and LCDR3 sequences, wherein the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NO:12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NO:16, 17 and 18, respectively. In the first CH, positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively; in the second CH, positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively; and In the first CH, the amino acid corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second CH, the amino acid corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R.

[0354] In some implementations, the binder comprises 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 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 with the amino acid sequence shown in SEQ ID NO: 26. iii) A second 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 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 with the amino acid sequence shown in SEQ ID NO: 20.

[0355] In some implementations, the binder comprises i) The first heavy chain, which contains the amino acid sequence shown in SEQ ID NO: 25, ii) A first light chain comprising the amino acid sequence shown in SEQ ID NO: 26. iii) A second chain comprising the amino acid sequence shown in SEQ ID NO: 19; and iv) The second light chain contains the amino acid sequence shown in SEQ ID NO: 20.

[0356] In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR3 sequence, the HCDR3 sequence containing the sequence shown in SEQ ID NO: 4. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR2 sequence, the HCDR2 sequence containing the sequence shown in SEQ ID NO: 93. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing an HCDR1 sequence, the HCDR1 sequence containing the sequence shown in SEQ ID NO: 2. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences contain the sequences shown in SEQ ID NO: 2, 93, and 4, respectively. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 64, 96, and 66, respectively. In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 74, 93, and 66, respectively.

[0357] In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR3 sequence, the LCDR3 sequence containing the sequence shown in SEQ ID NO: 8. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR2 sequence, the LCDR2 sequence containing the sequence shown in SEQ ID NO: 95. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing an LCDR1 sequence, the LCDR1 sequence containing the sequence shown in SEQ ID NO: 94. In some embodiments, the first antigen-binding region binding to EpCAM includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences contain the sequences shown in SEQ ID NO: 94, 95, and 8, respectively. In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences respectively contain sequences as shown in SEQ ID NO: 97, 98, and 8.

[0358] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 2, 93, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 94, 95, and 8, respectively.

[0359] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 64, 96, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 97, 98, and 8, respectively.

[0360] In some embodiments, the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 74, 93, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 94, 95, and 8, respectively.

[0361] In some embodiments, the first antigen-binding region binding to EpCAM includes a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region (VH) includes HCDR1, HCDR2, and HCDR3 sequences at positions 1-115 of SEQ ID NO: 27, and the light chain variable region (VL) includes LCDR1, LCDR2, and LCDR3 sequences at positions 1-108 of SEQ ID NO: 28.

[0362] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown at positions 1-115 of SEQ ID NO: 27.

[0363] In some embodiments, the first antigen-binding region that binds to EpCAM includes a heavy chain variable region (VH) containing the sequence shown in positions 1-115 of SEQ ID NO: 27.

[0364] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown at positions 1-108 of SEQ ID NO: 28.

[0365] In some embodiments, the first antigen-binding region that binds to EpCAM includes a light chain variable region (VL) containing sequences as shown in positions 1-108 of SEQ ID NO: 28.

[0366] In some embodiments, the first antigen-binding region binding 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 with the sequences shown 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 with the sequences shown in SEQ ID NO: 28 positions 1-108. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in SEQ ID NO: 27 positions 1-115, and the VL comprises a sequence having at least 80% identity with the sequences shown in SEQ ID NO: 28 positions 1-108. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in positions 1-115 of SEQ ID NO: 27, and the VL comprises a sequence having at least 90% identity with the sequences shown in positions 1-108 of SEQ ID NO: 28. In some embodiments, the first antigen-binding region binding 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 with the sequences shown in positions 1-115 of SEQ ID NO: 27, and the VL comprises a sequence having at least 95% identity with the sequences shown in positions 1-108 of SEQ ID NO: 28.

[0367] 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 as shown in positions 1-115 of SEQ ID NO: 27, and the VL comprises a sequence as shown in positions 1-108 of SEQ ID NO: 28.

[0368] In some embodiments, the first antigen-binding region that binds to EpCAM comprises heavy chain and light chain variable regions of an antibody, which competes with antibodies comprising the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding and / or for the specificity of antibodies containing the heavy chain variable region (VH) and / or light chain variable region (VL) as shown above for EpCAM binding.

[0369] In some implementations, the second antigen-binding region that binds to CD137 is shown above.

[0370] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 2, 93, and 4, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 94, 95, and 8; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0371] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 64, 96, and 66, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 97, 98, and 8; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 69, 70 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 72, 73 and 18, respectively.

[0372] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 74, 93, and 66, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 94, 95, and 8; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 75, 13 and 71, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

[0373] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the HCDR1, HCDR2, and HCDR3 sequences at positions 1-115 of SEQ ID NO: 27, and the light chain variable region (VL) comprises the LCDR1, LCDR2, and LCDR3 sequences at positions 1-108 of SEQ ID NO: 28; 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 heavy chain variable region (VH) comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 11, and the light chain variable region (VL) comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 15.

[0374] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 positions 1-115 of SEQ ID NO: 27, and the 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 positions 1-108 of SEQ ID NO: 28; and b) The second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the 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 SEQ ID NO: 11, and the 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 SEQ ID NO: 15.

[0375] In some implementation schemes, a) The first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence shown in positions 1-115 of SEQ ID NO: 27, and the VL comprises the sequence shown in positions 1-108 of SEQ ID NO: 28; 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 shown in SEQ ID NO: 11, and the VL comprises the sequence shown in SEQ ID NO: 15.

[0376] In some embodiments, the binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region that binds to EpCAM, and the second binding arm comprising a second antigen-binding region that binds to CD137, wherein... The first connecting arm includes: i) Peptides containing a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) A polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), The first VH comprises the first HCDR1, HCDR2 and HCDR3 sequences, and the first VL comprises the first LCDR1, LCDR2 and LCDR3 sequences, wherein the first HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 2, 93 and 4, respectively, and the first LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NO: 94, 95 and 8, respectively. And the second connecting arm includes iii) Peptides containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) Peptides containing a second light chain variable region (VL) and a second light chain constant region (CL), The second VH comprises the second HCDR1, HCDR2 and HCDR3 sequences, and the second VL comprises the second LCDR1, LCDR2 and LCDR3 sequences, wherein the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NO:12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NO:16, 17 and 18, respectively. In the first CH, positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively; in the second CH, positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively; and In the first CH, the amino acid corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second CH, the amino acid corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R.

[0377] In some implementations, the binder comprises 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 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 with the amino acid sequence shown in SEQ ID NO: 28. iii) A second 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 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 with the amino acid sequence shown in SEQ ID NO: 20.

[0378] In some implementations, the binder comprises i) The first heavy chain, which contains the amino acid sequence shown in SEQ ID NO: 27, ii) A first light chain comprising the amino acid sequence shown in SEQ ID NO: 28. iii) A second chain comprising the amino acid sequence shown in SEQ ID NO: 19; and iv) The second light chain contains the amino acid sequence shown in SEQ ID NO: 20.

[0379] In some embodiments, the first antigen-binding region binding to EpCAM comprises a heavy chain and a light chain variable region of an antibody, the antibody competing with antibodies comprising a heavy chain variable region or a light chain variable region or a combination thereof of the first antigen-binding region binding to EpCAM as described above for EpCAM binding and / or having specificity for EpCAM against antibodies comprising a heavy chain variable region or a light chain variable region or a combination thereof of the first antigen-binding region binding to EpCAM as described above; and the second antigen-binding region binding to CD137 comprises a heavy chain and a light chain variable region of an antibody, the antibody competing with antibodies comprising a heavy chain variable region or a light chain variable region or a combination thereof of the second antigen-binding region binding to CD137 as described above for CD137 binding and / or having specificity for CD137 against antibodies comprising a heavy chain variable region or a light chain variable region or a combination thereof of the second antigen-binding region binding to CD137 as described above.

[0380] In some implementations, the variable region comprises three complementary decision regions (CDR1, CDR2, and CDR3) and four frame regions (FR1, FR2, FR3, and FR4).

[0381] In some embodiments, the complementary determining regions and the framework regions are arranged from the amino-terminus to the carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0382] In some implementations, the binder is in the form of a full-length antibody or an antibody fragment.

[0383] In some implementations, the binder is a multispecific binder, such as a bispecific binder.

[0384] In some implementations, the binder is a multispecific antibody, such as a bispecific antibody.

[0385] In some implementations, the binder comprises (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.

[0386] In some implementations, the binder comprises 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 a second antigen-binding region that binds to CD137.

[0387] In some embodiments, the binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region that binds to EpCAM, and the second binding arm comprising a second antigen-binding region that binds to CD137, wherein... The first connecting arm includes: i) Peptides containing a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) A polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL); And the second connecting arm includes iii) Peptides containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) A polypeptide containing a second light chain variable region (VL) and a second light chain constant region (CL).

[0388] In some embodiments, the binder is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region that binds to EpCAM, and the second binding arm comprising a second antigen-binding region that binds to CD137, wherein... The first connecting arm includes: i) The first heavy chain containing the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and ii) A first light chain comprising a first light chain variable region (VL) and a first light chain constant region (CL); And the second connecting arm includes i) A second heavy chain containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH); and ii) A second light chain containing a second light chain variable region (VL) and a second light chain constant region (CL).

[0389] 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, both 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, both the first and second binding arms are derived from monoclonal antibodies.

[0390] In some embodiments, each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) includes one or more of the constant heavy chain 1 (CH1) region, the hinge region, the constant heavy chain 2 (CH2) region and the constant heavy chain 3 (CH3) region, preferably including at least the hinge region, the CH2 region and the CH3 region.

[0391] In some implementations, each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) contains a CH3 region, and the two CH3 regions contain asymmetric mutations.

[0392] In some embodiments, in the first heavy chain constant region (CH), at least one amino acid at a position corresponding to T366, L368, K370, D399, F405, Y407 and K409 of the human IgG1 heavy chain according to EU numbers has been substituted, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to T366, L368, K370, D399, F405, Y407 and K409 of the human IgG1 heavy chain according to EU numbers has been substituted, and wherein the substitutions in the first heavy chain and the second heavy chain do not occur at the same positions.

[0393] In some embodiments, (i) in the first heavy chain constant region (CH), the amino acid at position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second heavy chain constant region (CH), the amino acid at position K409 in the human IgG1 heavy chain according to the EU number is R, or (ii) in the first heavy chain constant region (CH), the amino acid at position K409 in the human IgG1 heavy chain according to the EU number is R, and in the second heavy chain constant region (CH), the amino acid at position F405 in the human IgG1 heavy chain according to the EU number is L.

[0394] In some implementations, the binder induces Fc-mediated effector function to a lesser extent compared to another antibody containing the same first antigen-binding region and second antigen-binding region, as well as two heavy chain constant regions (CH) containing the human IgG1 hinge region, CH2 region, and CH3 region.

[0395] In some embodiments, the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to the same antibody except that it contains unmodified first heavy chain constant regions (CH) and second heavy chain constant regions (CH).

[0396] In some embodiments, each of the unmodified first heavy chain constant region (CH) and second heavy chain constant region (CH) contains the amino acid sequence shown in SEQ ID NO: 47.

[0397] In some implementations, the Fc-mediated effector function is measured by binding to the Fcγ receptor, binding to C1q, or inducing Fc-mediated FcR crosslinking.

[0398] In some implementations, the Fc-mediated effector function is measured by combining it with C1q.

[0399] In some embodiments, the first heavy chain constant region and the second heavy chain constant region have been modified to reduce the binding of C1q to the antibody compared to the 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.

[0400] In some embodiments, in at least one of the first heavy chain constant regions (CH) and the second heavy chain constant regions (CH), one or more amino acids at positions corresponding to L234, L235, D265, N297, P331 and G236 in the human IgG1 heavy chain according to EU number are not L, L, D, N, P and G, respectively.

[0401] In some implementations, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering in the first and second heavy chains are F and E, respectively.

[0402] In some embodiments, the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbers in the first heavy chain constant region (HC) and / or the second heavy chain constant region (HC) are F, E, and A, respectively, and / or the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to EU numbers in the first heavy chain constant region (HC) and / or the second heavy chain constant region (HC) are F, E, and R, respectively.

[0403] In some implementation schemes, (i) In the first heavy chain constant region and the second heavy chain constant region, the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to the EU number are F, E and A, respectively; (ii) In the first and second heavy chain constant regions, the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to EU numbers are F, E, and R, respectively; or (iii) In one of the first heavy chain constant region and the second heavy chain constant region, the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to the EU number are F, E and A, respectively, and in the other of the first heavy chain constant region and the second heavy chain constant region, the positions corresponding to positions L234, L235 and G236 in the human IgG1 heavy chain according to the EU number are F, E and R, respectively.

[0404] In some embodiments, the positions of the first heavy chain constant region and the second heavy chain constant region corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU number are F and E, respectively, and wherein (i) the position of the first heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and the position of the second heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R, or (ii) the position of the first heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R, and the position of the second heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L.

[0405] In some embodiments, in the second heavy chain constant region (HC), the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively, and in the first heavy chain constant region (HC), the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively, and wherein (i) the position in the first heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and the position in the second heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R, or (ii) the position in the first heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R, and the position in the second heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L.

[0406] In some embodiments, in the second heavy chain constant region (HC), the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively, and in the first heavy chain constant region (HC), the positions corresponding to L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively, and wherein the position in the first heavy chain constant region corresponding to F405 in the human IgG1 heavy chain according to the EU number is L, and the position in the second heavy chain constant region corresponding to K409 in the human IgG1 heavy chain according to the EU number is R.

[0407] In some embodiments, the constant region of the first heavy chain and / or the second heavy chain contains 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: 47.

[0408] In some implementation schemes, a) The constant region of the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 54; and b) The constant region of the second heavy chain contains the amino acid sequence shown in SEQ ID NO: 52.

[0409] In some embodiments, the binder comprises a constant region of the kappa (κ) light chain.

[0410] In some embodiments, the binder comprises a constant region of a lambda (λ) light chain.

[0411] In some implementations, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0412] In some implementations, the second light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0413] In some implementation schemes, (i) The first light chain constant region and the second light chain constant region are kappa (κ) light chain constant regions. (ii) The first light chain constant region and the second light chain constant region are lambda(λ) light chain constant regions. (iii) The first light chain constant region is the kappa (κ) light chain constant region, and the second light chain constant region is the lambda (λ) light chain constant region, or (iv) The first light chain constant region is the lambda (λ) light chain constant region, and the second light chain constant region is the kappa (κ) light chain constant region.

[0414] 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 with the amino acid sequence shown in SEQ ID NO:55.

[0415] 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 with the amino acid sequence shown in SEQ ID NO: 56.

[0416] In some embodiments, the binder has an isotype selected from IgG1, IgG2, IgG3, and IgG4. In one embodiment, the isotype is selected from human IgG1, human IgG2, human IgG3, and human IgG4.

[0417] In some implementations, the binding agent is a full-length IgG1 antibody.

[0418] In some implementations, the binding agent is an antibody of the IgG1m(f) allotype.

[0419] The binding agents disclosed herein can, in principle, be any isotype of antibody. The choice of isotype is typically guided by the desired Fc-mediated effector function, such as ADCC induction, or requires an antibody without Fc-mediated effector function (“inert” antibody). Exemplary isotypes include IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant region κ or λ can be used. The effector function of the antibodies described herein can be altered by isotype conversion to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies for various therapeutic uses. In one embodiment, both heavy chains of the antibody described herein are of the IgG1 isotype, such as IgG1κ. Optionally, the heavy chains can be modified in the hinge region and / or CH3 region, as described elsewhere herein.

[0420] Preferably, each of the antigen-binding regions comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each of the variable regions comprises three CDR sequences, namely CDR1, CDR2, and CDR3, and four frame sequences, namely FR1, FR2, FR3, and FR4. Furthermore, preferably, the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0421] In one embodiment, the binder is a full-length antibody, such as a full-length IgG1 antibody. For example, in one embodiment, the binder (e.g., a bispecific antibody) comprises two half-molecules, each containing an antigen-binding region.

[0422] Many different forms of bispecific antibodies are known in the art and have been reviewed by Kontermann; DrugDiscov Today, 2015 Jul;20(7):838-47 and; MAbs, 2012 Mar-Apr;4(2):182-97. All of these forms are covered herein. Bispecific antibodies according to this disclosure are not limited to any particular bispecific form or method of manufacture.

[0423] Examples of bispecific antibody molecules that can be used in this disclosure include (i) monoclonal antibodies having two arms containing different antigen-binding regions; (ii) single-chain antibodies that are specific for two different epitopes, for example, by two scFvs tandemly linked by an additional peptide linker; (iii) bivariate domain antibodies (DVD-Ig) wherein each light chain and heavy chain contains two variable domains tandemly linked by short peptide bonds (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')2 fragments; (v) Tandab, which is a fusion of two single-chain biantibodies to produce a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) flexible antibodies, which are combinations of scFvs with biantibodies to produce multivalent molecules; (vii) so-called "dock and lock" antibodies. (viii) The so-called scorpion molecule, which is based on the dimerization and docking domain in protein kinase A, and when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; and (ix) the biantibody.

[0424] In one embodiment, the binding agent described herein is a bispecific antibody or a cross-body antibody. In one embodiment, the binding agent is a bispecific antibody obtained through controlled Fab-arm exchange (as described in WO2011131746 (Genmab)).

[0425] Examples of different classes of binders include, but are not limited to: (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual-targeting molecules, wherein each side of the molecule contains a Fab fragment or a portion of a Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, wherein a full-length IgG antibody is fused with an additional Fab fragment or a portion of a Fab fragment; (iv) Fc fusion molecules, wherein a single-chain Fv molecule or a stable biantibody is fused with a heavy chain constant domain, Fc region, or a portion thereof; (v) Fab fusion molecules, wherein different Fab fragments are fused together with a heavy chain constant domain, Fc region, or a portion thereof; and (vi) antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and biantibodies, wherein different single-chain Fv molecules or different biantibodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused together or fused with another protein or carrier molecule to a heavy chain constant region, Fc region, or a portion thereof.

[0426] Examples of IgG-like molecules with complementary CH3 domains include, but are not limited to, the Triomab / Quadroma molecule (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), the so-called Knobs-into-Holes molecule (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatically matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), and the LUZ-Y molecule (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869). Epub 2012 Nov 1), DIG and PIG molecules (Pharmabcine, WO2010134666, WO2014081202), SEEDbody (EMD Serono, WO2007110205), Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron, WO201015792), Bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecule (Xencor, WO2011028952), bivalent bispecific antibody (WO2009080254), and DuoBody® molecule (Genmab, WO2011131746).

[0427] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, dual-targeting (DT)-Ig molecules (WO2009058383), two-in-one antibodies (Genentech; Bostrom, et al 2009. Science 323, 1610–1614.), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), using a common light chain approach (Crucell / Merus, US7,262,028), κλ bodies (NovImmune, WO2012023053), and CovX bodies (CovX / Pfizer; Doppalapudi, VR, et al 2007. Bioorg. Med. Chem. Lett.). 17,501–506.

[0428] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig molecules (Abbott, US7,612,181), dual domain biheaded antibodies (Unilever; Sanofi Aventis, WO20100226923), 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), BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), and scFv fusion molecules (Changzhou Adam Biotech Inc, CN). 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0429] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Pearce et al., Biochem MolBiol 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, WO2010111625), dual affinity redirection technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538), and bis(ScFv)2-Fab molecules (National Research Center for Antibody Medicine – China).

[0430] Examples of Fab fusion 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.), docking and locking (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7.), and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[0431] Examples of ScFv-based, bispecific, and domain-based antibodies include, but are not limited to, bispecific T cell enzymatic (BiTE) molecules (Micromet, WO2005061547), tandem bispecific antibody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66., dual affinity redirection (DART) molecules (MacroGenics, WO2008157379, WO2010080538), single-chain bispecific antibody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO2010059315), and COMBODY molecules (Epigen Biotech, Zhu et 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 domain-only antibodies.

[0432] In one embodiment, the bispecific antibody of this disclosure comprises a first Fc sequence and a second Fc sequence, the first Fc sequence comprising a first CH3 region and the second Fc sequence comprising a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different, and such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the respective homodimeric interaction between the first CH3 region and the second CH3 region. WO2011131746 and WO2013060867 (Genmab) provide further details on these interactions and how to achieve them, which are incorporated herein by reference.

[0433] As further described herein, a specific method based on a homodimer-initiated EpCAM antibody and a homodimer-initiated CD137 antibody containing only a few conserved asymmetric mutations in the CH3 region can be used to obtain stable bispecific EpCAMxCD137 antibodies in high yield. The asymmetric mutation indicates that the sequences of the first and second CH3 regions contain amino acid substitutions at different positions.

[0434] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first CH3 region and a second CH3 region, the first CH3 region having an amino acid substitution at positions selected from human IgG1 heavy chain 366, 368, 370, 399, 405, 407 and 409, and the second CH3 region having an amino acid substitution at positions selected from human IgG1 heavy chain 366, 368, 370, 399, 405, 407 and 409, and wherein the first CH3 region and the second CH3 region are not substituted at the same positions.

[0435] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises sequences of a first CH3 region and a second CH3 region, said sequences containing an asymmetric mutation, i.e., a mutation at different positions in the two CH3 regions, for example, a mutation at position 405 in one CH3 region 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.

[0436] In one embodiment, the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein each of the first and second heavy chains comprises at least a hinge region, a CH2 region, and a CH3 region, wherein (i) in the first heavy chain, the amino acid at position F405 corresponding to the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at position K409 corresponding to the human IgG1 heavy chain is R, or (ii) in the first heavy chain, the amino acid at position K409 corresponding to the human IgG1 heavy chain is R, and in the second heavy chain, the amino acid at position F405 corresponding to the human IgG1 heavy chain is L.

[0437] Conventional methods such as hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used to prepare the bispecific antibodies described herein. The co-expression of two antibodies, composed of different heavy and light chains, in host cells results in a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be separated by, for example, affinity chromatography or similar methods.

[0438] When co-expressing different antibody constructs, strategies that favor the formation of functional bispecific products can also be used, such as the method described by Lindhofer et al. (1995 J Immunol 155:219). The fusion of rat and mouse hybridomas producing different antibodies results in a limited number of heterodimeric proteins due to preferred species-restricted heavy / light chain pairing. Another strategy to promote heterodimer formation over homodimer formation is the "knob-into-hole" strategy, in which a protrusion is introduced on the first heavy chain polypeptide and a corresponding cavity is introduced on the second heavy chain polypeptide, so that the protrusion can be positioned in the cavity at the interface of the two heavy chains, thereby promoting heterodimer formation and inhibiting homodimer formation. The "protrusion" is constructed by replacing the small amino acid side chain from the first polypeptide interface with a larger side chain. A compensating "cavity" of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (US Patent 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies that favor heterodimer formation when different antibody domains are co-expressed in host cells. In these methods, one or more residues constituting the CH3-CH3 interface in the two CH3 domains are replaced with charged amino acids, thus making homodimer formation electrostatically unfavorable and heterodimerization electrostatically favorable. WO2007110205 (Merck) describes another strategy in which the difference between the IgA and IgG CH3 domains is utilized to promote heterodimerization.

[0439] Another in vitro method for generating bispecific antibodies has been described in WO2008119353 (Genmab), in which the bispecific antibody is formed by the exchange of "Fab-arms" or "half-molecules" (exchange of heavy chain and linked light chain) during incubation of two monospecific IgG4 or IgG4-like antibodies under reducing conditions. The resulting product is a bispecific antibody with two Fab arms, which may contain different sequences.

[0440] Preferred methods for preparing the bispecific EpCAMxCD137 antibody of this disclosure include the methods described in WO2011131746 and WO2013060867 (Genmab), which include the following steps: a) Provide a first antibody comprising an Fc region, wherein the Fc region comprises a first CH3 region; b) Provide a second antibody comprising a second Fc region, wherein the 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 CH3 region and the second CH3 region are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction between the first CH3 region and the second CH3 region. c) Incubate the first antibody and the second antibody together under reducing conditions; and d) Obtain the bispecific EpCAMxCD137.

[0441] Similarly, a method for preparing an antibody according to this disclosure is provided, comprising the following steps: a) Culture host cells that produce a first antibody, the first antibody containing an antigen-binding region as defined herein that is capable of binding to human EpCAM, and purify the first antibody from the culture; b) Culture host cells that produce a second antibody containing an antigen-binding region as defined herein that is capable of binding to human CD137, and purify the second antibody from the culture; c) Incubate the first antibody and the second antibody together under reducing conditions sufficient to cause disulfide isomerization of cysteine ​​in the hinge region, and d) Obtain the bispecific antibody.

[0442] In one embodiment of this disclosure, the first antibody and the second antibody are incubated together under reducing conditions sufficient to cause disulfide isomerization of cysteine ​​in the hinge region, wherein the heterodimeric interaction between the first antibody and the second antibody in the resulting heterodimeric antibody is such that no Fab-arm exchange occurs after 24 hours at 37°C under 0.5 mM GSH.

[0443] Unrestricted by theory, in step c), the heavy-chain disulfide bonds in the hinge region of the parent antibody are reduced, and the resulting cysteine ​​is then able to form an inter-heavy-chain disulfide bond with a cysteine ​​residue of another parent antibody molecule (initially with different specificity). In one embodiment of the method, the reduction conditions in step c) include the addition of a reducing agent, such as those selected from 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In another embodiment, step c) includes recovery conditions to become non-reducing or less reducing, for example by removing the reducing agent, such as by desalting.

[0444] As described above, in one embodiment, the sequences of the first CH3 region and the second CH3 region of the homodimer initiating antibody are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the individual homodimer interaction between the first CH3 region and the second CH3 region. WO2011131746 and WO2013060867 (Genmab) provide further details on these interactions and how to achieve them, which are incorporated herein by reference in their entirety.

[0445] Specifically, the above-described method, based on two homodimer-initiating antibodies that bind to EpCAM and CD137 respectively and contain only a few asymmetric mutations in the CH3 region, can be used to obtain stable bispecific EpCAMxCD137 antibodies in high yield. The asymmetric mutations indicate that the sequences in the first and second CH3 regions contain amino acid substitutions at different positions.

[0446] In some embodiments, in addition to the antigen-binding region, the binder according to this disclosure also includes an Fc region consisting of Fc sequences of two heavy chains.

[0447] The first Fc sequence and the second Fc sequence can each be 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 Fc sequence and the second Fc sequence is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, each of the first Fc sequence and the second Fc sequence is an IgG4 isotype or derived therefrom, optionally having one or more mutations or modifications. In another embodiment, one of the Fc sequences is an IgG1 isotype and the other is an IgG4 isotype, or derived from these respective isotypes, optionally having one or more mutations or modifications.

[0448] In one implementation, one or both Fc sequences are defective effector functions. For example, the Fc sequence may be an IgG1 isotype, or a non-IgG1 isotype, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate the ability to mediate effector functions such as ADCC.

[0449] As used herein, the term "effective function" includes any function mediated by components of the immune system that results in, for example, the killing of diseased cells such as tumor cells, or the inhibition of tumor growth and / or the suppression of tumor development, including the inhibition of tumor spread and metastasis. Preferably, in the context of this disclosure, effector functions are T-cell-mediated effector functions. Such functions include ADCC, ADCP, or CDC.

[0450] Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0451] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to the killing of antibody-coated target cells by cytotoxic effector cells through a non-phagocytic process, characterized by the release of the contents of cytotoxic granules or the expression of cell death-inducing molecules. ADCC is independent of the immune complement system, which also cleaves targets but does not require any other cells. ADCC is triggered by the interaction of target-binding antibodies (belonging to the IgG, IgA, or IgE class) with certain Fc receptors (FcRs), glycoproteins present on the surface of effector cells that bind to the Fc region of immunoglobulins (Ig). Effector cells mediating ADCC include natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils, and dendritic cells. ADCC is a rapid effector mechanism whose potency depends on many parameters (density and stability of antigens on the target cell surface; antibody affinity; and FcR binding affinity). ADCC involving human IgG1 (the most commonly used IgG subclass in therapeutic antibodies) is highly dependent on the glycosylation profile of its Fc moiety and the polymorphism of the Fcγ receptor.

[0452] Antibody-dependent phagocytosis (ADCP)

[0453] ADCP is a key mechanism of action in many antibody therapies. It is defined as a highly regulated process in which antibodies eliminate their bound targets by attaching their Fc domains to specific receptors on phagocytes and triggering phagocytosis. Unlike ADCC, ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRIIa, FcγRI, and FcγRIIIa, with FcγRIIa (CD32a) on macrophages representing the primary pathway.

[0454] Complement-dependent cytotoxicity (CDC)

[0455] CDC is another antibody-guided cell-killing mechanism. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also highly effective in guiding CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of the antigen-antibody complex leads to the C15 activation of participating antibody molecules such as IgG molecules. H2. Multiple C1q binding sites (C1q is one of the three subcomponents of complement C1) are exposed in close proximity on the 2-domain. Preferably, these exposed C1q binding sites transform previously low-affinity C1q-IgG interactions into high-affinity C1q-IgG interactions, triggering a cascade of events involving a series of other complement proteins and leading to the proteolytic release of effector cell chemokines / activators C3a and C5a. Preferably, the complement cascade terminates in the formation of a membrane attack complex that creates pores in the cell membrane, facilitating the free movement of water and solutes into and out of the cell.

[0456] The antibodies described herein may contain modifications in their Fc regions. When an antibody contains such modifications, it can be an inert or non-activating antibody. As used herein, the terms "inert," "dormant," or "non-activating" refer to an Fc region that cannot bind to any Fcγ receptor, cannot induce Fc-mediated FcR crosslinking, or cannot induce FcR-mediated target antigen crosslinking through the two Fc regions of a single antibody, or cannot bind C1q. The inertness of the Fc region of humanized or chimeric EpCAM or CD137 antibodies can be advantageously tested using monospecific forms of antibodies.

[0457] Several variants can be constructed to render the Fc region of the antibody inactive for interaction with the Fcγ (gamma) receptor and C1q, for use in therapeutic antibody development. Examples of such variants are described in this article.

[0458] Therefore, in one embodiment of the antibody described herein, the antibody comprises a first heavy chain and a second heavy chain, wherein one or both heavy chains are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an identical antibody except that it comprises an unmodified first and second heavy chain. The Fc-mediated effector function can be determined, measured by binding to an Fcγ receptor, by binding to C1q, or by inducing Fc-mediated FcR crosslinking.

[0459] In another such embodiment, the constant sequences of the heavy and light chains have been modified such that the binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the unmodified antibody, wherein C1q binding is determined by ELISA.

[0460] Therefore, amino acids in the Fc region, which play a dominant role in the interaction with C1q and Fcγ receptors, can be modified.

[0461] For example, in IgG1 isotype antibodies, examples of amino acid positions that can be modified include positions L234, L235, G236, D265, and P331. Combinations of these, such as L234F / L235E / D265A or L234F / L235E / G236R, can lead to a significant reduction in binding to human CD64, CD32, CD16, and C1q. Such modifications and their effects are described, for example, in WO2022189667.

[0462] Amino acid substitutions at L234F and L235E can lead to the loss of interaction between the Fc region and the Fcγ receptor and C1q (Canfield et al., 1991, J. Exp. Med. (173):1483-91; Duncan et al., 1988, Nature (332):738-40). Therefore, in one embodiment, the amino acids corresponding to the L234 and L235 positions can be F and E, respectively. Amino acid substitutions at D265A or G236R can reduce binding to all Fcγ receptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604; Wilkinson et al., 2021, PLOS 1 (16(12)) e0260954). Therefore, in one embodiment, the amino acid corresponding to the D265 position can be A, or the amino acid corresponding to the G236 position can be R.

[0463] In one embodiment of this disclosure, the amino acids at positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively, in the first and second heavy chains.

[0464] In one embodiment, in one or both of the first and second heavy chains, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0465] In one embodiment, in one or both of the first and second heavy chains, the amino acids corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain are F, E, and R, respectively.

[0466] The L234F-L235E-D265A inactive mutation (also referred to herein as FEA or the FEA form) has shown excellent safety profiles and a strong ability to inhibit Fc-mediated effector function. However, it has been observed that antibodies carrying the FEA mutation can exhibit some residual CDC for IgG1 antibodies, which are potent inducers of complement-dependent cytotoxicity (CDC). Furthermore, recombinant antibodies with the FEA form have been observed to exhibit increased glycosylation heterogeneity due to additional processing of their N-glycans compared to the wild-type IgG1 Fc region, and these antibodies have also shown greater sensitivity to aggregation induced by low pH conditions. Meanwhile, when the mutations L234F, L235E, and G236R are combined in IgG1 antibodies (also referred to herein as FER or the FER form), this results in an improved inert form that avoids potential residual CDC activity, provides wild-type-like glycosylation, and improves tolerance to low pH conditions. Therefore, the FER form is a highly advantageous inactive antibody form that is well-suited for clinical development and clinical use. For bispecific antibodies, this FER inert form, relative to the inert form substitution, can also be combined in heterodimeric form. For example, a bispecific antibody may comprise one chain carrying the inert form substitution, while the other chain may contain a different inert form substitution, such as FEA. Therefore, the FER inert form is well-suited for combination, for example, with existing candidate antibodies that have undergone clinical development without requiring redesign and re-performance of all necessary assays, thus allowing for the rapid generation of bispecific antibodies using techniques such as controlled Fab arm exchange. Such modifications and their effects are described, for example, in WO2022189667.

[0467] In a particularly preferred embodiment, in one of the first heavy chain and the second heavy chain, the amino acids corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively, and in the other of the first heavy chain and the second heavy chain, the amino acids corresponding to positions L234, L235 and G236 in the human IgG1 heavy chain are F, E and R, respectively.

[0468] In another particularly preferred embodiment, the binder is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions of the first heavy chain and the second heavy chain corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU number are F and E, respectively, and wherein (i) the position of the first heavy chain corresponding to position F405 in the human IgG1 heavy chain according to EU number is L, and the position of the second heavy chain corresponding to position K409 in the human IgG1 heavy chain according to EU number is R, or (ii) the position of the first heavy chain corresponding to position K409 in the human IgG1 heavy chain according to EU number is R, and the position of the second heavy chain corresponding to position F405 in the human IgG1 heavy chain according to EU number is L.

[0469] In another particularly preferred embodiment, the binder is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein (1) The first and second heavy chains correspond to positions F, E, and A in the human IgG1 heavy chain according to EU numbering, specifically positions L234, L235, and D265. (2) The positions of the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to EU numbering are F, E, and R, respectively. (3) The positions of one of the first and second heavy chains corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively, and the positions of the other of the first and second heavy chains corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively. And wherein (i) the position of the first heavy chain corresponding to F405 in the human IgG1 heavy chain according to the EU number is L, and the position of the second heavy chain corresponding to K409 in the human IgG1 heavy chain according to the EU number is R, or (ii) the position of the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to the EU number is R, and the position of the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to the EU number is L.

[0470] Antibody variants with combinations of three amino acid substitutions L234F, L235E, and D265A, as well as additional K409R or F405L mutations, are named in this paper with the suffixes “FEAR” or “FEAL”, respectively.

[0471] Antibody variants with combinations of three amino acid substitutions L234F, L235E, and G236R, as well as additional K409R or F405L mutations, are named in this paper with the suffixes “FERR” or “FERL”, respectively.

[0472] In a preferred embodiment, the bispecific antibody described herein comprises: (i) Half-molecule antibodies derived from IgG1-EpCAM-FEAL and half-molecule antibodies derived from IgG1-CD137-FEAR, (ii) Half-molecule antibodies derived from IgG1-EpCAM-FEAR and half-molecule antibodies derived from IgG1-CD137-FEAL, (iii) Half-molecule antibodies derived from IgG1-EpCAM-FERL and half-molecule antibodies derived from IgG1-CD137-FEAR, or (iv) Half-molecule antibodies derived from IgG1-EpCAM-FEAR and half-molecule antibodies derived from IgG1-CD137-FERL.

[0473] In another embodiment, the binder or antibody described herein is linked or conjugated to one or more therapeutic motifs, such as cytokines, immunosuppressants, immunostimulatory molecules, and / or radioisotopes. Such conjugates are referred to herein as “immunoconjugates” or “pharmaceutical conjugates.” Immunoconjugates comprising one or more cytotoxins are referred to as “immunotoxins.”

[0474] In one embodiment, the first Fc sequence and / or the second Fc sequence are conjugated to a drug or prodrug, or contain a receptor group for the drug or prodrug. Such a receptor group may be, for example, a non-natural amino acid.

[0475] In one embodiment of the binder that binds to EpCAM and CD137, EpCAM is human EpCAM, particularly human EpCAM containing the sequence shown in SEQ ID NO: 59. In one embodiment of the binder that binds to EpCAM and CD137, CD137 is human CD137, particularly human CD137 containing the sequence shown in SEQ ID NO: 62. In one embodiment, EpCAM is human EpCAM, and CD137 is human CD137. In one embodiment, EpCAM is human EpCAM containing the sequence shown in SEQ ID NO: 59, and CD137 is human CD137 containing the sequence shown in SEQ ID NO: 62.

[0476] In one embodiment of the binder, a) The first binding region that binds to human EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 5; and b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 11, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 15.

[0477] In one embodiment of the binder, a) The first binding region that binds to human EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 2, 3 and 4, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 6, 7 and 8, respectively; and b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 12, 13 and 14, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 16, 17 and 18, respectively.

[0478] In one embodiment of the binder, a) The first binding region that binds to human EpCAM comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 9, and the light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 10, and b) The second binding region that binds to human CD137 comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 19, and the light chain comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 20.

[0479] In one embodiment of the binder, a) The first binding region that binds to human EpCAM comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 10, and b) The second binding region that binds to human CD137 comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 19 and the light chain comprising the amino acid sequence shown in SEQ ID NO: 20.

[0480] In one embodiment of the binder, a) The first binding region that binds to human EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) contains an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, and the light chain variable region (VL) contains an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 5, and b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 11, and the light chain variable region (VL) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 15.

[0481] In one embodiment of the binder, a) The first binding region that binds to human EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 5, and b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 11 and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 15.

[0482] Specifically, the conjugate can be an antibody, such as a multispecific antibody, for example, a bispecific antibody. Furthermore, the conjugate can be in the form of a full-length antibody or an antibody fragment.

[0483] Further optimization of the binding agent is to use human antibodies or humanized antibodies.

[0484] Each variable region may contain three complementary determinant regions (CDR1, CDR2, and CDR3) and four frame regions (FR1, FR2, FR3, and FR4).

[0485] The complementarity-determining regions (CDRs) and framework regions (FRs) can be arranged from the amino-terminus to the carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0486] In one embodiment, the binder is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises i) A polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and ii) A polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL); And the second connecting arm includes iii) A polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and iv) A polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL).

[0487] In one embodiment, the binder comprises i) a first heavy chain and a first light chain comprising an antigen-binding region capable of binding to EpCAM, the first heavy chain comprising a first heavy chain constant region and the first light chain comprising a first light chain constant region; and ii) a second heavy chain and a second light chain comprising an antigen-binding region capable of binding to CD137, the second heavy chain comprising a second heavy chain constant region and the second light chain comprising a second light chain constant region.

[0488] Each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) may contain a CH3 region, wherein both CH3 regions contain asymmetric mutations. An asymmetric mutation means that the sequences of the first and second CH3 regions contain amino acid substitutions at different positions. For example, one of the first and second CH3 regions contains a mutation at position 405 in the human IgG1 heavy chain according to EU numbering, and the other of the first and second CH3 regions contains a mutation at position 409 in the human IgG1 heavy chain according to EU numbering.

[0489] In the first heavy chain constant region (CH), at least one amino acid at a position corresponding to T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU designations may be substituted, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU designations may be substituted. In a particular embodiment, the first and second heavy chains are not substituted at the same position (i.e., the first and second heavy chains contain asymmetric mutations).

[0490] In one embodiment of the binder, (i) in the first heavy chain constant region (CH), the amino acid at position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second heavy chain constant region (CH), the amino acid at position K409 in the human IgG1 heavy chain according to the EU number is R, or (ii) in the first heavy chain, the amino acid at position K409 in the human IgG1 heavy chain according to the EU number is R, and in the second heavy chain, the amino acid at position F405 in the human IgG1 heavy chain according to the EU number is L.

[0491] In one embodiment, the binder induces Fc-mediated effector function to a lesser extent compared to another antibody containing the same first antigen-binding region and second antigen-binding region, as well as two heavy chain constant regions (CH) containing the human IgG1 hinge region, CH2 region, and CH3 region.

[0492] In a particular embodiment of the binder, the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody that contains only unmodified first and second heavy chain constant regions (CH). Specifically, each or both of the unmodified first and second heavy chain constant regions (CH) may contain the amino acid sequence shown in SEQ ID NO: 47, consist of the amino acid sequence shown in SEQ ID NO: 47, or consist substantially of the amino acid sequence shown in SEQ ID NO: 47.

[0493] The function of Fc-mediated effectors can be determined by measuring the binding of the binder to the Fcγ receptor, its binding to C1q, or the induction of Fc-mediated Fcγ receptor crosslinking. In particular, the function of Fc-mediated effectors can be determined by measuring the binding of the binder to C1q.

[0494] The first and second heavy chain constant regions of the binder may be modified to reduce the binding of C1q to the antibody compared to the 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.

[0495] In one embodiment of the binder, in at least one of the first heavy chain constant region (CH) and the second heavy chain constant region (CH), one or more amino acids at positions corresponding to L234, L235, G236, D265, N297 and P331 in the human IgG1 heavy chain according to EU number are not L, L, G, D, N and P, respectively.

[0496] In one embodiment of the binder, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU number can be F and E, respectively, in the first and second heavy chains.

[0497] Specifically, in the first heavy chain constant region (HC) and / or the second heavy chain constant region (HC), the positions corresponding to L234, L235, and G236 in the human IgG1 heavy chain according to the EU number can be F, E, and R, respectively, and / or in the first heavy chain constant region (HC) and / or the second heavy chain constant region (HC), the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to the EU number can be F, E, and A, respectively. In one embodiment, in one of the first heavy chain constant region (HC) and the second heavy chain constant region (HC), the positions corresponding to L234, L235, and G236 in the human IgG1 heavy chain according to the EU number can be F, E, and R, respectively, and in the other of the first heavy chain constant region (HC) and the second heavy chain constant region (HC), the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to the EU number can be F, E, and A, respectively. In one embodiment, in the first heavy chain constant region (HC), the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number can be F, E, and R, respectively, and in the second heavy chain constant region (HC), the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number can be F, E, and A, respectively.

[0498] In one embodiment of the binder, the positions of the first heavy chain constant region and the second heavy chain constant region corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU number are F and E, respectively, wherein (i) the position of the first heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and the position of the second heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R, or (ii) the position of the first heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R, and the position of the second heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L.

[0499] In one embodiment of the binder, the positions of the first heavy chain constant region and the second heavy chain constant region corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU number are F, E, and A, respectively; the positions of the first heavy chain constant region and the second heavy chain constant region corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to EU number are F, E, and R, respectively; or one of the first heavy chain constant regions and the second heavy chain constant region corresponds to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU number as F, E, and A, respectively, and the other of the first heavy chain constant region and the second heavy chain constant region corresponds to positions L234, L235, and G236 in the human IgG1 heavy chain according to EU number as F, E, and R, respectively, wherein (i) the position of the first heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to EU number is L, and the position of the second heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to EU number is R, or (ii) The position of the first heavy chain corresponding to K409 in the human IgG1 heavy chain according to the EU number is R, and the position of the second heavy chain corresponding to F405 in the human IgG1 heavy chain according to the EU number is L.

[0500] In one embodiment, the binder comprises a constant region of the kappa (κ) light chain.

[0501] In one implementation, the binder comprises a constant region of a lambda (λ) light chain.

[0502] In one embodiment of the binder, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0503] In one embodiment of the binder, the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0504] In one embodiment of the binder, the first light chain constant region is a kappa (κ) light chain constant region, and the second light chain constant region is a kappa (κ) light chain constant region. In another embodiment of the binder, the first light chain constant region is a lambda (λ) light chain constant region, and the second light chain constant region is a lambda (λ) light chain constant region. In yet another embodiment of the binder, 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 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.

[0505] In one embodiment, the binder (particularly an antibody) has an isotype selected from IgG1, IgG2, IgG3, and IgG4. Specifically, the binder may be a full-length IgG1 antibody. In a preferred embodiment, the binder (particularly an antibody) is an IgG1m(f) allotype.

[0506] PD-1 axis binding antagonists "Immune checkpoints" refer to regulatory factors of the immune system, particularly co-stimulatory and inhibitory signals that regulate the amplitude and quality of T-cell activity. In some embodiments, immune checkpoints are inhibitory signals. In some embodiments, the interaction between inhibitory signals D-1 and PD-L1 and / or PD-1 and PD-L2 is involved.

[0507] PD-1 is primarily expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isotypes, and species homologs of hPD-1, and analogs that share at least one common epitope with hPD-1. "Programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2). As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. Similarly, the term "PD-L2" as used herein includes human PD-L2 (hPD-L2), variants, isotypes, and species homologs of hPD-L2, and analogs that share at least one common epitope with hPD-L2. The interaction between PD-1 and its ligands leads to a reduction in tumor-infiltrating lymphocytes, decreased T-cell receptor-mediated proliferation, and immune escape from cancer cells. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is also blocked.

[0508] As used herein, the term "immune checkpoint modulator" or "checkpoint regulator" refers to a molecule or compound that modulates the function of one or more checkpoint proteins. Immune checkpoint modulators are generally able to modulate the magnitude and / or duration of self-tolerance and / or immune responses. Preferably, immune checkpoint modulators modulate the function of one or more human checkpoint proteins and are therefore "human checkpoint modulators." Specifically, human checkpoint modulators are immune checkpoint inhibitors.

[0509] As used herein, "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or negatively regulates the expression of one or more checkpoint proteins, or completely or partially reduces, inhibits, interferes with, or negatively regulates the expression of one or more checkpoint proteins. In some embodiments, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In some embodiments, the immune checkpoint inhibitor binds to one or more molecules that regulate checkpoint proteins.

[0510] In some embodiments, immune checkpoint inhibitors block inhibitory signals associated with immune checkpoints. In some embodiments, immune checkpoint inhibitors are antibodies or fragments thereof that disrupt inhibitory signaling associated with immune checkpoints. In some embodiments, immune checkpoint inhibitors are small molecule inhibitors that disrupt inhibitory signaling. In some embodiments, immune checkpoint inhibitors are peptide-based inhibitors that disrupt inhibitory signaling.

[0511] In some implementations, immune checkpoint inhibitors are antibodies, fragments thereof, or antibody mimics that prevent interactions between checkpoint blocking proteins.

[0512] In some embodiments, as described herein, inhibition or blockage of inhibitory immune checkpoint signaling leads to prevention or reversal of immunosuppression and the establishment or enhancement of T-cell immunity. In some embodiments, as described herein, inhibition of immune checkpoint signaling reduces or suppresses dysfunction of the immune system. In some embodiments, as described herein, inhibition of immune checkpoint signaling reduces the degree of dysfunction of dysfunctional immune cells. In some embodiments, as described herein, inhibition of immune checkpoint signaling reduces the degree of dysfunction of dysfunctional T cells.

[0513] In some implementations, inhibitory immunomodulators (immune checkpoint blockers) are components of the PD-1 / PD-L1 / PD-L2 signaling pathway.

[0514] In some implementations, the inhibitory immunomodulator (immune checkpoint blocker) is a PD(L)-1 axis binding antagonist.

[0515] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction between a PD-1 axis binding pair and one or more of its binding pairs (such as PD-1, PD-L1, or PD-L2) to eliminate T-cell dysfunction caused by signal transduction along the PD-1 signaling axis – resulting in the restoration or enhancement of T-cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0516] In some embodiments, a PD-1 axis binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding pairs. In one specific embodiment, a PD-1 axis binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 axis binding antagonists include anti-PD-1, anti-PD-L1, and anti-PD-L2 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In some embodiments, a PD-1 axis binding antagonist reduces negative co-stimulatory signaling mediated by or through cell surface proteins expressed on T lymphocytes, thereby reducing the degree of dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-1 axis binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 axis binding antagonists are provided below.

[0517] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more binding pairs (such as PD-L1 and PD-L2). In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 with one or more binding pairs. In one specific embodiment, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In some embodiments, a PD-1 binding antagonist reduces negative co-stimulatory signaling mediated by or through cell surface proteins expressed on T lymphocytes, thereby reducing the degree of dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some implementations, the PD-1 binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 binding antagonists are provided below.

[0518] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more binding pairs (such as PD-1, B7-1). In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding pair. In one particular aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more binding pairs (such as PD-1, B7-1). In some embodiments, PD-L1 binding antagonists reduce PD-L1 signaling mediated by negative co-stimulatory signals, which are mediated by or through cell surface proteins expressed on T lymphocytes, thereby reducing the degree of dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, PD-L1 binding antagonists are anti-PD-L1 antibodies. Specific examples of PD-L1 binding antagonists are provided below.

[0519] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L2 with one or more binding pairs (such as PD-1). In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 with one or more binding pairs. In one particular aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L2 with one or more binding pairs (such as PD-1). In some embodiments, a PD-L2 binding antagonist reduces negative co-stimulatory signaling mediated by or through cell surface proteins expressed on T lymphocytes, thereby reducing the degree of dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some implementations, the PD-L2 binding antagonist is an immunoadhesive.

[0520] In some embodiments, the PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PD-L1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PD-L2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0521] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding pair. In one specific embodiment, the PD-1 ligand binding pair is PD-L1 and / or PD-L2.

[0522] In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding pair. In one particular aspect, the PD-L1 binding pair is PD-1 and / or B7-1.

[0523] In some implementations, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding pair. In one particular aspect, the PD-L2 binding pair is PD-1.

[0524] Antagonists can be antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, or oligopeptides.

[0525] In some implementations, the PD-1 axis binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).

[0526] Exemplary PD-1 axis-binding antagonists include, but are not limited to, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606 and US 2015 / 0079109), cimiprimab (Regeneron; see WO2015 / 112800) and lambrolizumab (e.g., disclosed in WO2008 / 156712 as hPD109A and its humanized derivatives h409A1, h409A16 and h409A17), AB137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (AffymetrixeBioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; see WO 20...

Claims

1. A method for treating or preventing a disease or condition in a subject, the method comprising: (i) The subject is administered a binding agent comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, and (ii) Administer a PD-1 axis binding antagonist to the subject. The administration of the binding agent is performed before, simultaneously with, or after the administration of the PD-1 axis binding antagonist.

2. The method of claim 1, wherein the EpCAM is human EpCAM and / or wherein the CD137 is human CD137.

3. The method of claim 1 or 2, wherein the first antigen-binding region that binds to EpCAM binds to EpCAM expressed on tumor cells.

4. The method of any one of claims 1-3, wherein the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) containing an HCDR3 sequence, said HCDR3 sequence comprising the sequence shown in SEQ ID NO:

4.

5. The method of any one of claims 1-4, wherein the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) containing an HCDR2 sequence, said HCDR2 sequence comprising the sequence shown in SEQ ID NO:

3.

6. The method of any one of claims 1-5, wherein the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) containing an HCDR1 sequence, said HCDR1 sequence comprising the sequence shown in SEQ ID NO:

2.

7. The method of any one of claims 1-6, wherein the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 2, 3 and 4, respectively.

8. The method of any one of claims 1-7, wherein the first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising an LCDR3 sequence comprising the sequence shown in SEQ ID NO:

8.

9. The method of any one of claims 1-8, wherein the first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising an LCDR2 sequence comprising the sequence shown in SEQ ID NO:

7.

10. The method of any one of claims 1-9, wherein the first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) containing an LCDR1 sequence, said LCDR1 sequence comprising the sequence shown in SEQ ID NO:

6.

11. The method of any one of claims 1-10, wherein the first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 6, 7 and 8, respectively.

12. The method of any one of claims 1-11, wherein the first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise sequences as shown in SEQ ID NO: 2, 3, and 4, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise sequences as shown in SEQ ID NO: 6, 7, and 8.

13. The method of any one of claims 1-12, wherein the first antigen-binding region binding 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 with the sequence shown in SEQ ID NO:

1.

14. The method of any one of claims 1-13, wherein the first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:

1.

15. The method of any one of claims 1-14, wherein the first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO:

5.

16. The method of any one of claims 1-15, wherein the first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:

5.

17. The method of any one of claims 1-16, wherein the first antigen-binding region binding 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: 1, and the VL comprises the sequence shown in SEQ ID NO:

5.

18. The method of any one of claims 1-17, wherein the first antigen-binding region binding to EpCAM comprises a heavy chain and a light chain variable region of an antibody, said antibody competing with antibodies comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) as described in any one of claims 5-18 for EpCAM binding and / or having specificity for EpCAM with an antibody comprising a heavy chain variable region (VH) and / or a light chain variable region (VL) as described in any one of claims 5-18.

19. The method of any one of claims 1-18, wherein the second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) containing an HCDR3 sequence, said HCDR3 sequence comprising the sequence shown in SEQ ID NO:

14.

20. The method of any one of claims 1-19, wherein the second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) containing an HCDR2 sequence, said HCDR2 sequence comprising the sequence shown in SEQ ID NO:

13.

21. The method of any one of claims 1-20, wherein the second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) containing an HCDR1 sequence, said HCDR1 sequence comprising the sequence shown in SEQ ID NO:

12.

22. The method of any one of claims 1-21, wherein the second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 12, 13 and 14, respectively.

23. The method of any one of claims 1-22, wherein the second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising an LCDR3 sequence comprising the sequence shown in SEQ ID NO:

18.

24. The method of any one of claims 1-23, wherein the second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising an LCDR2 sequence comprising the sequence shown in SEQ ID NO:

17.

25. The method of any one of claims 1-24, wherein the second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising an LCDR1 sequence comprising the sequence shown in SEQ ID NO:

16.

26. The method of any one of claims 1-25, wherein the second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

27. The method of any one of claims 1-26, wherein the second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise sequences as shown in SEQ ID NO: 12, 13, and 14, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise sequences as shown in SEQ ID NO: 16, 17, and 18.

28. The method of any one of claims 1-27, wherein the second antigen-binding region binding 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 with the sequence shown in SEQ ID NO:

11.

29. The method of any one of claims 1-28, wherein the second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:

11.

30. The method of any one of claims 1-29, wherein the second antigen-binding region binding 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 with the sequence shown in SEQ ID NO:

15.

31. The method of any one of claims 1-30, wherein the second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:

15.

32. The method of any one of claims 1-31, wherein the second antigen-binding region binding to CD137 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: 11, and the VL comprises the sequence shown in SEQ ID NO:

15.

33. The method of any one of claims 1-32, wherein the second antigen-binding region binding to CD137 comprises a heavy chain and a light chain variable region of an antibody, said antibody competing with an antibody comprising the heavy chain variable region and / or the light chain variable region as described in any one of claims 20-33 for CD137 binding and / or having specificity for CD137 with an antibody comprising the heavy chain variable region and / or the light chain variable region as described in any one of claims 20-33.

34. The method of any one of claims 1-33, wherein a) The first antigen-binding region binding to EpCAM comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 2, 3, and 4, and the LCDR1, LCDR2, and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 6, 7, and 8; and b) The second antigen-binding region binding to CD137 comprises: a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region (VL) containing LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences comprise sequences as shown in SEQ ID NO: 12, 13 and 14, respectively, and the LCDR1, LCDR2 and LCDR3 sequences comprise sequences as shown in SEQ ID NO: 16, 17 and 18, respectively.

35. The method of any one of claims 1-34, wherein the binding agent is in the form of a full-length antibody or an antibody fragment.

36. The method of any one of claims 1-35, wherein the binder comprises an Fc region.

37. The method of any one of claims 1-36, wherein the binder is a multispecific binder such as a bispecific binder.

38. The method of any one of claims 1-37, wherein the binder is a bispecific divalent binder.

39. The method of any one of claims 1-38, wherein the binder does not contain an antigen-binding region that binds to EpCAM other than the first antigen-binding region that binds to EpCAM, and does not contain an antigen-binding region that binds to CD137 other than the second antigen-binding region that binds to CD137.

40. The method of any one of claims 1-39, wherein the binding agent is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region binding to EpCAM, and the second binding arm comprising a second antigen-binding region binding to CD137, wherein The first connecting arm includes i) Peptides containing a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) A polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL); And the second connecting arm includes iii) Peptides containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) A polypeptide containing a second light chain variable region (VL) and a second light chain constant region (CL).

41. The method of claim 40, wherein The first VH comprises first HCDR1, HCDR2, and HCDR3 sequences, and the first VL comprises first LCDR1, LCDR2, and LCDR3 sequences, wherein the first HCDR1, HCDR2, and HCDR3 sequences respectively comprise sequences as shown in SEQ ID NO: 2, 3, and 4, and the first LCDR1, LCDR2, and LCDR3 sequences respectively comprise sequences as shown in SEQ ID NO: 6, 7, and 8; and The second VH comprises the second HCDR1, HCDR2 and HCDR3 sequences, and the second VL comprises the second LCDR1, LCDR2 and LCDR3 sequences, wherein the second HCDR1, HCDR2 and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 12, 13 and 14, respectively, and the second LCDR1, LCDR2 and LCDR3 sequences comprise the sequences shown in SEQ ID NO: 16, 17 and 18, respectively.

42. The method of claim 40 or 41, wherein 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 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 with the amino acid sequence shown in SEQ ID NO: 5; and The second VH contains 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 contains 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.

43. The method of any one of claims 40-42, wherein The first VH contains the amino acid sequence shown in SEQ ID NO: 1, and the first VL contains the amino acid sequence shown in SEQ ID NO: 5; and The second VH contains the amino acid sequence shown in SEQ ID NO: 11, and the second VL contains the amino acid sequence shown in SEQ ID NO:

15.

44. The method of any one of claims 40-43, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises one or more of a constant heavy chain 1 (CH1) region, a hinge region, a constant heavy chain 2 (CH2) region and a constant heavy chain 3 (CH3) region, preferably comprising at least a hinge region, a CH2 region and a CH3 region.

45. The method of any one of claims 40-44, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises a CH3 region, and wherein the two CH3 regions comprise an asymmetric mutation.

46. ​​The method of any one of claims 40-45, wherein (i) in the first heavy chain constant region (CH), the amino acid at position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second heavy chain constant region (CH), the amino acid at position K409 in the human IgG1 heavy chain according to the EU number is R, or (ii) in the first heavy chain constant region (CH), the amino acid at position K409 in the human IgG1 heavy chain according to the EU number is R, and in the second heavy chain constant region (CH), the amino acid at position F405 in the human IgG1 heavy chain according to the EU number is L.

47. The method of any one of claims 40-46, wherein the binder induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first antigen-binding region and second antigen-binding region and two heavy chain constant regions (CH) comprising a human IgG1 hinge region, a CH2 region and a CH3 region.

48. The method of any one of claims 40-47, wherein the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an identical antibody except that it contains an unmodified first heavy chain constant region (CH) and a second heavy chain constant region (CH).

49. The method of any one of claims 40-48, wherein in at least one of the first heavy chain constant region (CH) and the second heavy chain constant region (CH), one or more amino acids at positions corresponding to L234, L235, D265, N297, P331 and G236 in the human IgG1 heavy chain according to EU number are not L, L, D, N, P and G, respectively.

50. The method of claim 49, wherein in the first heavy chain and the second heavy chain, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU number are F and E, respectively.

51. The method of claim 49 or 50, wherein in the first heavy chain constant region (HC) and / or the second heavy chain constant region (HC), the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to EU numbers are F, E and A, respectively, and / or in the first heavy chain constant region (HC) and / or the second heavy chain constant region (HC), the positions corresponding to positions L234, L235 and G236 in the human IgG1 heavy chain according to EU numbers are F, E and R, respectively.

52. The method of any one of claims 49-51, wherein (i) In the first heavy chain constant region and the second heavy chain constant region, the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to the EU number are F, E and A, respectively; (ii) In the first and second heavy chain constant regions, the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to EU numbers are F, E, and R, respectively; or (iii) In one of the first heavy chain constant region and the second heavy chain constant region, the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to the EU number are F, E and A, respectively, and in the other of the first heavy chain constant region and the second heavy chain constant region, the positions corresponding to positions L234, L235 and G236 in the human IgG1 heavy chain according to the EU number are F, E and R, respectively.

53. The method of any one of claims 49-52, wherein the positions of the first heavy chain constant region and the second heavy chain constant region corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU number are F and E, respectively, and wherein (i) the position of the first heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to EU number is L, and the position of the second heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to EU number is R, or (ii) the position of the first heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to EU number is R, and the position of the second heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to EU number is L.

54. The method of any one of claims 49-53, wherein in the second heavy chain constant region (HC), the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU number are F, E, and A, respectively, and in the first heavy chain constant region (HC), the positions corresponding to positions L234, L235, and G236 in the human IgG1 heavy chain according to EU number are F, E, and R, respectively, and wherein (i) the position in the first heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to EU number is L, and the position in the second heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to EU number is R, or (ii) the position in the first heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to EU number is R, and the position in the second heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to EU number is L.

55. The method of any one of claims 49-54, wherein in the second heavy chain constant region (HC), the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to EU number are F, E and A, respectively, and in the first heavy chain constant region (HC), the positions corresponding to positions L234, L235 and G236 in the human IgG1 heavy chain according to EU number are F, E and R, respectively, and wherein the position in the first heavy chain constant region corresponding to position F405 in the human IgG1 heavy chain according to EU number is L, and the position in the second heavy chain constant region corresponding to position K409 in the human IgG1 heavy chain according to EU number is R.

56. The method of any one of claims 40-55, wherein a) The constant region of the first heavy chain contains the amino acid sequence shown in SEQ ID NO: 54; and b) The constant region of the second heavy chain contains the amino acid sequence shown in SEQ ID NO:

52.

57. The method of any one of claims 1-56, wherein the binding agent is an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising a first antigen-binding region binding to EpCAM, and the second binding arm comprising a second antigen-binding region binding to CD137, wherein The first connecting arm includes i) Peptides containing a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) A polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), The first VH comprises the first HCDR1, HCDR2 and HCDR3 sequences, and the first VL comprises the first LCDR1, LCDR2 and LCDR3 sequences, wherein the first HCDR1, HCDR2 and HCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 2, 3 and 4, and the first LCDR1, LCDR2 and LCDR3 sequences respectively comprise the sequences shown in SEQ ID NO: 6, 7 and 8; And the second connecting arm includes iii) Peptides containing a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) Peptides containing a second light chain variable region (VL) and a second light chain constant region (CL), The second VH comprises the second HCDR1, HCDR2, and HCDR3 sequences, and the second VL comprises the second LCDR1, LCDR2, and LCDR3 sequences, wherein the second HCDR1, HCDR2, and HCDR3 sequences comprise the sequences shown in SEQ ID NO: 12, 13, and 14, respectively, and the second LCDR1, LCDR2, and LCDR3 sequences comprise the sequences shown in SEQ ID NO: 16, 17, and 18, respectively. In the first CH, positions L234, L235, and G236 in the human IgG1 heavy chain according to the EU number are F, E, and R, respectively; in the second CH, positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU number are F, E, and A, respectively; and In the first CH, the amino acid corresponding to position F405 in the human IgG1 heavy chain according to the EU number is L, and in the second CH, the amino acid corresponding to position K409 in the human IgG1 heavy chain according to the EU number is R.

58. The method of any one of claims 1-57, wherein the binder comprises 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 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 with the amino acid sequence shown in SEQ ID NO:

10. iii) A second 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 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 with the amino acid sequence shown in SEQ ID NO:

20.

59. The method of any one of claims 1-58, wherein the binder comprises i) The first heavy chain, which contains the amino acid sequence shown in SEQ ID NO: 9, ii) A first light chain comprising the amino acid sequence shown in SEQ ID NO:

10. iii) A second chain comprising the amino acid sequence shown in SEQ ID NO: 19; and iv) The second light chain contains the amino acid sequence shown in SEQ ID NO:

20.

60. The method of any one of claims 1-59, wherein the PD-1 axis binding antagonist comprises a PD-1 binding antagonist.

61. The method of claim 60, wherein the PD-1 binding antagonist comprises an anti-PD-1 antibody.

62. The method of claim 61, wherein the anti-PD-1 antibody is selected from IgG1-PD1, nivolumab, cimiprimab, dotalimab, or pembrolizumab, optionally wherein IgG1-PD1 comprises (a) a heavy chain variable region (VH) comprising: CDR-1 comprising the amino acid sequence GFSLYSYN (SEQ ID NO: 99), CDR-2 comprising the amino acid sequence ISGGTIG (SEQ ID NO: 100), and CDR-3 comprising the amino acid ARAFYDDYDYNV (SEQ ID NO: 101), and (b) a light chain variable region (VL) comprising: CDR-1 comprising the amino acid sequence QSVYGNNQ (SEQ ID NO: 102), CDR-2 comprising the amino acid sequence QAS (SEQ ID NO: 103), and CDR-3 comprising the amino acid sequence AGGYSSSSDTT (SEQ ID NO: 104).

63. The method of claim 61 or 62, wherein the PD-1 antibody is IgG1-PD1.

64. The method of any one of claims 1-59, wherein the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist.

65. The method of claim 64, wherein the PD-L1 binding antagonist comprises an anti-PD-L1 antibody.

66. The method of claim 65, wherein the anti-PD-L1 antibody is selected from atezolizumab, avelumab, or durvalumab.

67. The method of any one of claims 1-59, wherein the PD-1 axis binding antagonist comprises a PD-L2 binding antagonist.

68. The method of claim 67, wherein the PD-L2 binding antagonist comprises an anti-PD-L2 antibody.

69. The method of claim 68, wherein the anti-PD-L2 antibody is selected from OT17B10 and PDL2 / 1850.

70. The method of any one of claims 1-69, wherein the disease or symptom involves disordered cell growth.

71. The method of claim 70, wherein the disordered cell growth is cancer or tumor.

72. The method of claim 71, wherein the cancer or tumor is a solid tumor.

73. The method of claim 71 or 72, wherein the cancer, tumor, or solid tumor is selected from cholangiocarcinoma, gastric / gastroesophageal junction (GEJ) cancer, melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, urothelial carcinoma, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Merkel cell carcinoma, and mesothelioma.

74. The method of any one of claims 1-73, wherein the disease or condition is bile duct cancer (cholangiocarcinoma) or gastroesophageal junction (GEJ).

75. The method of any one of claims 1-74, wherein the subject is a mammalian subject.

76. The method of claim 75, wherein the mammalian subject is a human subject.

77. The method of any one of claims 1-76, wherein the subject has not received prior treatment for the disease or condition.

78. The method of any one of claims 1-76, wherein the subject has received prior treatment for the disease or condition.

79. The method of claim 77, wherein the subject has not received prior treatment with a checkpoint inhibitor.

80. The method of any one of claims 1-79, wherein the method further comprises administering one or more additional therapeutic agents to the subject.

81. The method of claim 80, wherein the one or more additional therapeutic agents comprise one or more chemotherapeutic agents, such as platinum-based compounds (e.g., cisplatin, oxaliplatin, and carboplatin), taxane-based compounds (e.g., paclitaxel and nab-paclitaxel), nucleoside analogs (e.g., 5-fluorouracil and gemcitabine) and combinations thereof (e.g., cisplatin / carboplatin + 5-fluorouracil or nab-paclitaxel + gemcitabine).

82. The method of any one of claims 1-81, wherein administering the binding agent to the subject comprises administering one or more nucleic acids encoding the binding agent.

83. The method of any one of claims 1-82, wherein administering a PD-1 axis binding antagonist to the subject comprises administering one or more nucleic acids encoding the PD-1 axis binding antagonist.

84. The method of any one of claims 1-83, wherein administering the binder and PD-1 axis binding antagonist to the subject comprises administering one or more nucleic acids encoding the binder and the PD-1 axis binding antagonist.

85. The method of any one of claims 82-84, wherein at least one of the nucleic acids is RNA.

86. A polynucleotide or a group of polynucleotides encoding a binding agent and / or a PD-1 axis binding antagonist as defined in any one of claims 1-69.

87. A reagent kit comprising one or more vials: (i) A binding agent comprising a first antigen-binding region that binds to EpCAM and a second antigen-binding region that binds to CD137, or one or more nucleic acids encoding said binding agent. (ii) A PD-1 axis binding antagonist, or one or more nucleic acids encoding said PD-1 axis binding antagonist, and (iii) One or more therapeutic agents that may be present.

88. The kit of claim 87, wherein the kit further comprises instructions for using the kit to treat or prevent a disease or condition of a subject.

89. The kit of claim 87 or 88, wherein the binding agent or encoding nucleic acid and the PD-1 axis binding antagonist or encoding nucleic acid are in separate vials.

90. The kit of any one of claims 87-89, a method for treating or preventing a disease or condition in a subject, preferably wherein the disease or condition is a tumor, a solid tumor, or cancer, wherein the method comprises (i) administering a binding agent to the subject, and (ii) administering a PD-1 axis binding antagonist to the subject, wherein the administration of the binding agent is prior to, concurrent with, or after the administration of the PD-1 axis binding antagonist.

91. The kit of any one of claims 87-89, a method for reducing or preventing the progression of a tumor, solid tumor, or cancer in a subject, wherein the method comprises (i) administering a binding agent to the subject, and (ii) administering a PD-1 axis binding antagonist to the subject, wherein the administration of the binding agent is prior to, concurrent with, or after the administration of the PD-1 axis binding antagonist.

92. The kit of any one of claims 87-89 or the kit used in claim 90 or 91, wherein the binder is defined in any one of claims 1-59.

93. The kit of any one of claims 87-89 or the kit used in any one of claims 90-92, wherein the PD-1 axis binding antagonist is defined in any one of claims 60-69.

94. The kit of any one of claims 87-89 or the kit used in any one of claims 90-93, wherein the one or more therapeutic agents are as defined in claim 81.

95. The kit of any one of claims 87-89 or the kit used in any one of claims 90-94, wherein the tumor, solid tumor or cancer is as defined in claim 73 or 74.

96. The kit of any one of claims 87-89 is used in the method of any one of claims 1-85.

97. A conjugate comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, for treating or preventing a disease or condition of a subject, the method comprising administering the conjugate to the subject in combination with a PD-1 axis binding antagonist, wherein the administration of the conjugate is prior to, concurrent with, or after the administration of the PD-1 axis binding antagonist.

98. A method of treating or preventing a disease or condition in a subject, the method comprising administering the PD-1 axis binding antagonist to the subject in combination with a binder, the binder comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, wherein the administration of the PD-1 axis binding antagonist is prior to, concurrent with, or after the administration of the binder.

99. A conjugate comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, and a PD-1 axis binding antagonist, for treating or preventing a disease or condition in a subject, the method comprising administering the conjugate and the PD-1 axis binding antagonist to the subject, wherein the administration of the conjugate is prior to, concurrent with, or after the administration of the PD-1 axis binding antagonist.

100. The binder and / or PD-1 axis binding antagonist used in any one of claims 97-99, wherein the method for treating or preventing a disease or condition of the subject is a method for reducing or preventing tumor progression in the subject or for treating cancer in the subject.

101. Use of a binder comprising a first antigen-binding region that binds to EpCAM and a second antigen-binding region that binds to CD137 in the preparation of a medicament, in combination with a PD-1 axis binding antagonist, for the treatment or prevention of a disease or condition in a subject.

102. Use of a PD-1 axis binding antagonist in the preparation, in combination with a binding agent, in a medicament for the treatment or prevention of a disease or condition in a subject, said binding agent comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137.

103. (i) the use of a binder comprising a first antigen-binding region that binds to EpCAM and a second antigen-binding region that binds to CD137, and (ii) the use of a PD-1 axis binding antagonist in the preparation of a medicament for the treatment or prevention of a disease or condition of a subject.

104. Use according to any one of claims 101-103, wherein the disease or ailment is a tumor, a solid tumor, or cancer.

105. A medical article comprising a binder as defined in any one of claims 1-104 and a PD-1 axis binding antagonist.

106. The medical article of claim 105, used in any one of claims 1-85.

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