Multispecific anti-gene binding proteins against EpCAM

Abstract

The present invention provides antigen-binding proteins specific to epithelial cell adhesion molecules (EpCAM), their variants and fragments, and multispecific antigen-binding proteins such as bispecific T cell engagers (BiTEs) that bind to EpCAM and other targets. The invention also provides compositions comprising antigen-binding proteins, their variants and fragments, cells expressing / secreting them, therapeutic methods involving antigen-binding proteins, their variants and fragments, and other uses. In one embodiment, the cells are immune cells, selected from the group including, for example, T cells, macrophages, monocytes, and NK cells. In a more specific embodiment, the cells are CAR T cells.

Description

[Technical Field]

[0001] This disclosure relates to antigen-binding proteins, variants, and fragments specific to epithelial cell adhesion molecules (EpCAM). In particular, this disclosure includes the nucleotide and amino acid sequences, or antigen-binding moieties thereof, of six human antibodies, five llama antibodies, and one humanized llama antibody that specifically target human EpCAM both in solution and on the cell surface. Also provided are multispecific antigen-binding proteins, such as bispecific T cell engagers (BiTEs), that bind to EpCAM and other targets. [Background technology]

[0002] In 2020, an estimated 19.3 million new cancer cases were reported worldwide, and the incidence of cancer is projected to increase to 28.4 million by 2040. Approximately 90% of adult cancer patients have solid tumors. Solid tumors, such as lung cancer, liver cancer, stomach cancer, and breast cancer in women, are among the leading causes of cancer-related death. The high global incidence of cancer, coupled with poor outcomes and survival rates with standard treatment modalities such as surgery, chemotherapy, and radiation therapy, highlights a strong need for the development of more effective cancer treatment options.

[0003] Advanced therapies such as antibody-based and cell-based immunotherapies offer a new paradigm in oncology and hold immense potential as more effective treatments for cancer compared to conventional strategies. As a result, antibody and cell therapies are attracting increasing attention and investment as more promising standard treatments for cancer. However, even with potent cytotoxic mechanisms such as bispecific T cell engagers (BiTEs) and antibody-drug conjugates (ADCs), antibody-based targeted therapies rarely cure patients with solid tumors. Cell therapies such as chimeric antigen receptor (CAR) T have been highly successful in hematological malignancies but have shown limited efficacy in treating solid tumors. Among the complex challenges each approach faces, one essential problem is the heterogeneous distribution of tumor markers in solid tumor cell populations and the evasion of antigens by treatment. To overcome these challenges, researching next-generation immunotherapies to prevent cancer recurrence is a medically urgent task.

[0004] Overexpression of cancer antigens such as GPC3, Her2, and Claudin18.2 is observed in various cancer types and is known as a promising target for immunotherapy using antibodies and CAR-T cells. However, clinical studies have shown that targeting known tumor antigens alone has limited efficacy. Epithelial cell adhesion molecules (EpCAMs) are transmembrane glycoproteins widely expressed in almost all epithelium and epithelial neoplasms and represent a different category of biomarkers. Additional targeting of EpCAMs could be an attractive therapeutic regime for overcoming the challenge of tumor heterogeneity.

[0005] However, because EpCAM is widely expressed at low levels in normal epithelium, anti-EpCAM T cell engagers have been shown to be highly toxic to normal tissues. As a result, high-affinity anti-EpCAM BiTE antibodies such as solitomab and catumaxomab have shown dose-limiting toxicity and have not been approved by the FDA.

[0006] Therefore, there is an urgent need to develop low-affinity anti-EpCAM clones that can be used as T cell engagers targeting EpCAM and preserve normal epithelial cells with low EpCAM levels. Summary of the Invention

[0007] In previous methods in antibody discovery, it often starts with immunizing animals such as mice and rabbits with antigen proteins multiple times. As a result, usually, high-affinity conjugates derived from non-human sources are identified. After evaluating their properties extensively, a special step of humanization to avoid immunogenicity is usually required for further development. However, the humanization process always involves complex antibody engineering, and empirically obtained humanized clones may greatly lose the binding affinity and stability of the original clones. The inventors were able to identify low-affinity EpCAM conjugates in a simple manner using an in-house naive human Fab phage display library.

[0008] The fully human anti-EpCAM antibodies (1B5, 1C1, 1C11, 1D4, 1E4, and 1H6) of the present disclosure showed strong cytotoxic activity when used for target-specific cell killing in the BiTE format, despite having relatively low binding affinity. Therefore, in application examples using the BiTE format, the problems of immunogenicity and high toxicity / low tolerance caused by high-affinity non-human anti-EpCAM antibodies shown in most prior art fields can be overcome.

[0009] On the other hand, conventional scFv-formatted BiTEs often have problems of low stability and low expression yield due to difficult proper folding, but BiTEs in the nanobody format have the following excellent advantages: i) High stability; ii) High production yield; and iii) Small gene size packaged in a lentiviral system for achieving high T cell transduction efficiency

[0010] Therefore, the inventors also produced anti-EpCAM nanobodies of llama-derived single-domain antibody fragments (VHH) against EpCAM that can be fused to many different anti-CD3 T cell agonist antibodies via a flexible linker. These BiTE antibodies can be used to locally target EpCAM. As an example, by using CAR-T cells that secrete anti-EpCAM BiTE, the inventors were able to achieve not only T cell proliferation directed against specific tumor antigens but also limited expression of the BiTE targeting the more widely expressed tumor antigen EpCAM in tumors to avoid the toxicity caused by conventional systemic delivery.

[0011] In one aspect, there is provided an antigen-binding protein, an antigen-binding variant or an antigen-binding fragment that specifically binds to epithelial cell adhesion molecule (EpCAM), wherein the antigen-binding protein, its variant or fragment comprises a heavy chain variable region and / or a light chain variable region selected from the group consisting of:

[0012] (i) a heavy chain variable region comprising: CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, and, CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto (ii) a heavy chain variable region comprising: CDR-H1 comprising GDSISSNSVA (SEQ ID NO: 5) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, <{0000105}>CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TYYRSKWYS (sequence number: 6), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to AREVEGSSYDAFDI (Sequence ID: 7) (iii) Light chain variable region including the following: CDR-L1 includes the following: • QSLLHSNGYNY (sequence number: 9) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. • QSLLHSNRYNY (Sequence ID: 17), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it, or • Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSISDF (sequence number: 19): CDR-L2 includes the following: • LGS (Sequence ID: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or • AAS (Sequence ID: 20) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-L3 includes the following: • MQALQTPYT (sequence number: 11) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. • MQGLQSPWT (Sequence ID: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. • QQSYIMPDT (Sequence ID: 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or • Sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to MQGLQTPYT (sequence number: 23); and, (iv) Heavy chain variable region including the following: CDR-H1 includes the following: • GSIFSGND (sequence number: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, or • Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSSERFTS (sequence number: 29). CDR-H2 includes the following: • ITSGGST (sequence number: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, or • ITNGGST (Sequence ID: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and, CDR-H3 includes the following: • A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHH (sequence number: 27), • A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (sequence number: 31), or • A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHL (sequence number: 33).

[0013] In one embodiment, an antigen-binding protein, its variant, or fragment includes a heavy chain variable region selected from the group consisting of:

[0014] (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ARSLGGRFRY (SEQ ID NO: 3) (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TYYRSKWYS (sequence number: 6), and CDR-H3 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to AREVEGSSYDAFDI (Sequence ID: 7); and, Includes the following light chain variable region: CDR-L1 includes the following: • QSLLHSNGYNY (sequence number: 9) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. • QSLLHSNRYNY (Sequence ID: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it, or • Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSISDF (sequence number: 19). CDR-L2 includes the following: · LGS (Sequence ID: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, • AAS (Sequence ID: 20) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-L3 includes the following: • A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MQALQTPYT (SEQID number: 11), • Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to MQGLQSPWT (Sequence ID: 15). • QQSYIMPDT (Sequence ID: 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or • A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to MQGLQTPYT (sequence number: 23).

[0015] In one embodiment, an antigen-binding protein, its variant, or binding fragment includes a heavy chain variable region comprising:

[0016] CDR-H1 includes the following: • GSIFSGND (sequence number: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, or • Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSSERFTS (sequence number: 29). CDR-H2 includes the following: • ITSGGST (sequence number: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, or • ITNGGST (Sequence ID: 30) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and, CDR-H3 includes the following: • A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHH (sequence number: 27), • A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (sequence number: 31), or • A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHL (sequence number: 33).

[0017] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a heavy chain variable region and / or a light chain variable region selected from the group consisting of:

[0018] (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ARSLGGRFRY (SEQ ID NO: 3) (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TYYRSKWYS (sequence number: 6), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to AREVEGSSYDAFDI (Sequence ID: 7) (iii) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (iv) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (v) and below: Light chain variable region: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (vi) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSISDF (sequence number: 19), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with AAS (sequence number: 20), and CDR-L3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QQSYIMPDT (SEQ ID NO: 21). Light chain variable region including (vii) and below: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (viii) and below: Heavy chain variable region: CDR-H1 containing GSIFSGND (sequence number: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITSGGST (sequence number: 26), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHH (Sequence ID: 27). Heavy chain variable region including (ix) and below: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSSERFTS (sequence number: 29), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITNGGST (SEQ ID NO: 30), and CDR-H3 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (SEQ ID NO: 31); and, Heavy chain variable region including (x) and below: CDR-H1 containing GSIFSGND (sequence number: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it; CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITSGGST (sequence number: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0019] In one embodiment, an antigen-binding protein, its variant, or fragment includes a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0020] (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and, The light chain variable region includes the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and, The light chain variable region includes the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (iii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and, The light chain variable region includes the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (iv) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and, The light chain variable region includes the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and (v) Heavy chain variable region including the following CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TYYRSKWYS (sequence number: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and The light chain variable region includes the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSISDF (sequence number: 19), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with AAS (sequence number: 20), and CDR-L3 containing QQSYIMPDT (Sequence ID: 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0021] In one embodiment, an antigen-binding protein, its variant, or fragment includes a heavy chain variable region selected from the group consisting of:

[0022] (i) Heavy chain variable region including the following: CDR-H1 containing GSIFSGND (sequence number: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITSGGST (sequence number: 26), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHH (Sequence ID: 27). (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSSERFTS (sequence number: 29), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITNGGST (SEQ ID NO: 30), and CDR-H3 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (SEQ ID NO: 31); and, (iii) Heavy chain variable region including the following: CDR-H1 containing GSIFSGND (sequence number: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, CDR-H2 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ITSGGST (sequence number: 26), CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0023] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of:

[0024] (i) Heavy chain variable domains including the following: VQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (SEQ ID NOs: 4 - 1B6, 1C1, 1C11, 1D4 and 1H6), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (ii) Heavy chain variable domains including the following: QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQG™ (SEQ ID NO: 8), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iii) Light chain variable domains including the following: DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK (SEQ ID NO: 12), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iv) Light chain variable domains including the following: EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT FGQGTK (SEQ ID NO: 16), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (v) Light chain variable domains including the following: DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK (SEQ ID NO: 18), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (vi) Light chain variable domains including the following: DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDTFGQGTK (SEQ ID NO: 22), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Light chain variable domains including (vii) and below: DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT FGQGTK (SEQ ID NO: 24), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (viii) and below: Heavy chain variable domains: QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (SEQ ID NO: 28), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains including (ix) and below: QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ (SEQ ID NO: 32), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. Heavy chain variable domains including (x) below: QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL WGQGTQ (SEQ ID NO: 34), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains including (xi) and below: QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (SEQ ID NO: 35), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains including (xii) and below: QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (SEQ ID NO: 36), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions; and, Heavy chain variable domains including the following: QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions.

[0025] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a heavy chain variable domain and a light chain variable domain selected from the group consisting of:

[0026] (i) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (SEQ ID NO: 4), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, Light chain variable domains including the following: DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK (SEQ ID NO: 12), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (ii) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRYWGQGTL (SEQ ID NO: 4), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, Light chain variable domains including the following: EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT FGQGTK (SEQ ID NO: 16), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iii) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (SEQ ID NO: 4), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, Light chain variable domains including the following: DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT A light chain variable domain containing FGQGTK (SEQ ID NO: 18), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iv) Heavy chain variable domains including the following: QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQG™ (SEQ ID NO: 8), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, Light chain variable domains including the following: DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT FGQGTK (SEQ ID NO: 22), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, or (v) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (SEQ ID NO: 4), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, Light chain variable domains including the following: DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYTFGQGTK (SEQ ID NO: 24), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions.

[0027] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a single-domain heavy chain variable domain having the following sequence:

[0028] (i) QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (SEQ ID NO: 28), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, (ii) QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ (SEQ ID NO: 32), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, (iii) QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, (iv) QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (SEQ ID NO: 35), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, (v) QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (SEQ ID NO: 36), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, (vi) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH WGQGTL (SEQ ID NO: 37), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions.

[0029] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a light chain constant domain having the following sequence:

[0030] (i) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, or (ii) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (SEQ ID NO: 14), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions.

[0031] In one embodiment, the antigen-binding protein, its variant, or fragment is an IgG antibody, and more particularly, an IgG1 antibody.

[0032] In one embodiment, the antigen-binding protein, its variant, or fragment is a multispecific antigen-binding protein such as a bispecific antibody, its variant, or fragment.

[0033] In one embodiment, a multispecific antigen-binding protein, its variant, or fragment binds to an immunomarker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0034] In one embodiment, the antigen-binding protein, its variant, or fragment is a bispecific T cell engager (BiTE).

[0035] In one embodiment, the bispecific T cell engager (BiTE) comprises an anti-EpCAM heavy chain antibody variable region (i.e., VHH) or a single-strand variable fragment (scFv).

[0036] In one embodiment, the antigen-binding protein, its variant, or fragment includes an Fc region.

[0037] In one aspect, a polynucleotide encoding an antigen-binding protein, a variant thereof, or a fragment thereof, as described in any one of the claims is provided.

[0038] In one embodiment, the heavy chain variable domain is encoded by a nucleotide sequence comprising:

[0039] (i) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGGAGATTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (ii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC Sequence TGGGGCCAAGGGACAATG (Sequence ID: 45), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or have 10 to 20 nucleic acid substitutions; (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 65), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (Sequence ID: 69), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (Sequence ID: 71), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 72), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 73), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (viii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (Sequence ID: 74), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; and / or, The light chain variable domain is encoded by a nucleotide sequence that includes the following: (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 49), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID: 53), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 55), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (iv) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10 to 20 nucleic acid substitutions, or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 61), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions.

[0040] In one embodiment, the heavy chain and light chain variable domains are encoded by nucleotide sequences selected from the group consisting of:

[0041] (i) Heavy chain variable domain encoded by a nucleotide sequence including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (SEQ ID NO: 49), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (ii) Heavy chain variable domain encoded by a nucleotide sequence including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, Light chain variable domain encoded by the following nucleotide sequence: GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or have 10 to 20 nucleic acid substitutions. (iii) Heavy chain variable domains encoded by nucleotide sequences including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (SEQ ID NO: 55), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (iv) Heavy chain variable domains encoded by nucleotide sequences including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or have 10 to 20 nucleic acid substitutions; and, (v) Heavy chain variable domains encoded by nucleotide sequences including the following: CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (Sequence ID: 45), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, Light chain variable domain encoded by the following nucleotide sequence: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10 to 20 nucleic acid substitutions.

[0042] In one embodiment, the single-domain heavy chain variable domain is encoded by a nucleotide sequence comprising:

[0043] (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 65), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (ii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (Sequence ID: 69), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (Sequence ID: 71), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 72), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 73), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, (vi) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10 to 20 nucleic acid substitutions.

[0044] In one embodiment, the light chain constant domain is encoded by a nucleotide sequence comprising:

[0045] (i) CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (Sequence ID: 50), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (ii) CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT (Sequence ID: 51), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10 to 20 nucleic acid substitutions.

[0046] A vector expressing the above polynucleotide is provided in one phase.

[0047] In one aspect, a host cell containing the above vector is provided.

[0048] In one aspect, cells expressing / secreting the above-mentioned antigen-binding protein, its variant, or fragment are provided.

[0049] In one aspect, cells that express / secrete EpCAM-specific immune cell engagers are provided.

[0050] In one embodiment, the immune cell engager is selected from the group including T cell engagers, NK cell engagers, monocyte engagers, and macrophage engagers.

[0051] In one embodiment, the immune cell engager is a bispecific T cell engager (BiTE), which may be an inducible BiTE, a non-inducible BiTE, or a constitutively expressed BiTE containing the antigen-binding protein, its variants, or fragments.

[0052] In one embodiment, the cells are immune cells, selected from the group including, for example, T cells, macrophages, monocytes, and NK cells.

[0053] In one embodiment, the immune cells are T cells, particularly CAR T cells.

[0054] In one embodiment, the cells are stem cells, selected from the group including, for example, mesenchymal stem cells, neural stem cells, and pluripotent stem cells such as induced pluripotent stem cells (iPSCs).

[0055] In one aspect, a composition comprising the above-mentioned antigen-binding protein, its variant or fragment, and / or cells is provided.

[0056] In one aspect, the above-mentioned antigen-binding proteins, their variants or fragments, cells or compositions are provided for use in the treatment of diseases.

[0057] In one aspect, a method is provided for treating a disease in a subject in need thereof, comprising administering the subject the above-mentioned antigen-binding protein, its variant or fragment, cells or composition.

[0058] In one aspect, the use of the above-mentioned antigen-binding protein, its variant or fragment, cell or composition in the manufacture of a pharmaceutical product for the prevention and / or treatment of a disease is provided.

[0059] In one embodiment, the disease is a proliferative disorder such as a tumor or cancer.

[0060] In one aspect, a method is provided for determining the diagnosis / prognosis or presence of an epithelial solid tumor, comprising detecting high EpCAM expression on the solid tumor using the above-mentioned antigen-binding protein, its variant or fragment or composition.

[0061] Definition In this specification, the term "antigen-binding protein" is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies, as long as they exhibit the desired antigen-binding activity.

[0062] As used herein, the term “antibody” refers to a whole (i.e., full-length) antibody (i.e., comprising two heavy chains and two light chains) and its functionally active fragment (i.e., a molecule containing an antigen-binding domain that specifically binds to an antigen, also called an antibody fragment or antigen-binding fragment). As described herein with respect to antibodies, the same applies to antibody fragments unless otherwise indicated in the context. The term “antibody” encompasses monovalent antibodies, i.e., antibodies containing only one antigen-binding domain (e.g., a one-arm antibody with a full-length heavy chain and a full-length light chain linked together, also called a “half-antibody”), and polyvalent antibodies, i.e., antibodies containing two or more antigen-binding domains, e.g., a bivalent antibody.

[0063] As used herein, the term "antigen-binding fragment" refers to a functionally active antibody-binding fragment, including but not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies, scFv, Fv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, triabody, tetrabody, and any of the epitope-binding fragments described above.

[0064] As used herein, "binding fragment" refers to a fragment that can bind to a target peptide or antigen with sufficient affinity and that can be characterized as being specific to the peptide or antigen.

[0065] The term "monoclonal antibody" (or "mAb") refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, each individual in a monoclonal antibody preparation is identical except for any possible trace variations (e.g., naturally occurring variations). Nevertheless, certain differences in protein sequences related to post-translational modifications (e.g., cleavage of the heavy chain C-terminal lysine, deamidation of asparagine residues, and / or isomerization of asparagine residues) may exist between the various different antibody molecules present in the composition. Unlike polyclonal antibody preparations, each monoclonal antibody in a monoclonal antibody preparation is directed towards a single determinant on an antigen.

[0066] As used herein, the term "diabody" refers to two Fv pairs, a first VH / VL pair, and a further VH / VL pair having two Fv linkers such that the VH of the first Fv is connected to the VL of the second Fv, and the VL of the first Fv is connected to the VH of the second Fv.

[0067] As used herein, the term “tribody” (also known as Fab(scFv)2) refers to a Fab fragment having a first scFv attached to the C-terminus of the light chain and a second scFv attached to the C-terminus of the heavy chain. As used herein, the term “tetrabody” refers to a form similar to a diabody, containing four Fvs and four inter-Fv linkers.

[0068] The term "multivalent antibody" refers to an antibody that contains two or more antigen-binding domains, such as a bivalent antibody.

[0069] The term "Fv" refers to the pair of two variable domains in a full-length antibody, such as a cooperative variable domain like a cognitive pair or affinity maturation variable domain, i.e., the VH and VL pair. The term "scFv" refers to a single-chain variable fragment, which is a fusion protein in which the variable regions of the heavy and light chains of an immunoglobulin are linked by a short linker peptide of 10 to approximately 25 amino acids. As used herein, the term "bis-scFv" refers to a bispecific scFv.

[0070] As used herein, the term "dsscFv" or "disulfide-stabilized single-strand variable fragment" refers to a single-strand variable fragment stabilized by a peptide linker between the VH variable domain and the VL variable domain, and further containing an interdomain disulfide bond between VH and VL.

[0071] The term "DVD-Ig" (also known as dual V-domain IgG) refers to a full-length antibody that has a total of four variable domains, one at the N-terminus of the heavy chain and one at the N-terminus of the light chain.

[0072] As used herein, the term "Fab" refers to an antibody fragment comprising a light chain fragment containing a VL (variable light chain) domain and a constant domain (CL) of the light chain, and a VH (variable heavy chain) domain and a first constant domain (CHI) of the heavy chain. Dimers of Fab' according to this disclosure produce, for example, F(ab')2, in which dimerization can occur via a hinge. The term "F(ab')" refers to a monovalent fragment of a single light chain homodimer obtained by pepsin digestion of IgG, followed by reduction of the light chain disulfide bond. As described herein, the term "F(ab')2" refers to a fragment of IgG prepared by pepsin digestion of IgG. Since the F(ab')2 fragment is a disulfide-bonded homodimer of two light chain dimers, it retains a divalent epitope bond like whole IgG, but is smaller in size than whole IgG because it lacks a heavy chain. Since F(ab')2 and F(ab') fragments do not bind to immunoglobulin receptors on cells, they are useful for specifically staining the targets of primary antibodies.

[0073] As used herein, the term "DiFab" refers to two Fab molecules linked via the C-terminus of a heavy chain, or two Fab' molecules linked via one or more disulfide bonds in their hinge region.

[0074] As used herein, the term "nanobody" refers to a single-domain antibody (sdAb) having an antibody fragment consisting of a single monomer variable antibody domain.

[0075] The term “antigen-binding variant” refers to a polypeptide, for example, an antibody comprising VH and / or VL having the desired properties described herein and having at least about 80% amino acid sequence identity with the VH and / or VL of a reference antibody. Such antibody variants include, for example, antibodies in which one or more amino acid residues are added to or deleted from the VH and / or VL domains. Typically, antibody variants have at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, as described herein. Optionally, a variant antibody has one or fewer conserved amino acid substitutions compared to the antibody sequence provided herein, and alternatively, it has about two, three, four, five, six, seven, eight, nine, or ten or fewer conserved amino acid substitutions compared to the antibody sequence provided herein.

[0076] In the context of antibodies, the term "specific" as used herein is intended to mean an antibody that recognizes only a specific antigen, or an antibody that has a significantly higher binding affinity to a specific antigen compared to binding to a nonspecific antigen, for example, at least 5, 6, 7, 8, 9, or 10 times higher.

[0077] In the context of antibodies, the term “epitope” or “binding site” refers to the site (or portion) on an antigen to which an antibody paratope binds or recognizes. Epitopes can be formed from consecutive amino acids (often also called “linear epitopes”) or from discontinuous amino acids formed by the tertiary folding of proteins (often also called “conformal epitopes”). Epitopes formed from consecutive amino acids are usually retained even when exposed to denaturing solvents, while epitopes formed by folding are usually lost when treated with denaturing solvents. Epitopes typically contain at least three, and more commonly at least five to ten, amino acids in a unique spatial conformation. Epitopes are usually composed of chemically active molecular surface groups such as amino acids and sugar side chains, and typically have a specific three-dimensional structure and charge properties.

[0078] The "class" of an antibody refers to the type of constant domain or region in its heavy chain. There are five main classes of antibodies: for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0079] The term "chimeric antibody" (or its antigen-binding fragment) refers to an antibody molecule (or its antigen-binding fragment) in which (a) the constant region or part thereof has been modified, substituted, or exchanged, and the antigen-binding site (variable region) is linked to the constant region of a different or modified class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or (b) the variable region or part thereof has been changed, substituted, or exchanged with a variable region having a different or modified antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with the constant region of human immunoglobulin. By replacing it with a human constant region, the chimeric antibody can have reduced antigenicity in humans compared to the original mouse antibody while retaining its specificity for recognizing the antigen.

[0080] The term "chimeric antigen receptor" refers to a receptor protein that has been engineered to give T cells a new ability to target specific antigens. A chimeric antigen receptor combines both antigen-binding and T-cell activation functions into a single receptor. CAR T-cell therapy uses CAR-engineered T cells to treat cancer. In CAR T-cell immunotherapy, T cells are modified to recognize cancer cells in order to more effectively target and destroy them. CAR T cells can be obtained from the patient's own T cells (autologous) or from T cells of another healthy donor (allogeneic). After isolation from a person, these T cells are genetically engineered to express a specific CAR and programmed to target antigens present on the tumor surface. For safety, CAR T cells are genetically engineered to be specific to antigens expressed in tumors but not on healthy cells. CAR T cells destroy cells through widespread cell proliferation, increasing their toxicity to other living cells (cytotoxicity) and causing increased secretion of factors that affect other cells, such as cytokines, interleukins, and growth factors. The surface of CAR T cells can have one of two types of co-receptors, CD4 and CD8, each with different and interacting cytotoxic effects.

[0081] As used herein, the terms “human antibody” or “humanized antibody” (or its antigen-binding fragment) are intended to include antibodies (or their antigen-binding fragments) having a variable region in which both the framework region and the CDR region are derived from human sequences. Antibodies or immunoglobulins are classified into classes IgA, IgD, IgE, IgG, and IgM based on the amino acid sequence of the constant region of their heavy chain. Some of these are further divided into subclasses (subtypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Therefore, when an antibody molecule is intended for therapeutic use and antibody effector function is required, the human IgG constant region domains of the IgG1 and IgG3 isotypes can be used, in particular. Alternatively, when the antibody molecule is for therapeutic purposes and antibody effector function is not required, the IgG2 and IgG4 isotypes can be used. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences. Humanized antibodies (or their antigen-binding fragments) retain the reactivity of non-human antibodies while exhibiting low immunogenicity in humans. This can be achieved, for example, by retaining the non-human CDR region while replacing the rest of the antibody (i.e., the framework portion in the constant and variable regions) with the corresponding human portion. Further modifications of the framework region can be made not only within the human framework sequence but also within the CDR sequence derived from germline cells of other mammalian species. The humanized antibodies of this disclosure may include amino acid residues not encoded by the human sequence (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro, or somatic mutations in vivo, or conservative substitutions to enhance stability or production). In this definition of humanized antibodies, humanized antibodies containing non-human antigen-binding residues are specifically excluded. Human antibodies can be produced using various techniques known in the art, such as phage display libraries or antigen administration to transgenic animals modified to produce such antibodies in response to antigen challenge (the endogenous loci of the animal have been deactivated), such as immunoxenogenic mice via human B-cell hybridoma technology.

[0082] As used herein, the term “recombinant humanized antibody” includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from host cells transformed to express humanized antibodies, for example, transfectomas, or antibodies prepared, expressed, produced or isolated by any other means of splicing all or part of the sequence of a human immunoglobulin gene with another DNA sequence.

[0083] The term "isolated" throughout this specification means that an antibody or polynucleotide exists in a physical environment different from the environment in which it may exist in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule isolated from the natural environment or synthesized. Isolated nucleic acids include, for example, synthetic DNA, cDNA, genomic DNA, or combinations thereof, produced by chemical processes. An isolated antibody refers to an antibody that is substantially free of other cellular material and / or chemical substances.

[0084] The term “complementarity-determining region” (“CDR”) refers to an amino acid sequence with boundaries determined using one of many well-known schemes, including those described by Kabat (i.e., the “Kabat” numbering scheme); Al-Lazikani (the “Chothia” numbering scheme); and ImMunoGenTics (IMGT) numbering (the “IMGT” numbering scheme). The term “complementarity-determining region” (“CDR”) refers to a hypervariable region containing a binding domain that interacts with an antigen. Antibodies typically contain six CDRs: three in the VH (HI, H2, H3) and three in the VL (LI, L2, L3).

[0085] As used herein, the term “sequence identity” refers to the percentage of sequence identity determined by the antibody sequence that is most alignable according to the Kabat numbering rules. After alignment, when a target antibody region (e.g., the entire maturation variable region of the heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the target antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acids in both the target antibody region and the reference antibody region by the total number of aligned positions in the two regions (without counting gaps), and multiplying by 100 to convert it to a percentage.

[0086] "Conservative substitutions" can be made, for example, based on the similarity of polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the amino acid residues involved. The 20 naturally occurring amino acids can be classified into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0087] As used herein, “conservative substitution” is defined as the exchange of an amino acid by another amino acid listed within the same group as the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the thus modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0088] In this specification, "non-conservative substitution" is defined as the exchange of an amino acid by another amino acid listed in a different group from the six standard amino acid groups (1) to (6) shown above.

[0089] The term "affinity" refers to the strength of all non-covalent interactions between an antibody and its target protein. Unless otherwise specified, the term "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule for its binding partner can generally be expressed as the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0090] As used herein, the term "K , , D ,

[0094] ,

[0095] , , " refers to the dissociation constant obtained from the ratio of K d to K a (i.e., K d / K a ) and expressed as molar concentration (M). K d and K a refer to the dissociation rate and association rate of a specific antigen-antibody interaction, respectively. The K D value of an antibody can be determined using methods established in the art. As used herein, the term "low affinity" refers to a K D of 100 nM or greater.

[0091] As used herein, the term "moderate affinity" refers to a K[[ID==27]] D in the range of 10 nM to 100 nM.

[0092] As used herein, the term "high affinity" refers to a K D in the range of 1 to 10 nM.

[0093] As used herein, the term "very high affinity" refers to a K D of 1 nM or less.

[0094] As used herein, the term "EC50" refers to the concentration of an antibody or antigen-binding protein / portion thereof that induces a response that is 50% of the maximum response (i.e., midway between the maximum response and the baseline) in an in vivo or in vitro assay.

[0095] As used herein, the terms “multispecific” or “multispecific antibody” refer to an antibody having at least two binding domains, i.e., two or more binding domains, for example, two or three binding domains, wherein at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. Multispecific antibodies are generally monovalent for each specificity (antigen). The multispecific antibodies described herein include monovalent and polyvalent, for example, bivalent, trivalent, and tetravalent multispecific antibodies.

[0096] As used herein, the terms “bispecificity” or “bispecific antibody” refer to an antibody that has two antigen specificities, or an antibody that has the ability to bind to two target antigens / sites simultaneously.

[0097] As used herein, “bispecific T cell engagers” (BiTEs) refer to a group of artificial bispecific monoclonal antibodies that induce the host immune system, such as cytotoxic activity of T cells against target cells (e.g., cancer cells). A BiTE is a fusion protein consisting of two single-chain variable fragments (scFvs) of different antibodies, i.e., amino acid sequences from four different genes, linked together on a single peptide chain of approximately 55 kDa. One scFv binds to an immune cell (e.g., a T cell via the CD3 receptor), and the other binds to the target of interest (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs form a link between immune cells (e.g., T cells) and target cells (e.g., tumor cells). This allows immune cells (e.g., T cells) to exert cytotoxic activity against tumor cells. For example, if the immune cell is a T cell, the T cell exerts cytotoxic activity by entering tumor cells and producing proteins such as perforin and granzyme, which initiate apoptosis.

[0098] The term "immune cells" refers to a type of specialized cell that plays a crucial role in the body's defense against infections and foreign substances. Immune cells are part of the immune system and are responsible for identifying and eliminating harmful pathogens such as bacteria, viruses, and parasites, as well as abnormal cells and cancer cells. As used herein, "immune cells" refers to all cells of the immune system, including but not limited to T cells, helper T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), granulocytes (basophils, eosinophils, neutrophils, etc.), mast cells, monocytes, and macrophages.

[0099] As used herein, "nanobody having only heavy chains" refers to a heavy-chain antibody based on a nanobody. A heavy-chain antibody is an antibody consisting of two heavy chains and lacking the two light chains typically found in antibodies.

[0100] In this specification, “vector” means any molecule or composition having the ability to carry a nucleic acid sequence to a suitable host cell in which the encoded polypeptide can be synthesized. Typically, and preferably, a vector is a nucleic acid manipulated to incorporate a desired nucleic acid sequence (e.g., the nucleic acids of this disclosure) using recombinant DNA techniques known in the art. An expression vector typically comprises one or more of the following components (if not already provided by a nucleic acid molecule): a promoter, one or more enhancer sequences, an origin of replication, a termination sequence, a complete intron sequence including donor and acceptor splice sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0101] Vectors are typically selected to be functional in the host cell in which they are used (the vector is compatible with the host cell's mechanisms so that gene amplification and / or gene expression can occur). The vectors described herein may be expression vectors and / or cloning vectors.

[0102] The term “host cell,” as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the progeny cells of such a cell. Such progeny cells may not be identical to the parent cell in fact, as certain modifications may occur in later generations due to either mutation or environmental influences, but they still fall within the scope of the term “host cell” as used herein.

[0103] The terms “treating,” “treat,” and “therapy,” as well as their synonyms, refer to both therapeutic actions and preventive or prophylactic measures, the purpose of which is to prevent or delay (alleviate) medical conditions, including but not limited to diseases, symptoms, and disorders. Medical conditions also include the body’s response to diseases or disorders, such as inflammation. Those who require such treatment include not only those who already have a medical condition, but also those who are susceptible to it or who should be prevented from developing a medical condition.

[0104] As used herein, the term “subject” includes patients and non-patients. The term “patient” refers to an individual who has or is likely to have a medical condition, and the term “non-patient” refers to an individual who does not have or is not likely to have a medical condition. “Non-patients” include healthy individuals, disease-free individuals, and / or individuals who do not have a medical condition. The term “subject” includes humans and animals. Animals include, but are not limited to, mammals (e.g., non-human primates, dogs, mice, etc.). “Murine” refers to mammals of the Muridae and / or Leporidae families, such as mice, rats, and rabbits.

[0105] As used herein, the terms “prevent” and / or “reduce the severity of symptoms” refer to a process that delays the onset of the disease, reduces the severity of symptoms, reduces and / or prevents weight loss, prevents death, halts exacerbation, halts further exacerbation, and / or improves at least one sign or symptom of the disease.

[0106] The term "and / or," for example, "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be taken as providing explicit support for both meanings or either meaning.

[0107] Furthermore, the term “substantially” in this specification is understood to include, but not be bound by, “entirely” or “completely” whenever it is used. Additionally, terms such as “comprising” and “comprises” are intended to be non-restrictive descriptive language in that they always include, in addition to other components not explicitly mentioned, the elements / components referred to after such terms. For example, when “comprising” is used, a reference to “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting” and “consist” are understood, in appropriate contexts, as a subset of terms such as “comprising” and “comprise.” Thus, embodiments disclosed herein using terms such as “comprising” and “comprise” are understood to provide teachings of corresponding embodiments using terms such as “consisting” and “consist.” Furthermore, whenever terms such as "about" or "approximately" are used, they typically mean a reasonable variation, for example, a variation of ±5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, or a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

[0108] Furthermore, this specification may disclose certain values ​​as ranges. Numerical values ​​indicating the endpoints of a range are intended to illustrate preferred ranges. Whenever a range is described, it is intended to cover and teach all possible sub-ranges, as well as the individual numerical values ​​within that range. That is, the endpoints of a range should not be interpreted as inflexible limitations. For example, a description of a range from 1% to 5% is intended to specifically disclose sub-ranges such as 1% to 2%, 1% to 3%, 1% to 4%, and 2% to 3%, as well as the individual numerical values ​​within that range, such as 1%, 2%, 3%, 4%, and 5%. It should be understood that individual numerical values ​​within a range include integers, fractions, and decimals. Furthermore, whenever a range is described, it is also intended that the range covers and teaches values ​​up to two decimal places or significant figures (where applicable) beyond the endpoints of the indicated numerical values. For example, a description of a range from 1% to 5% is intended to encompass the ranges of 1.00% to 5.00% and 1.0% to 5.0%, as well as the intermediate values ​​spanning these ranges (e.g., 1.01%, 1.02%…4.98%, 4.99%, 5.00%, and 1.1%, 1.2%…4.8%, 4.9%, 5.0%). The specific intent of disclosure described above is applicable to any depth / breadth of range.

[0109] As used herein, "at least 95% identical" is intended to refer to an amino acid sequence that is 95% or more identical to a reference sequence, such as a 96%, 97%, 98%, or 99% identical sequence, over its entire length. Software programs can be used to calculate the percentage of identity.

[0110] Furthermore, in some embodiments, this disclosure may disclose a method and / or process as a specific set of steps. However, unless specifically required, it should be understood that such method or process should not be limited to the specific sequence of steps disclosed. Other orders of steps are also possible. The specific order of steps disclosed herein should not be construed as an unreasonable limitation. Unless specifically required, the methods and / or processes disclosed herein should not be limited to steps performed in the order described. The order of steps may vary, and these variations are also within the scope of this disclosure.

[0111] Furthermore, while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may be excluded in other alternative embodiments, and it is understood that this disclosure provides support for such exclusions and related alternative embodiments. [Brief explanation of the drawing]

[0112]

Figure 1

Figure 2

Figure 3A - D

Figure 3E - G

Figure 4A

Figure 4B - C

Figure 5

Figure 6A - B

Figure 6C - F

Figure 7A - D

Figure 7E

Figure 8A

Figure 8B - E

Figure 9

Figure 10A

Figure 10B - D

Figure 11A - C

Figure 11D - G

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Mode for Carrying Out the Invention

[0113] Those skilled in the art will understand that other variations and / or modifications can be made to one embodiment disclosed herein without departing from the spirit or scope of the disclosure described herein. For example, in the description herein, the features of different exemplary embodiments can be mixed, combined, exchanged, incorporated, adopted, modified, included, etc. across different exemplary embodiments. Therefore, this embodiment should be considered exemplary in every respect and not restrictive.

[0114] In one aspect, there is provided an antigen-binding protein, antigen-binding variant, and / or antigen-binding fragment thereof that specifically binds to epithelial cell adhesion molecule (EpCAM).

[0115] In one embodiment, the antigen-binding protein, its variant or fragment is a monoclonal antibody.

[0116] In one embodiment, the antigen-binding protein or its variant is a full-length antibody.

[0117] In one embodiment, the antigen-binding fragment is selected from the group consisting of Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibody, VHH, scFv, Fv, bivalent antibody, trivalent antibody, tetravalent antibody, Bis-scFv, diabody, triabody, tetrabody, epitope-binding fragment, and the like.

[0118] In one embodiment, the antigen-binding protein, its variant or fragment is an IgG, IgA, IgD, IgE or IgM antibody.

[0119] In one embodiment, the antigen-binding protein, its variant or fragment is a chimeric antibody, human antibody / humanized antibody, recombinant humanized antibody, animal-derived antibody (such as llama antibody), and the like.

[0120] In one embodiment, the antigen-binding protein, its variant or fragment is a human antibody. In one embodiment, the antigen-binding protein, its variant or fragment is a llama antibody. In one embodiment, the antigen-binding protein, its variant or fragment is a humanized llama antibody.

[0121] In one embodiment, the antigen-binding protein, its variant or fragment contains one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 37.

[0122] In one embodiment, the antigen-binding protein, its variant or fragment is encoded by one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 38 to 74.

[0123] In one embodiment, the antigen-binding protein, its variant or fragment contains a heavy chain variable region and a light chain variable region encoded by a nucleic acid sequence selected from the following group:

[0124] (i) A heavy chain variable region containing the following: (1B6 and 1C1) CDR-H1 containing ggaggcaccttcagcagctatgct (SEQ ID NO: 38), CDR-H2 containing atcatccctatctttggtacagca (SEQ ID NO: 39), and CDR-H3 containing gcgagatcgttgggtgggagatttcgctac (SEQ ID NO: 40); and, The light chain variable region includes the following: CDR-L1 containing cagagcctcctgcatagtaatggatacaactat (SEQ ID NO: 46), CDR-L2 containing ttgggttct (SEQ ID NO: 47), and CDR-L3 containing atgcaagctctacaaactccgtacact (SEQ ID NO: 48) (ii) Heavy chain variable region including the following: (1C11) CDR-H1 containing ggaggcaccttcagcagctatgct (SEQ ID NO: 38), CDR-H2 containing atcatccctatctttggtacagca (SEQ ID NO: 39), and CDR-H3 containing gcgagatcgttgggtgggagatttcgctac (SEQ ID NO: 40); and, The light chain variable region includes the following: CDR-L1 containing cagagcctcctgcatagtaatggatacaactat (SEQ ID NO: 46), CDR-L2 containing ttgggttct (SEQ ID NO: 47), and CDR-L3 containing atgcaaggtctacaaagtccctggacg (SEQ ID NO: 52) (iii) Heavy chain variable region including the following: (1D4) CDR-H1 containing ggaggcaccttcagcagctatgct (SEQ ID NO: 38), CDR-H2 containing atcatccctatctttggtacagca (SEQ ID NO: 39), and CDR-H3 containing gcgagatcgttgggtgggagatttcgctac (SEQ ID NO: 40); and, The light chain variable region includes the following: CDR-L1 containing cagagcctcctgcatagtaatagatacaactat (SEQ ID NO: 54), CDR-L2 containing ttgggttct (SEQ ID NO: 47), and CDR-L3 containing atgcaagctctacaaactccgtacact (SEQ ID NO: 48) (iv) Heavy chain variable region including the following: (1H6) CDR-H1 containing ggaggcaccttcagcagctatgct (SEQ ID NO: 38), CDR-H2 containing atcatccctatctttggtacagca (SEQ ID NO: 39), and CDR-H3 containing gcgagatcgttgggtgggagatttcgctac (SEQ ID NO: 40); and, The light chain variable region includes the following: CDR-L1 (SEQ ID NO: 46) containing cagagcctcctgcatagtaatggatacaactat, CDR-L2 containing ttgggttct (SEQ ID NO: 47), and CDR-L3 containing atgcaaggtctacagactccgtacact (SEQ ID NO: 60); and, (v) Heavy chain variable region including the following: (1E4) CDR-H1 (SEQ ID NO: 42) containing ggggacagtatctctagtaacagtgttgct, CDR-H2 containing acatactacaggtccaagtggtacagt (SEQ ID NO: 43), and CDR-H3 containing gcaagagaagttgagggcagcagctatgatgcttttgatatc (SEQ ID NO: 44); and, The following is included in the light chain variable region: (1E4) CDR-L1 (SEQ ID NO: 56) containing C cagagtattagcgacttt, CDR-L2 containing gctgcatcg (sequence number: 57), and A CDR-L3 containing ttacattatgcccgacact (SEQ ID NO: 58), or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0125] In one embodiment, the antigen-binding protein, its variant or fragment comprises a heavy chain variable region and a light chain variable region encoded by a nucleic acid sequence selected from the group consisting of:

[0126] (i) A heavy chain variable region comprising: (1A5-VHH, 2C4-VHH, 2D10-VHH and hu2C4-VHH) A CDR-H1 containing ggaagcatcttcagtggcaatgac (SEQ ID NO: 62), A CDR-H2 containing attactagcggtggtagtaca (SEQ ID NO: 63), and A CDR-H3 containing acaaacggaagatggtcaggcgatacttactatgcccatcac (SEQ ID NO: 64) (ii) A heavy chain variable region comprising: (1B8-VHH) A CDR-H1 containing ggaagctccgaaagattcacatca (SEQ ID NO: 66), A CDR-H2 containing attactaatggtggtagcaca (SEQ ID NO: 67), and A CDR-H3 containing atggcgggtacgtcc (SEQ ID NO: 68); and, (iii) A heavy chain variable region comprising: (2B7-VHH) A CDR-H1 containing ggaagcatcttcagtggcaatgac (SEQ ID NO: 62), A CDR-H2 containing attactagcggtggtagtaca (SEQ ID NO: 63), and A CDR-H3 containing acaaacggaagatggtcaggcgatacttactatgcccatctc (SEQ ID NO: 70), Or a fragment, variant, or fragment having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to it.

[0127] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a heavy chain variable domain encoded by a nucleotide sequence including:

[0128] (i) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41 - Clones 1B6, 1C1, 1C11, 1D4, 1H6), or, (ii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (Sequence ID: 45-Clone 1E4) A sequence that is at least 60% identical to or to the same, and / or has 10 to 20 nucleic acid substitutions.

[0129] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a light chain variable domain encoded by a nucleotide sequence including:

[0130] (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 49-Clones 1B6 and 1C1), or, (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID: 53-Clone 1C11), or, (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 55-Clone 1D4), or, (iv) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence ID: 59-Clone 1E4), or, (v) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 61-Clone 1H6) A sequence that is at least 60% identical to or to the same, and / or has 10 to 20 nucleic acid substitutions.

[0131] In one embodiment, an antigen-binding protein, its variant, or fragment comprises a single-domain heavy chain variable domain encoded by a nucleotide sequence including:

[0132] (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 65-Clone 1A5) (ii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (Sequence ID: 69-Clone 1B8) (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 72 - Clone 2C4), (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 73 - Clone 2D10), or, (vi) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (Sequence ID: 74-clone hu2C4-VHH) A sequence that is at least 60% identical to or to the same, and / or has 10 to 20 nucleic acid substitutions.

[0133] In one embodiment, an antigen-binding protein, a variant thereof, or a fragment comprises a light chain constant domain encoded by a nucleotide sequence including:

[0134] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (Sequence ID: 50-Clone 1B6-Light Chain Constant Domain), or, CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT (Sequence ID: 51-Clone 1C1-Light Chain Steady-State Domain), or, In contrast, a sequence that is at least 60% identical and / or has 10 to 20 nucleic acid substitutions.

[0135] In some embodiments, “antibody variant” means an antibody or its antigen-binding fragment comprising VH and / or VL, wherein the non-CDR region of the antibody or its antigen-binding fragment has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity as described herein.

[0136] In one embodiment, the antigen-binding protein is an antibody. In some embodiments, the antigen-binding protein may include an isolated antibody.

[0137] In some embodiments, the antigen-binding protein comprises a sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, the antigen-binding protein comprises at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, and at least Approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% of any of the sequences disclosed herein. It is the same as (for example, for any one of the sequences disclosed herein, about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%). or approximately 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The percentage of sequence identity can be determined using methods known in the art, such as programs like BLAST, NBLAST, or XBLAST.

[0138] In some embodiments, the antigen-binding protein comprises a sequence or amino acid region that differs from the sequence disclosed herein by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more amino acids or nucleic acid bases, or is encoded by a nucleotide region.

[0139] In some embodiments, the antigen-binding protein comprises an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, the antigen-binding protein comprises an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, one or more amino acid mutations may be selected independently of substitutions, insertions, deletions, and truncations.

[0140] In some embodiments, the amino acid mutation is an amino acid substitution, which may include conserved substitutions and / or non-conservative substitutions.

[0141] In various embodiments, substitutions involve substituting an amino acid with a natural amino acid such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, but are not limited to these. In various embodiments, substitutions do not involve substitution with cysteine. Therefore, in various embodiments, substitutions involve substituting an amino acid with a natural amino acid such as alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, but are not limited to these.

[0142] In various embodiments, the substitution may be a conservative substitution, replacing one amino acid with another having similar properties. For example, replacing one amino acid with another amino acid from the same group. In various embodiments, the substitution may include substitution with an amino acid having different properties. In various embodiments, the substitution is made without affecting the biological activity of the antigen-binding protein, its variant, or fragment, as described herein. In various embodiments, the substitution increases the binding affinity to EpCAM.

[0143] In some embodiments, substitutions may also include non-classical amino acids. Examples of non-classical amino acids include, but are not limited to, designer amino acids such as selenocysteine, pyrrolidine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, C-α-methylamino acids, N-α-methylamino acids, and amino acid analogs in general.

[0144] In some embodiments, amino acid mutations may be present in the CDR (e.g., CDR1, CDR2, or CDR3 region) of the antigen-binding protein. In other embodiments, amino acid modifications may be present in the framework region (FR) (e.g., FR1, FR2, FR3, or FR4 region) of the antigen-binding protein.

[0145] Modification of amino acid sequences can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis.

[0146] In some embodiments, the mutation does not substantially reduce the ability of the antigen-binding protein to specifically bind to its target. In some embodiments, the mutation does not substantially reduce the ability of the antigen-binding protein to specifically bind to its target, and functionally modulates the antigen-binding protein without functionally modifying (e.g., partially or completely neutralizing) the target.

[0147] In one embodiment, an antigen-binding protein, its variant, or fragment binds to EpCAM with low affinity, moderate affinity, or high affinity.

[0148] In one embodiment, an antigen-binding protein, its variant, or fragment has a low to moderate affinity (K D It binds to EpCAM at a range of 1 to 100 nM.

[0149] In one embodiment, an antigen-binding protein, its variant, or fragment has a moderate affinity K in the range of 10-100 nM. D They are joined together.

[0150] In one embodiment, an antigen-binding protein, its variant, or fragment binds to EpCAM with low affinity.

[0151] In some embodiments, the binding affinity of the antigen-binding protein of this disclosure to the full-length and / or mature and / or isoform and / or splice variant and / or fragment and / or monomer and / or dimer and / or other naturally occurring or synthetic analogs, variants, or variants (including monomer and / or dimer forms) of the antigen-binding protein is determined by the equilibrium dissociation constant (K). D) can be described by. In some examples, the antigen-binding protein is the full-length and / or mature and / or isoform and / or splice variant and / or fragment and / or other naturally occurring or synthetic analogs, variants, or variants (including monomers and / or dimers) of the antigen-binding protein, and K D The particle sizes are approximately less than 1 μM, approximately 900 nM, approximately 800 nM, approximately 700 nM, approximately 600 nM, approximately 500 nM, approximately 400 nM, approximately 300 nM, approximately 200 nM, approximately 100 nM, approximately 90 nM, approximately 80 nM, approximately 70 nM, approximately 60 nM, approximately 50 nM, approximately 40 nM, approximately 30 nM, approximately 20 nM, approximately 10 nM, or approximately 5 nM, or approximately 1 nM, approximately 50 pM, approximately 40 pM, approximately 30 pM, approximately 20 pM, approximately 10 pM, approximately 5 pM, or approximately 1 pM.

[0152] The binding affinity of the antigen-binding protein / fragment of this disclosure was measured by Bio-layer Interferometry (BLI) analysis (results are shown in Example 4). Favorably, all clones tested showed nanomolar-scale binding affinity to EpCAM. Surprisingly, the Koff rates of two prior art, clinically tested, high-affinity anti-EpCAM antibodies, 3622W94 and ING-1, were 1.8 × 10⁶, respectively. -5 and 3.2 × 10 -5 In comparison, the anti-EpCAM IgG1 antibody of this disclosure has a much slower off-rate (6.134 × 10⁻¹⁴). -4 ~3.278 × 10 -3 ) showed a much lower binding affinity than 3622W94 and ING-1.

[0153] In some embodiments, the antigen-binding protein may be a nanobody.

[0154] In some embodiments, the multispecific antigen-binding protein, its variant, or fragment is a bispecific antibody. In some embodiments, the multispecific antigen-binding protein may be provided as a nanobody. In some embodiments, the multispecific antigen-binding protein may be provided as an Fc region.

[0155] In some examples, the second antigen-binding protein of an immune cell engager, its variant, or its binding fragment binds to an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0156] In some embodiments, multispecific antigen-binding proteins, their variants, or fragments are inducible bispecific T cell engagers. In some embodiments, the multispecific antigen-binding proteins are secreted from immune cells.

[0157] In some examples, the multispecific antigen-binding protein is an inducible bispecific T cell engager containing a heavy-chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv).

[0158] In some embodiments, the bispecific T cell engager (BiTE) is a nanobody (VHH) having only heavy chains.

[0159] In one embodiment, a bispecific T cell engager (BiTE) binds to two antigens; where the first antigen is EpCAM; and the second antigen is an immune cell marker.

[0160] In some embodiments, the second antigen is an immune cell marker involved in the activation of immune cells.

[0161] In some embodiments, the antigen-binding protein binds to EpCAM in solution and / or on the surface of host cells.

[0162] In one embodiment, the second antigen targeted by the bispecific T cell engager (BiTE) is selected from the group including CD3, NKG2D, CD28, CD16, and the like.

[0163] In one embodiment, a bispecific T cell engager (BiTE), which is an antigen-binding protein, a variant thereof, or a fragment, targets one or more antigens, where the first antigen is EpCAM and where the second antigen is CD3.

[0164] In some embodiments, CD3 may include CD3ζ, CD3ε, CD3γ, CD3δ, and so on.

[0165] As shown in the experimental data in Figure 3A, the Fab region of one arm from each of the six anti-EpCAM human antibody clones (clones 1B6, 1C1, 1C11, 1D4, 1E4, and 1H6 disclosed herein) was replaced with an anti-CD3 scFv fragment, a knob-in-hole mutation was introduced to facilitate correct heavy chain pairing, and an LALA mutation was introduced to remove Fcγ receptor binding.

[0166] In one embodiment, the antigen-binding protein, its variant, or fragment is a human anti-EpCAM BiTE (e.g., clones 1B6, 1C1, 1C11, 1D4, 1E4, 1H6 disclosed herein).

[0167] Advantageously, as shown in the experimental data of this disclosure, the BiTE antibodies using the six anti-EpCAM clones of this disclosure (clones 1B6, 1C1, 1C11, 1D4, 1E4, and 1H6) showed significantly lower absorbance at 450 nm and higher EC50 values ​​compared to the benchmark BiTE (anti-EpCAM arm of "MT110" with scFv fragment of anti-CD3 clone Okt3) at 0.666 nM, suggesting lower binding affinity to the antigen (Figure 3B). All six antibodies bound to EpCAM-expressing HT-29 cells (Figure 3C) and CD3-expressing human T cells (Figure 3E), but the binding EC50 to HT-29 cells was 5 to 20 times lower than that of the benchmark BiTE (MT110, Solitomab) (4.106 pM) (Figure 3G). Unlike benchmark BiTEs, which showed 10–20% nonspecific binding to EpCAM-negative HeyA8 cells at high concentrations of 0.5, 2, and 10 nM (Figure 3F), the six antibodies did not bind to HeyA8 cells at any concentration (Figure 3D), suggesting that these anti-EpCAM BiTEs can bind to cell surface antigens with high degree of specificity. All anti-EpCAM BiTE antibodies (clones 1B6, 1C1, 1C11, 1D4, and 1H6) were able to kill 70–100% of HT-29 cells within 72 hours at a low concentration of 0.1 nM (Figure 4B). The 1C1 and 1H6 BiTE antibodies showed minimum EC50 values ​​of 18.5 pM and 12.02 pM, respectively; while 1B6, 1C11, 1D4, and 1E4 showed low to moderate cytotoxicity (Figure 4C).

[0168] In some embodiments, the anti-EpCAM antibody is a nanobody (VHH) having only a heavy chain. In some embodiments, the nanobody having only a heavy chain includes anti-EpCAM BiTEs (1A5-VHH, 1B8-VHH, 2B7-VHH, 2C4-VHH, 2D10-VHH, etc.).

[0169] In some embodiments, anti-EpCAM BiTE may comprise a single-domain antibody fragment (VHH) against EpCAM that can be fused to another antigen-binding protein (such as an anti-CD3 T cell agonist antibody) via a flexible linker.

[0170] As shown in the experimental data of this disclosure, the five Lama anti-EpCAM BiTE clones of this disclosure (1A5-VHH, 1B8-VHH, 2B7-VHH, 2C4-VHH, and 2D10-VHH) exhibited EC50 values ​​equivalent to or lower than the known benchmark anti-EpCAM BiTE "MT110" in the art, in binding to both immobilized biotinylated EpCAM antigen (Figure 6B) and EpCAM-expressing antigen (Figure 6F), suggesting high binding affinity to the antigen. All four clones (1A5-VHH, 2B7-VHH, 2C4-VHH, and 2D10-VHH) bound strongly to EpCAM-expressing HT-29 cells, as well as to human T cells (CD3-positive) (Figure 6E). The binding EC50 to HT-29 cells was in the range of 10-20 pM, and even at the highest concentration of 10 nM, none of the antibodies bound to EpCAM-negative and CD-negative HeyA8 cells, demonstrating excellent binding specificity (Figure 6D). Four anti-EpCAM VHH BiTE antibodies (1A5-VHH, 2B7-VHH, 2C4-VHH, and 2D10-VHH) were able to kill 90-100% of EpCAM-positive HT-29 (Figure 7A), HepG2 (Figure 7B), and Hep3B (Figure 7C) cells within 96 hours at a low concentration of 10 pM, while EpCAM-negative HeyA8 cells were preserved.

[0171] While we do not wish to be constrained by theory, the antigen-binding fragments of VHH antibodies are advantageously much smaller than those of conventional antibodies, possessing superior properties such as high tissue penetration, high solubility, stability, and resistance to heat denaturation.

[0172] In one embodiment, the antigen-binding protein, its variant, or fragment is a humanized anti-EpCAM BiTE (e.g., hu2C4-VHH as disclosed herein).

[0173] As shown in the experimental results of this disclosure, the humanized clone hu2C4-VHH was constructed by pairing the EpCAM BiTE antibody with a well-characterized anti-CD3 Okt3. Humanization resulted in a tenfold decrease in binding affinity to the antigen protein (Figure 8A) and approximately 2.5-fold weakening of cell binding (Figures 8B and 8E). Binding to the CD3 arm was not affected by the humanization of the VHH arm (Figure 8D), but hu2C4-VHH / Okt3 showed high binding specificity, as it did not bind to EpCAM-negative HeyA8 cells, similar to its parent clone (Figure 8C). The EC50 of hu2C4-VHH / Okt3 remained within the pM range (Figures 9A and 9C). Similar to 2C4-VHH / Okt3 BiTE, hu2C4-VHH / Okt3 also did not kill EpCAM-negative HeyA8 cells (Figure 9B), and unlike the benchmark BiTE (MT110), it did not induce the secretion of interferon-γ (Figure 9D) or IL-2 (Figure 9E) during co-culture.

[0174] In one embodiment, the antigen-binding protein, its variant, or fragment comprises an immunoglobulin Fc fragment (region / domain), a protein capable of extending the half-life of the recombinant / fusion polypeptide (such as albumin, human albumin), a linker capable of increasing the binding titer (e.g., a rigid linker or a flexible linker such as a GGGGS repeat), or a combination thereof. The immunoglobulin Fc fragment is an IgG Fc fragment such as a human IgG Fc fragment.

[0175] In various embodiments, immunoglobulin fragments may, but are not limited to, Fc regions / domains, one or more CH regions / domains, Fab, Fab', F(ab')2, single-chain Fv(ScFv) and / or Fv fragments, hinge regions / domains, and fragments or portions thereof.

[0176] In some embodiments, antigen-binding proteins, their variants, or fragments may also include immunoglobulin molecules or parts of antibodies, for example, but not limited to, the constant heavy chain, variable heavy chain, constant light chain, variable light chain, hinge region, and / or Fc domain of Ig, as well as their variants. For example, the recombinant / fusion polypeptides described herein may be combined with a part of human IgG. In various embodiments, the type of immunoglobulin may include, but is not limited to, one or more types such as IgG, IgE, IgM, IgD, IgA, and IgY. In one embodiment, the type of immunoglobulin may be immunoglobulins of class IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or their subclasses.

[0177] In one embodiment, the immunoglobulin fragment is an IgG Fc fragment.

[0178] In another context, polynucleotides encoding immune cells and / or polypeptides and / or multispecific antigen-binding proteins described herein are provided.

[0179] In some examples, the sequence encoding the immune cell engager encodes a multispecific antigen-binding protein. In some examples, the multispecific antigen-binding protein is a bispecific antibody. In some examples, the sequence encoding the immune cell engager encodes an antigen-binding protein (anti-EpCAM antigen-binding protein), or a fragment or variant thereof, and an anti-immune cell antigen-binding protein.

[0180] In some examples, the sequence encoding the immune cell engager encodes a single variable domain located on a single-chain variable fragment (scFv) or a heavy chain (VHH).

[0181] In some examples, the sequence encoding the immune cell engager encodes either anti-EpCAM scFv or anti-EpCAM VHH.

[0182] In some embodiments, the sequence encoding the immune cell engager encodes an anti-immune cell antigen-binding protein that binds to an immune cell activation marker. In some embodiments, the immune cell activation marker may include, but is not limited to, CD3, NKG2D, CD4, CD8, CD16, CD64, and the like.

[0183] In some examples, the immune activation marker may be of the VHH type or scFv type. In some examples, the sequence encoding the immune cell engager encodes a CD3-binding antigen-binding protein (anti-CD3 antigen-binding protein), or a fragment or variant thereof. In some examples, the sequence encoding the immune cell engager encodes a single-chain variable fragment (scFv) type or a VHH type. In some examples, the sequence encoding the immune cell engager encodes anti-CD3 scFv or anti-CD3 single-domain VHH.

[0184] In some embodiments, the immune cell engager may include a His tag.

[0185] In some embodiments, the immune cell engager comprises an anti-EpCAM antigen-binding protein, a linker, an anti-CD3 scFv or anti-CD3 single-domain VHH, and a His tag. In some embodiments, the linker is a cleavable linker, which includes, but is not limited to, P2A, T2A, F2A, etc.

[0186] In some examples, the polynucleotide containing a sequence encoding an immune cell engager is a sequence encoding a bispecific T cell engager (BiTE). In some examples, the polynucleotide contains a sequence encoding BiTE that bispecifically binds to EpCAM and T cells. In some examples, the polynucleotide contains a sequence encoding an anti-GPC3 scFv CAR having a CD3 intracellular domain and a sequence encoding BiTE that binds to EpCAM and T cells.

[0187] In one embodiment, the polynucleotide comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 38 to 74.

[0188] In some embodiments, the polynucleotide sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences disclosed herein.

[0189] In one embodiment, the vector is an expression vector.

[0190] In some embodiments, the vector is selected from the group consisting of plasmids, viral particles, phages, baculoviruses, yeast plasmids, lipid-based vehicles, polymer microspheres, liposomes, and cell-based vehicles, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, viral vehicles, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, Formy viruses, cytomegaloviruses, Semryki forest viruses, poxviruses, pseudorabies viruses, RNA viral vectors, DNA viral vectors, and vectors derived from combinations of plasmids and phage DNA, further optionally, the polynucleotides are operably linked to expression control sequences(options) to specify peptide synthesis, and further optionally, the vector includes one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0191] In one embodiment, the vector is selected from the group including DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0192] In some embodiments, the vector is a lentiviral vector.

[0193] In some embodiments, the host cell contains the cloning vector or expression vector and / or a nucleic acid sequence encoding the antigen-binding protein, antibody and its binding fragment.

[0194] A host cell can be any type of cell that can be transformed or transfected with a nucleic acid or vector to produce an antigen-binding protein or its binding fragment / protein encoded by it. A host cell containing a nucleic acid or vector can be used to produce an antigen-binding protein or its binding fragment / protein, or a portion thereof (e.g., a heavy chain or light chain sequence encoded by the nucleic acid or vector). After introducing the nucleic acid or vector into the cell, the cell is cultured under conditions suitable for the expression of the encoded sequence. The antibody, antigen-binding protein, fragment, or portion of the antibody can then be isolated from the cell.

[0195] The host cell may be a prokaryotic host cell (such as E. coli) or a eukaryotic host cell (such as yeast cells, insect cells, or vertebrate cells). When cultured under appropriate conditions, the host cell expresses the antibody or its binding fragment, which can then be recovered from the culture medium (if the host cell secretes it into the culture medium) or directly from the host cell producing it (if it does not secrete it). The selection of an appropriate host cell depends on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule. The selection of the host cell also depends in part on whether the antibody or its binding fragment is post-transcriptionally modified (e.g., glycosylation and / or phosphorylation). Host cells can include bacterial cells, yeast cells, animal cells, such as mammalian cells and / or plant cells.

[0196] Suitable mammalian host cells include CHO cells, myeloma cells, and hybridoma cells. Many are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include Chinese hamster ovary cells (CHO) (ATCC number CCL61), human embryonic kidney (HEK) 293 or 293T cells (ATCC number CRL1573), 3T3 cells (ATCC number CCL92), or PER.C6 cells. Other cell types that can be used to express antibodies include lymphocyte cell lines, such as NSO myeloma cells and SP2 cells, and COS cells.

[0197] In one embodiment, a host cell expresses / secretes an antigen-binding protein, variant, or fragment thereof as disclosed herein.

[0198] In one aspect, cells that express / secrete EpCAM-specific immune cell engagers are provided.

[0199] In one embodiment, the cells are stem cells, selected from the group including, for example, mesenchymal stem cells, neural stem cells, and pluripotent stem cells such as induced pluripotent stem cells (iPSCs). Therefore, in one embodiment, the stem cells are mesenchymal stem cells. In one embodiment, the stem cells are neural stem cells. In one embodiment, the stem cells are pluripotent stem cells such as iPSCs.

[0200] In one embodiment, the cells are immune cells.

[0201] In one embodiment, immune cells express / secrete EpCAM-specific immune cell engagers. In one embodiment, the immune cell engagers are selected from the group including T cell engagers, NK cell engagers, monocyte engagers, and macrophage engagers.

[0202] In one embodiment, immune cells express / secrete bispecific T cell engagers (BiTEs) that are specific to EpCAM.

[0203] In one embodiment, immune cells express / secrete bispecific T cell engagers (BiTEs), such as inducible, non-inducible, or constitutively expressed BiTEs, which include antigen-binding proteins, variants, or fragments thereof, as disclosed herein.

[0204] Surprisingly, the inventors have demonstrated that immune cells, such as CAR T cells, can secrete EpCAM BiTE. See Examples 5-7. This was previously unknown. Advantageously, by engineering immune cells to express the EpCAM immune engager, the EpCAM immune engager can be secreted at a target site (e.g., a solid tumor site), thereby minimizing toxicity and / or side effects.

[0205] In some embodiments, immune cells include, but are not limited to, macrophages, dendritic cells, T cells, B cells, eosinophils, basophils, neutrophils, mast cells, natural killer T cells (NKT cells), natural killer cells (NK cells), macrophages, monocytes, and the like. In one embodiment, the immune cells are NK cells. In one embodiment, the immune cells are macrophages. In one embodiment, the immune cells are dendritic cells. In one embodiment, the immune cells are monocytes.

[0206] In one embodiment, the immune cells are T cells. In one embodiment, the immune cells are CAR T cells such as anti-GPC3, anti-HER2, or anti-CD19 CAR T cells. Therefore, in one embodiment, the CAR T cells are anti-GPC3 CAR T cells. In one embodiment, the CAR T cells are anti-HER2 CAR T cells. In one embodiment, the CAR T cells are anti-CD19 CAR T cells. In one embodiment, the immune cells are CAR T cells, CAR NK cells, CAR macrophages, or CAR monocytes.

[0207] In some embodiments, immune cells can bind to multiple host cell antigens. Therefore, in some embodiments, immune cells may further bind to one host cell antigen, two host cell antigens, three host cell antigens, four host cell antigens, and so on.

[0208] While we don't want to be constrained by theory, CAR T cells directly kill target cells, whereas anti-EpCAM BiTE exerts cytotoxicity by recruiting nearby immune cells (such as T cells). This approach physically induces bystander T cells near the tumor, simultaneously activating them to aid in tumor cell clearance. By utilizing the local secretion of anti-EpCAM BiTE by CAR T cells, non-drug targets can be converted into drug targets.

[0209] Since EpCAM is also defined as a cancer stem cell marker expressed in cancer progenitor cells and cancer stem cells, the secretion of anti-EpCAM BiTE by CAR T cells simultaneously contributes to the prevention of cancer relapse and recurrence by eliminating cancer stem cells and cancer progenitor cells.

[0210] In one embodiment, immune cells express an inducible bispecific T cell engager (BiTE) that includes a heavy chain variable region containing a heavy chain complementarity determining region:

[0211] (i) (In the case of clones 1B6, 1C1, 1C11, 1D4, and 1H6) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3), or (ii) (In the case of clone 1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7), and (iii) (In the case of clone 1A5-VHH) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (Sequence ID: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID: 27), or (iv) (Regarding clones 1B8-VHH, 2C4-VHH, 2D10-VHH, and hu2C4-VHH) CDR-H1 containing GSSERFTS (Sequence ID: 29), CDR-H2 containing ITNGGST (Sequence ID: 30), and CDR-H3 containing MAGTS (Sequence ID: 31), or (v) (In the case of clone 2B7-VHH) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (Sequence ID: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33), Or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical fragments, variants, or sequences, and, Light chain variable region including light chain complementarity determination region: (i) (In the case of clones 1B6 and 1C1) CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11), or (iii) (In the case of clone 1C11) CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and A CDR-L3 containing MQGLQSPWT (Sequence ID: 15), or (iii) (In the case of clone 1D4) CDR-L1 containing QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11), or (iv) (In the case of clone 1E4) CDR-L1 containing QSISDF (Sequence ID: 19), CDR-L2 containing AAS (Sequence ID: 20), and CDR-L3 containing QQSYIMPDT (Sequence ID: 21), or (v) (In the case of clone 1H6) CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (Sequence ID: 23), Or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical fragments, variants, or arrays.

[0212] In one embodiment, immune cells express inducible bispecific T cell engagers (BiTEs) that further bind to immune cell activators.

[0213] In one embodiment, immune cells express an inducible bispecific T cell engager (BiTE) that further binds to CD3.

[0214] Polypeptides containing multispecific antigen-binding proteins are also disclosed. In some examples, the multispecific antigen-binding protein is a bispecific antibody. In some examples, the polypeptide includes multispecific antigen-binding proteins that bind to EpCAM (epithelial cell adhesion molecule) and immune cells.

[0215] In some examples, the multispecific antigen-binding protein is a bispecific immune cell engager capable of binding to both antigens and immune cells.

[0216] In some examples, the polypeptide comprises an anti-EpCAM antigen-binding protein. In some examples, the polypeptide comprises a single-domain anti-EpCAM antibody, optionally an anti-EpCAM H-chain antibody variable region (i.e., VHH).

[0217] In some examples, the polypeptide comprises an anti-immune cell antigen-binding protein. In some examples, the polypeptide comprises an anti-immune cell antigen-binding protein that binds to an immune cell activation marker. In some examples, the immune cell activation marker is CD3, NKG2D, CD4, CD8, CD16, CD64, etc. In some examples, the polypeptide binds to CD3. In some examples, CD3 may include CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0218] In some examples, the polypeptide comprises an anti-CD3 antigen-binding protein. In some examples, the polypeptide comprises a single-chain variable fragment (anti-CD3 scFv) of an anti-CD3 antibody.

[0219] In some examples, the polypeptide is a bispecific antibody / antigen-binding protein. In some examples, the bispecific antibody / antigen-binding protein is a bispecific T cell engager (BiTE). In some examples, the bispecific T cell engager (BiTE) binds to two antigens, where the first antigen is EpCAM and the second antigen is an immune cell marker. In some examples, the second antigen is an immune cell marker involved in the activation of immune cells. In some examples, the second antigen targeted by the bispecific T cell engager (BiTE) may include, but is not limited to, CD3, NKG2D, CD28, CD16, CD64, etc. In some examples, the polypeptide contains BiTE that bispecifically binds to EpCAM and T cells. In some examples, the polypeptide contains BiTE that bispecifically binds to EpCAM and CD3. In some examples, the EpCAM targeted by BiTE is modified with anti-EpCAM VHH paired with anti-CD3 scFv.

[0220] In some embodiments, anti-EpCAM VHH is paired with anti-CD3 scFv, which may include clone Okt3 (Nb01-013A) or anti-CD3 clone (MT110) (Nb01-013B) used by another anti-EpCAM in the art.

[0221] In one aspect, a method for producing / generating an antigen-binding protein, a variant thereof, or a fragment disclosed herein is provided, the method comprising:

[0222] (a) A step of using a library for screening antigen-binding proteins; and, (b) A step of cloning antigen-binding proteins into antibody format.

[0223] As shown in the experimental data of this disclosure, 21 clones exhibiting a positive Fab supernatant binding signal to biotinylated hEpCAM-His protein and specific binding to EpCAM-positive HT-29 cells were sequenced, and six unique sequences (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6) were identified. Clones 1B6, 1C1, 1C11, 1D4, and 1H6 shared the same sequence in the heavy chain variable region, but differed in the light chain sequences of the variable region. However, while 1B6 and 1C1 shared the same sequence in the light chain variable region, there was a difference of 3 amino acids in the κ light chain constant region.

[0224] As shown in the experimental data of this disclosure, five unique clones (1A5, 1B8, 2B7, 2C4, and 2D10) were identified that specifically bind to gastric cancer cells (such as AGS cell lines).

[0225] In one embodiment, the disclosed anti-EpCAM human Fab / VHH clone is used to construct an anti-EpCAM BiTE antibody.

[0226] In one aspect, a method is provided for producing / generating an antigen-binding protein, a variant thereof, or a fragment disclosed herein, comprising expressing a polynucleotide described herein in a host cell.

[0227] In one aspect, a pharmaceutical composition comprising an antigen-binding protein, a variant thereof, or a fragment thereof, as disclosed herein, is provided.

[0228] In one embodiment, the composition is a prophylactic and / or therapeutic composition.

[0229] In some embodiments, the composition comprises one or more pharmaceutically acceptable agents. Examples of pharmaceutically acceptable agents for use in the pharmaceutical compositions of the present invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, fragrances and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonic agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobial agents and surfactants.

[0230] Pharmaceutically acceptable salts may be used, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate, or organic salts like acetate, propionate, malonate, and benzoate.

[0231] Pharmaceutically acceptable carriers in therapeutic compositions may further include liquids such as water, saline, glycerol, and ethanol. Furthermore, auxiliary substances such as wetting agents, emulsifiers, and pH buffers may be present in such compositions. Such carriers enable the formulation of pharmaceutical compositions for patient administration as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions. A thorough discussion of pharmaceutically acceptable carriers is presented in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991).

[0232] In one embodiment, the composition further comprises a pharmaceutically acceptable excipient, buffer, or carrier.

[0233] In one embodiment, the composition is intended for use in therapeutics / pharmaceuticals / vaccines.

[0234] In one aspect, a method is provided for preventing and / or reducing the severity of symptoms caused by a disease in a subject that requires it. The method includes the step of administering an antigen-binding protein, a variant or fragment thereof, or a composition disclosed herein to a subject.

[0235] In one embodiment, antigen-binding proteins, their variants or fragments, or compositions thereof are administered to a subject by a mode of administration known in the Art, including but not limited to intramuscular, subcutaneous, intravenous, intraarterial, intra-articular, intraperitoneal, nasal, or parenteral administration.

[0236] In one embodiment, the subject is a mammal such as a monkey, rabbit, mouse, rat, pig, or dog. In another embodiment, the subject is a human.

[0237] In one aspect, the proteins, compositions, therapeutic methods, manufacturing methods, or other methods described herein are provided.

[0238] [array] CDR is bold (CDR1); bold and italic (CDR2); or bold, italic and underlined ( CDR3 The highlighted residues / bases show the difference between the 1B6 and 1C1 light chain constant domains.

[0239] JPEG2026519826000001.jpg97139

[0240] JPEG2026519826000002.jpg197143

[0241] JPEG2026519826000003.jpg181129

[0242] JPEG2026519826000004.jpg203140

[0243] JPEG2026519826000005.jpg202141

[0244] JPEG2026519826000006.jpg208138 [Examples]

[0245] The exemplary embodiments of this disclosure will be well understood and readily apparent to those skilled in the art from the following discussion, together with the drawings where applicable. It should be understood that other modifications may be made without departing from the scope of the invention. The exemplary embodiments are not necessarily mutually exclusive, as in some embodiments one or more embodiments may be combined to form new exemplary embodiments. No embodiment should be construed as limiting the scope of this disclosure.

[0246] Example 1 - Discovery of fully human anti-EpCAM antibodies from a naive human Fab phage display library. To identify cell lines that can be used to characterize the properties of the anti-EpCAM antibody associated with the present invention, the inventors analyzed surface EpCAM antigen expression using commercially available anti-EpCAM antibodies (Figure 1). As expected, EpCAM showed high expression in all cells tested in epithelial morphology, including lung adenocarcinoma cell line HT-29, gastric adenocarcinoma cell line AGS, hepatoblastoma cell line HepG2, hepatocellular carcinoma cell line Hep3B, and breast cancer cell line MCF-7. However, EpCAM was not expressed in non-epithelial cells such as human T cells, ovarian cell line HeyA8, and two glioblastoma cell lines A172 and U-87.

[0247] Next, using biotinylated recombinant human EpCAM protein with a polyHis tag (hEpCAM-His), low-affinity EpCAM conjugates were isolated from a naive library of Fab sequences constructed with SIgN using phage display technology. Of the 190 clones screened, the nucleotide sequences of 21 clones showing a positive Fab supernatant binding signal to biotinylated hEpCAM-His protein and specific binding to EpCAM-positive HT-29 cells were determined, and six unique sequences were identified: 1B6, 1C1, 1C11, 1D4, 1E4, and 1H6. Clones 1B6, 1C1, 1C11, 1D4, and 1H6 share the same sequence in the heavy chain variable region, but their light chain sequences in the variable region differ. However, clones 1B6 and 1C1 also share the same sequence in the light chain variable region, but there is a difference of 3 amino acids in the κ light chain constant region (see appendix). The results of cell binding analysis using Fab supernatant via binding ELISA and flow cytometry for six unique clones are shown in Figures 2A and 2B, respectively. Next, these Fab antibodies were cloned into IgG1 format, and their binding affinity to the antigen protein was determined by affinity binding ELISA (Figure 2C). The binding EC50 values ​​were 3–6 nM, which was higher than the binding EC50 of benchmark IgG (1.161 nM, using the anti-EpCAM arm sequence of "MT110"), suggesting that these anti-EpCAM antibodies derived from the human naive Fab library have relatively low binding affinity to the EpCAM antigen.

[0248] Example 2 - Use of anti-EpCAM bispecific T cell engagers Anti-EpCAM bispecific T cell engagers (BiTEs) were constructed using anti-EpCAM antibodies. In these constructs, the Fab region of one arm of six anti-EpCAM human antibody clones (1B6, 1C1, 1C11, 1D4, 1E4, 1H6) was replaced with an anti-CD3 scFv fragment, a knob-in-hole mutation was introduced to promote correct heavy chain pairing, and an LALA mutation was introduced to remove Fcγ receptor binding (Figure 3A). The binding of these anti-EpCAM BiTE antibodies to the antigen protein was first examined by affinity-binding ELISA. Compared to a benchmark BiTE showing a binding EC50 of 0.666 nM (constructed with the anti-EpCAM arm of "MT110" and the scFv fragment of anti-CD3 clone Okt3, the same structure as shown in Figure 3A), the BiTE antibodies using the six newly identified anti-EpCAM clones showed much lower absorbance at 450 nm and higher EC50 values, suggesting lower binding affinity to the antigen (Figure 3B).

[0249] Using HT-29 cells, HeyA8 cells, and human T cells, we measured the ability of these antibodies to specifically recognize and bind to both EpCAM and human CD3 molecules on the cell surface. HT-29 cells and human T cells constitutively express high levels of EpCAM and CD3, respectively, while HeyA8 cells do not express either EpCAM or CD3. In this assay, all six anti-EpCAM BiTE antibodies bound to EpCAM-expressing HT-29 cells (Figure 3C) and CD3-expressing human T cells (Figure 3E). The binding EC50 to HT-29 cells was 5 to 20 times lower than that of the benchmark BiTE (MT110, Solitomab, constructed with the same structure as shown in Figure 3A) (4.106 pM) (Figure 3G). However, unlike the benchmark BiTE, which showed 10–20% nonspecific binding to EpCAM-negative HeyA8 cells at high concentrations of 0.5, 2, and 10 nM (Figure 3F), these antibodies did not bind to HeyA8 cells at any of the concentrations tested (Figure 3D). This suggests that these anti-EpCAM bispecific T cell engagers can bind to cell surface antigens with high degree of specificity.

[0250] The efficacy of these anti-EpCAM BiTE antibodies was then tested in an antibody-dependent T cell-mediated cytotoxicity assay. Briefly, EpCAM-positive HT-29 cells (T: target cells) were seeded on an xCelligence E-plate and cultured overnight to measure cell index values. The following day, immediately after adding various concentrations of anti-EpCAM BiTE antibody, primary T cells (E: effector cells) isolated from human PBMCs were added to the target cells in an effector-to-target ratio (E:T = 6:1). The assay was continued for a further 96 hours, and target cell index values ​​were measured using the xCelligence RTCA system (Figure 4A). With the exception of clone 1E4, which induced deep cell lysis of target cells only at high concentrations of 10 nM and 1 nM, all other anti-EpCAM BiTE antibodies (clones 1B6, 1C1, 1C11, 1D4, and 1H6) were able to kill 70–100% of HT-29 cells within 72 hours at a low concentration of 0.1 nM (Figure 4B). Among the six clones, the BiTE antibodies of clones 1C1 and 1H6 showed the lowest EC50 values ​​of 18.5 pM and 12.02 pM, respectively; while 1B6, 1C11, 1D4, and 1E4 showed low to moderate cytotoxicity (Figure 4C).

[0251] In summary, the results of the assays described above, shown in Figures 1-4, demonstrate that highly specific human anti-EpCAM antibodies disclosed in the format of bispecific T cell engagers can induce a wide range of low-to-high titers in the induction of cell death in EpCAM-expressing cells, and (based on the results of in vitro binding and killing assays) may be usable for the treatment of solid tumors with an enhanced safety profile.

[0252] Example 3 - Discovery of anti-EpCAM llama antibodies from immunized llamas To discover anti-EpCAM llama VHH antibodies, the inventors immunized male alpacas with recombinant human EpCAM protein with a polyHis tag (hEpCAM-His) a total of six times at time intervals of 1-2 weeks. The llama immunization process was performed by VIB Nanobody Core at VUB (Vrije Universiteit Brussel) as a paid service. Subsequent humanization of the llama antibodies was performed using SIgN. Approximately 150 μg of protein was used for the two initial immunizations, and 100 μg of protein was used for the last four booster immunizations. After the six immunizations, peripheral blood lymphocytes were collected from the alpacas, RNA was extracted, and the VHH sequence was amplified using cDNA as a PCR template to construct a VHH library. To isolate EpCAM conjugates from the constructed llama VHH library, biotinylated recombinant human EpCAM protein with a polyHis tag (hEpCAM-His) was used with phage display technology. Of the 190 clones screened, the nucleotide sequences of eight clones showing a positive VHH supernatant binding signal to biotinylated hEpCAM-His protein and specific binding to EpCAM-positive AGS cells were determined, and five unique sequences were identified: 1A5, 1B8, 2B7, 2C4, and 2D10 (see sequences below). The results of binding ELISA for the five unique VHH clones and flow cytometry analysis of cell binding using the supernatant of crude cells are shown in Figure 5.

[0253] Similar to the anti-EpCAM human Fab clones mentioned earlier, five VHH clones of anti-EpCAM antibodies were also used to construct anti-EpCAM VHH BiTE antibodies (Figure 6A). The binding affinity of these anti-EpCAM VHH BiTE antibodies to the antigen protein was first examined using affinity-binding ELISA. Compared to the benchmark BiTE (Figure 3B), the BiTE antibodies using these five anti-EpCAM VHH clones showed equivalent or lower binding EC50 values, suggesting high binding affinity to the antigen (Figure 6B).

[0254] The ability of these antibodies to specifically recognize and bind to both EpCAM and human CD3 molecules on the cell surface was also measured using HT-29 (EpCAM-positive), HeyA8 (EpCAM-negative, CD3-negative), and human T cells (CD3-positive). With the exception of clone 1B8-VHH, the other four clones (1A5-VHH, 2B7-VHH, 2C4-VHH, and 2D10-VHH) all strongly bound to EpCAM-expressing HT-29 cells (Figure 6C) and human T cells (Figure 6E). The binding EC50 to HT-29 cells ranged from 10 to 20 pM, and even at the highest concentration of 10 nM, they did not bind to EpCAM-negative HeyA8 cells, indicating excellent binding specificity (Figure 6D).

[0255] These anti-EpCAM VHH BiTE antibodies were also tested for their killing effects in antibody-dependent T cell-mediated cytotoxicity assays. The results showed that, with the exception of clone 1B8-VHH, which induced deep cytolysis of HT-29 cells only at a maximum concentration of 10 nM, the other four anti-EpCAM VHH BiTE antibodies (1A5-VHH, 2B7-VHH, 2C4-VHH, 2D10-VHH) were able to kill 90-100% of EpCAM-positive HT-29 (Figure 7A), HepG2 (Figure 7B), and Hep3B (Figure 7C) cells within 96 hours at a low concentration of 10 pM, while EpCAM-negative HeyA8 cells (Figure 7D) were preserved. Of the five clones, clones 2B7-VHH, 2C4-VHH, and 2D10-VHH all showed very potent cytotoxic effects (Figure 7E), but 2C4-VHH and 2D10-VHH showed superior cytotoxic specificity compared to 2B7-VHH (Figure 7D).

[0256] Although 2C4-VHH and 2D10-VHH have the same CDR sequence, 2C4-VHH was selected as a lead VHH clone for further development because its skeleton was closer to the human VH sequence. A humanized clone, hu2C4-VHH, was constructed, and its antigen-binding ability was compared with that of the parent clone 2C4-VHH using a combination of a BiTE antibody and the well-characterized anti-CD3 agonist Okt3. Humanization reduced the binding affinity to the antigen protein tenfold (Figure 8A), and cell binding weakened by approximately 2.5 times (Figures 8B and 8E). Binding to the CD3 arm was not affected by the humanization of the VHH arm (Figure 8D). On the other hand, hu2C4-VHH / Okt3, like the parent clone, did not bind to EpCAM-negative HeyA8 cells, showing high binding specificity (Figure 8C).

[0257] Next, the killing power of the BiTE antibody using hu2C4-VHH / Okt3 was tested using EpCAM-positive MCF-7 cells. Interestingly, by using clone Okt3 as the anti-CD3 arm, the 2C4-VHH BiTE antibody became extremely potent, with a cell-killing EC50 of less than 0.1 pM. The humanized version of hu2C4-VHH / Okt3 BiTE showed a 144.1-fold decrease in killing power compared to the parent clone, but still maintained an EC50 within the picomolar range (Figures 9A and 9C), making hu2C4-VHH / Okt3 an excellent lead with potential for downstream applications. Similar to 2C4-VHH / Okt3 BiTE, hu2C4-VHH / Okt3 did not kill EpCAM-negative HeyA8 cells (Figure 9B), and unlike the benchmark BiTE (MT110 constructed with the same structure as shown in Figure 3A), it did not induce the secretion of interferon-γ (Figure 9D) or IL-2 (Figure 9E) during co-culture.

[0258] In summary, the BiTE antibody derived from llama VHH of the present invention (Figures 5-7) and its humanized counterpart hu2C4-VHH (Figures 8-9) both exhibit highly potent and specific killing activity directed towards EpCAM-expressing cells, and can be used to treat epithelial solid tumors when delivery is locally limited to the tumor site.

[0259] Example 4 - Experiment to determine the binding affinity of anti-EpCAM human IgG1 antibody and anti-EpCAM llama VHH antibody Anti-EpCAM human IgG 1 Antibody binding affinity The binding affinities of six anti-EpCAM IgG1 antibodies were measured by Bio-Layer Interferometry (BLI) analysis using the Octet RED96 system and are shown in Table 1. All six antibody clones exhibited nanomolar-scale binding affinities to recombinant EpCAM protein, ranging from the lowest affinity at 32.39 nM (clone 1H6) to the highest affinity at 1.432 nM (clone 1D11). The Koff rates of 3622W94 and ING-1, two high-affinity anti-EpCAM antibodies already undergoing clinical trials, were 1.8 x 10⁻⁶, respectively. -5 and 3.2x10 -5 In comparison, the off-rate of the anti-EpCAM IgG1 antibody of the present invention is much more gradual (6.134 x 10⁻¹⁴). -4 to 3.278 x 10 -3 ) showed a much lower binding affinity than 3622W94 (0.19 nM) and ING-1 (0.16 nM) (Reference - Munz et al. Cancer Cell International 2010, 10:44).

[0260] (Table 1) Binding affinity of anti-EpCAM human IgG1 antibody to human EpCAM protein (K D ), association rate constant, dissociation rate constant (K d Measurement by Octet BLI analysis of )

[0261] [Table 1]

[0262] Binding affinity of anti-EpCAM rama VHH antibody The binding affinities of five anti-EpCAM llama VHH antibodies were measured by Bio-Layer Interferometry (BLI) analysis using the Octet RED96 system and are shown in Table 2. All five VHH antibody clones were expressed as bivalent Fc fusion proteins for the assay and exhibited nanomolar-scale binding affinities to recombinant EpCAM protein, ranging from the lowest affinity of 9.715 nM (clone 2C4-VHH) to the highest affinity of 1.975 nM (clone 1B8-VHH).

[0263] (Table 2) Binding affinity (KD), binding rate constant, and dissociation rate constant (K) of anti-EpCAM rama VHH antibody (expressed as a bivalent Fc fusion) against human EpCAM protein. d Measurement by Octet BLI

[0264] [Table 2]

[0265] Example 5 - Experiment demonstrating that human CAR T cells secreting the anti-EpCAM BiTE molecule exhibited cytotoxicity, killing EpCAM-expressing cells in vitro. Using anti-EpCAM bispecific T cell engagers with 2C4-VHH paired with either the anti-CD3 clone Okt3 (named "Nb01-013A") or the anti-CD3 clone used in "MT110" (named "Nb01-013B"), His-tagged secretory anti-EpCAM BiTEs were constructed. These anti-EpCAM BiTEs were inserted into different CAR constructs against different tumor antigens, e.g., GPC3, CD19, or HER2.

[0266] To demonstrate that anti-EpCAM BiTE molecules can be secreted by human T cells and contribute to the cytotoxicity of EpCAM-expressing target cell killing, the inventors constructed anti-GPC3 CAR T cells that secrete different anti-EpCAM BiTE molecules ("MT110", "Nb01-013A", "Nb01-013B") and named them GE CAR-BiTE T cells. The inventors also constructed anti-CD19 CAR T cells that secrete different anti-EpCAM BiTE molecules ("Nb01-013A" or "Nb01-013B") and named them "19E CAR-BiTE T" cells (Figure 10A). For these GE CAR-BiTE T cells and 19E CAR-BiTE T cells, the inventors constructed Hep3B cells (GPC3 High , EpCAM High ), HT-29 cells (GPC3 Low , EpCAM High ) and HeyA8 cells (GPC3 -ve、 EpCAM -ve The in vitro killing effect was tested using ).

[0267] In in vitro killing assays using the xCelligence RTCA system, an E:T ratio of 1:1 (adjusted to CAR expression level 10%) was applied. All GE CAR-BiTE T cells did not kill HeyA8 (GPC3-ve, EpCAM-ve) cells at low E:T ratios (Figure 10D), but strong cytotoxicity was observed in killing EpCAM-positive cells Hep3B (Figure 10B) and HT-29 (Figure 10C). 19E CAR-BiTE T cells (using Nb01-013A as BiTE) were shown to kill EpCAM-positive cells such as Hep3B (GPC3-ve). High , EpCAM High (Figure 10B) and HT-29 (GPC3 Low , EpCAM HighPartial killing was observed against both (Figure 10C). This indicates that the amount of anti-EpCAM BiTE secreted by 19E CAR-BiTE T (Nb01-013A) cells exceeds the threshold for BiTE-mediated cell killing in the current in vitro setting and is independent of CAR-specific antigen expression levels. On the other hand, 19E CAR-BiTE T cells (using "Nb01-013B" as BiTE) showed partial killing of HT-29 (GPC3 Low , EpCAM High Since it showed only minimal cell killing, it was suggested that the BiTE of "Nb01-013B" is far less effective at mediating T cell killing than the BiTE of "Nb01-013A" (Figure 10C).

[0268] The inventors also constructed HE CAR-BiTE T cells by fusing anti-EpCAM BiTE (using the sequence "Nb01-013A") to anti-HER2 CAR ("Trastuzumab" clone 4D5 or their own anti-HER2 clone F5) lentiviral constructs via a P2A cleavable linker (Figure 11A). Following lentiviral introduction and expansion (Figure 11C), AGS(HER2) High , EpCAM High ) cells and MDA-MB468 (HER2 -ve , EpCAM HighThe in vitro killing power of anti-HER2 CAR T (4D5 or F5) and HE CAR-BiTE T (4D5 or F5) cells was tested using both types of cells (Figure 11B). HE CAR-BiTE T cells were shown to kill AGS cells at a much faster rate than anti-HER2 CAR alone (Figure 11D), suggesting that BiTE is involved in target cell killing. Surprisingly, anti-HER2 CAR T cells constructed from clone 4D5 had low levels of interferon-γ secretion (Figure 11F) and were unable to initiate efficient killing of AGS cells (Figure 11D), suggesting that the cytotoxic function of HE CAR-BiTE T cells (clone 4D5) is primarily contributed to by secreted anti-EpCAM BiTE. Even more interestingly, while anti-HER2 CAR T cells (clone 4D5) showed similar killing power against HER2-negative MDA-MB468 cells, anti-HER2 CAR T cells constructed using our patented clone F5 showed high specificity by preserving HER2-negative cells (Figures 11E and 11G).

[0269] Example 6 - Experiment demonstrating that anti-EpCAM BiTE secreted by CAR T cells can induce potent T cell-mediated killing of cancer cells. In another experiment, the culture supernatant containing anti-EpCAM BiTE molecules secreted by human GE CAR-BiTE T cells (using the "MT110" sequence) was collected and mixed with naive human T cells isolated from healthy donor PBMCs, and then Hep3B(GPC3) was added. High , EpCAM High The anti-EpCAM BiTE molecule was added to target cells (E:T ratio = 6:1). BiTE-mediated T cell-mediated cytotoxicity was measured over time by xCelligence impedance assay. The results showed that the anti-EpCAM BiTE molecule secreted by GE CAR-BiTE T (MT110) cells induced potent killing of Hep3B cells, but cultures with supernatant without the anti-EpCAM BiTE molecule (Mock T or CAR T) did not result in significant killing of Hep3B target cells (Figure 12).

[0270] Example 7 - Experiment demonstrating that secretion of anti-EpCAM rama VHH BiTE from different CAR-BiTE T cells can be detected in vitro cultures. Both anti-GPC3 CAR T(5C4) cells and GE CAR-BiTE T(Nb01-013A) cells were cultured in T cell proliferation medium containing IL-7 and IL-15 at a starting cell density of 0.5 million / ml. Cell culture supernatant was collected daily for four consecutive days, and the amount of secreted anti-EpCAM BiTE molecule Nb01-013A was then detected by ELISA. The results clearly showed that the amount of secreted Nb01-013A molecule increased over time, suggesting that anti-EpCAM VHH BiTE is continuously secreted and accumulated from GE CAR-BiTE T(Nb01-013A) cell cultures (Figure 13).

[0271] Similarly, the Nb01-013A BiTE molecule was also detected in in vitro cultured HE CAR-BiTE T(4D5) and HE CAR-BiTE T(F5) cells. Anti-HER2 CAR T(4D5 or F5) cells or HE CAR-BiTE T(4D5 or F5) cells were cultured in T cell proliferation medium containing IL-7 and IL-15 at a cell density of 1.5 million / ml. Cell culture supernatants were collected after 24 and 48 hours, and the amount of secreted anti-EpCAM BiTE molecule Nb01-013A was detected by ELISA. In both HE CAR-BiTE T(4D5) and HE CAR-BiTE T(F5) cultures, the amount of secreted Nb01-013A molecule increased over time, suggesting that anti-EpCAM VHH BiTE is continuously secreted and accumulated from both cultures (Figure 14).

[0272] Example 8 - Experiment demonstrating that human CAR T cells secreting anti-EpCAM BiTE molecules exhibit superior in vivo cytotoxicity in killing EpCAM-expressing tumors. In addition to characterizing the in vitro cytotoxicity of human CAR T cells secreting anti-EpCAM BiTE molecules (Figures 10 and 11), the inventors also evaluated the in vivo effectiveness of killing EpCAM-expressing tumors using a mouse xenograft model.

[0273] First, Hep3B (GPC3) in NSG mice. High , EpCAM HighUsing a xenograft model, anti-GPC3 CAR T cells secreting different anti-EpCAM BiTE molecules ("MT110", "Nb01-013A", "Nb01-013B") and anti-CD19 CAR T cells secreting different anti-EpCAM BiTE molecules ("Nb01-013A" or "Nb01-013B") were tested (Figure 15). Interestingly, Hep3B xenografts from mice treated with the two 19E CAR-BiTE T cells showed similar proliferation rates to the control groups ("tumor only" and "Mock T" groups), suggesting that the in vivo tumor control effect of CAR-BiTE T cells is dependent on CAR-specific antigen expression. Anti-GPC3 CAR T(5C4) cells were able to effectively control tumor growth in all mice tested, but after 3 weeks of tumor reduction, the remaining tumor began to grow again, suggesting that the tumor is related to GPC3 Negative This suggested that the cells were escaping from the cell population. In this experiment, we were unable to observe the effect of GE CAR-BiTE T(MT110) cells on controlling the Hep3B xenograft. This is because, from a humanitarian perspective, the in vivo expansion of GE CAR-BiTE T(MT110) cells reached a threshold (tumor size 3000 mm) before it could exert a significant tumor control effect. 3 This was because mice had to be voluntarily culled once they reached a certain level (exceeding a certain threshold). This may be due to variability in the T cell donors used to generate CAR T cells in different batches of the experiment. Nevertheless, GE CAR-BiTE T cells ("Nb01-013A" or "Nb01-013B" used as BiTE) generated using the same T cell donor showed a much stronger tumor control effect, suggesting that replacing the anti-EpCAM arm with the anti-EpCAM 2C4-VHH of this disclosure significantly improved the in vivo proliferation and in vivo killing power of GE CAR-BiTE T cells. Mice in the GE CAR-BiTE T (Nb01-013A) group achieved efficient tumor control in all mice tested. Furthermore, three mice remained healthy for more than 90 days after treatment.

[0274] The in vivo efficacy of human CAR T cells secreting the anti-EpCAM BiTE molecule was demonstrated by treating AGS(Her2) with anti-HER2 CAR T cells secreting Nb01-013A. High , EpCAM High The cells were tested using a second xenograft model (Figure 16). Similar to the in vitro data (Figure 11), AGS showed resistance to 4D5 CAR-T cells. While CAR T (4D5) failed to control tumor growth, other groups treated with CAR T (F5) or HE CAR-BiTE T cell therapy showed tumor control effects in vivo. CAR T (F5) treatment showed effective control of tumor growth in all five mice. However, after 5-6 weeks of tumor reduction, the remaining tumors began to grow again, reaching a tumor size of 1000 mm. 3 Having reached the above, Her2 Negative Tumor escape from the cell population was suggested. Effective tumor control was observed in 3 mice from the HE CAR-BiTE T (4D5) group and 2 mice from the HE CAR-BiTE T (F5) group, respectively, and these mice remained tumor-free up to 181 days after T cell injection. Negative EpCAM Positive The study demonstrated an extension of tumor control by removing tumor cells.

[0275] In summary, this example demonstrates that human CAR T cells secreting a self-developed anti-EpCAM 2C4-VHH-derived anti-EpCAM BiTE molecule exhibited excellent in vivo cytotoxicity in killing EpCAM-expressing tumors.

[0276] application The antigen-binding proteins, variants, and fragments of this disclosure are useful for one or more of the following: • As a diagnostic antibody for solid tumors derived from EpCAM-overexpressing epithelium; • Anti-EpCAM BiTEs incorporating the human Fab clones of this disclosure can be used to treat EpCAM-overexpressing solid tumors due to their low affinity for the EpCAM antigen protein; and Anti-EpCAM BiTE incorporating rama VHH clones or humanized VHH clones can be used as a locally secreted molecule in the treatment of EpCAM-positive solid tumors.

Claims

1. An antigen-binding protein, an antigen-binding variant thereof, or an antigen-binding fragment that specifically binds to epithelial cell adhesion molecules (EpCAM), wherein the antigen-binding protein, its variant, or fragment includes a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ARSLGGRFRY (SEQ ID NO: 3) (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to AREVEGSSYDAFDI (Sequence ID: 7) (iii) Light chain variable region including the following: CDR-L1 includes the following: - CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), - QSLLHSNRYNY (Sequence ID: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or - Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSISDF (Sequence ID: 19). CDR-L2 includes the following: - LGS (Sequence ID: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or - AAS (Sequence ID: 20) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and, CDR-L3 includes the following: - MQALQTPYT (SEQID number: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. - MQGLQSPWT (Sequence ID: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to QQSYIMPDT (Sequence ID: 21), or - MQGLQTPYT (Sequence ID: 23) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and, (iv) Heavy chain variable region including the following: CDR-H1 includes the following: - GSIFSGND (Sequence ID: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSSERFTS (SEQ ID NO: 29), CDR-H2 includes the following: - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGGST (Sequence ID: 26), or - ITNGGST (Sequence ID: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and, CDR-H3 includes the following: - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHH (Sequence ID: 27), - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (SEQ ID NO: 31), or - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TNGRWSGDTYYAHL (SEQ ID NO: 33).

2. An antigen-binding protein, a variant thereof, or a fragment according to claim 1, wherein the antigen-binding protein, a variant thereof, or a fragment comprises a heavy chain variable region selected from the group consisting of: (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ARSLGGRFRY (SEQ ID NO: 3) (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and, Includes the following light chain variable region: CDR-L1 includes the following: - QSLLHSNGYNY (Sequence ID: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. - QSLLHSNRYNY (Sequence ID: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or - QSISDF (Sequence ID: 19) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. CDR-L2 includes the following: - LGS (SEQID number: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or - AAS (Sequence ID: 20) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and, CDR-L3 includes the following: - MQALQTPYT (Sequence ID: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. - MQGLQSPWT (Sequence ID: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to QQSYIMPDT (Sequence ID: 21), or - A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to MQGLQTPYT (Sequence ID: 23).

3. An antigen-binding protein, a variant thereof, or a fragment according to claim 1, wherein the antigen-binding protein, a variant thereof, or a fragment comprises the following heavy chain variable region: CDR-H1 includes the following: - GSIFSGND (Sequence ID: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or - Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSSERFTS (SEQ ID NO: 29). CDR-H2 includes the following: - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGGST (Sequence ID: 26), or - ITNGGST (Sequence ID: 30) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and, CDR-H3 includes the following: - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHH (Sequence ID: 27), - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (SEQ ID NO: 31), or - A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHL (SEQ ID NO: 33).

4. An antigen-binding protein, variant, or fragment according to any one of the prior claims, wherein the antigen-binding protein, variant, or fragment comprises a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ARSLGGRFRY (SEQ ID NO: 3) (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TYYRSKWYS (SEQID number: 6), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to AREVEGSSYDAFDI (Sequence ID: 7) (iii) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (iv) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (v) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (vi) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSISDF (Sequence ID: 19), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with AAS (Sequence ID: 20), and CDR-L3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QQSYIMPDT (SEQ ID NO: 21). (vii) Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (viii) The following is the heavy chain variable region: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITSGGST (Sequence ID: 26), and CDR-H3 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHH (Sequence ID: 27), (ix) Heavy chain variable region including the following: CDR-H1 containing GSSERFTS (SEQ ID NO: 29) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity, CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITNGGST (SEQ ID NO: 30), and CDR-H3 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (SEQ ID NO: 31); and, Heavy chain variable region including (x) and below: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITSGGST (Sequence ID: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

5. An antigen-binding protein, a variant thereof, or a fragment according to any one of the prior claims, wherein the antigen-binding protein, a variant thereof, or a fragment comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and, Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and, Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (iii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and, Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (iv) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and, Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and, (v) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and, Light chain variable region including the following: CDR-L1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to QSISDF (Sequence ID: 19), CDR-L2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with AAS (Sequence ID: 20), and CDR-L3 containing QQSYIMPDT (Sequence ID: 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

6. An antigen-binding protein, variant or fragment thereof according to any one of the prior claims, wherein the antigen-binding protein, variant or fragment comprises a heavy chain variable region selected from the group consisting of: (i) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITSGGST (Sequence ID: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID: 27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it. (ii) The following is the heavy chain variable region: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSSERFTS (SEQ ID NO: 29), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITNGGST (SEQ ID NO: 30), and CDR-H3 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with MAGTS (SEQ ID NO: 31); and, (iii) Heavy chain variable region including the following: CDR-H1 containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to ITSGGST (Sequence ID: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

7. An antigen-binding protein, variant, or fragment according to any one of the prior claims, wherein the antigen-binding protein, variant, or fragment comprises a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of: (i) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (Sequence IDs: 4 - 1B6, 1C1, 1C11, 1D4 and 1H6), or a fragment, variant or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (ii) Heavy chain variable domains including the following: QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. (iii) Light chain variable domains including the following: DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (Sequence ID No. 12), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iv) Light chain variable domains including the following: EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (v) Light chain variable domains including the following: DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (vi) Light chain variable domains including the following: DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Light chain variable domains including (vii) and below: DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24) or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. (viii) and below: Heavy chain variable domains: QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains including (ix) and below: QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains including (x) below: QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains including (xi) and below: QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains including (xii) and below: QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions; and, Heavy chain variable domains including the following: QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (Sequence ID: 37), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions.

8. An antigen-binding protein, variant or fragment thereof according to any one of the prior claims, wherein the antigen-binding protein, variant or fragment comprises a heavy chain variable domain and a light chain variable domain selected from the group consisting of: (i) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, and Light chain variable domains including the following: DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (ii) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, and Light chain variable domains including the following: EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (Sequence ID No: 16), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iii) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, and Light chain variable domains including the following: DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (Sequence ID No: 18), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iv) Heavy chain variable domains including the following: QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions thereof, and Light chain variable domains including the following: DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, (v) Heavy chain variable domains including the following: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, and Light chain variable domains including the following: DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions.

9. An antigen-binding protein, variant or fragment thereof according to any one of the prior claims, wherein the antigen-binding protein, variant or fragment comprises a single-domain heavy chain variable domain having the following sequence: (i) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions thereof, or (ii) QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, or (iii) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, or (iv) QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions thereof, or (v) QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions thereof, or (vi) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (Sequence ID: 37), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions.

10. An antigen-binding protein, variant or fragment thereof according to any one of the prior claims, wherein the antigen-binding protein, variant or fragment comprises a light chain constant domain having the following sequence: (i) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, or (ii) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (SEQ ID NO: 14), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions.

11. An antigen-binding protein, a variant thereof, or a fragment according to any one of the prior claims, wherein the antigen-binding protein, a variant thereof, or a fragment is an IgG antibody, in particular an IgG1 antibody.

12. An antigen-binding protein, a variant thereof, or a fragment according to any one of the prior claims, wherein the antigen-binding protein, a variant thereof, or a fragment is a multispecific antigen-binding protein such as a bispecific antibody, a variant thereof, or a fragment thereof.

13. The antigen-binding protein, variant, or fragment according to claim 12, wherein the multispecific antigen-binding protein, variant, or fragment thereof binds to an immunomarker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

14. The antigen-binding protein, variant, or fragment according to any one of the prior claims, wherein the antigen-binding protein, variant, or fragment thereof is a bispecific T cell engager (BiTE).

15. The antigen-binding protein, variant or fragment according to claim 14, wherein the bispecific T cell engager (BiTE) comprises an anti-EpCAM heavy chain antibody variable region (i.e., VHH) or a single-chain variable fragment (scFv).

16. The antigen-binding protein, variant, or fragment according to any one of the prior claims, wherein the antigen-binding protein, variant, or fragment comprises an Fc region.

17. A polynucleotide encoding an antigen-binding protein, a variant thereof, or a fragment, as described in any one of the prior claims.

18. The polynucleotide according to claim 17, wherein the heavy chain variable domain is encoded by a nucleotide sequence comprising: (i) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (Sequence ID: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (ii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (Sequence ID: 45), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (Sequence ID: 65), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (Sequence ID: 69), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (Sequence ID: 71), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (Sequence ID: 72), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (Sequence ID: 73), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; or, (viii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (Sequence ID: 74), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions; and / or The light chain variable domain is encoded by a nucleotide sequence that includes the following: (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (Sequence ID: 49), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (Sequence ID: 53), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (Sequence ID: 55), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or (iv) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (Sequence ID: 59), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (Sequence ID: 61), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions.

19. The polynucleotide according to claim 17 or 18, wherein the heavy chain and light chain variable domains are encoded by a nucleotide sequence selected from the group consisting of: (i) Heavy chain variable domain encoded by a nucleotide sequence including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (Sequence ID: 41), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions, and Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (Sequence ID: 49), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions. (ii) Heavy chain variable domain encoded by a nucleotide sequence including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (Sequence ID: 41), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions, and Light chain variable domain encoded by the following nucleotide sequence: GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (Sequence ID: 53), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (iii) Heavy chain variable domains encoded by nucleotide sequences including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (Sequence ID: 41), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions, and Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (Sequence ID: 55), or a sequence having 10 to 20 nucleic acid substitutions that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. (iv) Heavy chain variable domains encoded by nucleotide sequences including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (Sequence ID: 41), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions, and Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (Sequence ID: 61), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions; and, (v) Heavy chain variable domains encoded by nucleotide sequences including the following: CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (Sequence ID: 45), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions, and Light chain variable domain encoded by the following nucleotide sequence: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (Sequence ID: 59), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions.

20. The polynucleotide according to claim 17 or 18, wherein the single-domain heavy chain variable domain is encoded by a nucleotide sequence comprising: (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (Sequence ID: 65), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (ii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (Sequence ID: 69), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (Sequence ID: 71), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10 to 20 nucleic acid substitutions. (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (Sequence ID: 72), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions. (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (Sequence ID: 73), or a sequence having 10 to 20 nucleic acid substitutions that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, or (vi) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (Sequence ID: 74), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions.

21. The polynucleotide according to any one of claims 17 to 19, wherein the light chain constant domain is encoded by a nucleotide sequence comprising: (i) CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (Sequence ID: 50), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions, (ii) CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT (Sequence ID: 51), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10 to 20 nucleic acid substitutions.

22. A vector for expressing a polynucleotide according to any one of claims 17 to 21.

23. A host cell comprising the vector according to claim 22.

24. A cell expressing / secreting an antigen-binding protein, a fragment thereof, or a variant according to any one of claims 1 to 16.

25. Cells that express / secrete EpCAM-specific immune cell engagers.

26. The cell according to claim 25, wherein the immune cell engager is selected from the group including T cell engagers, NK cell engagers, monocyte engagers, and macrophage engagers.

27. The cell according to claim 25 or 26, wherein the immune cell engager is a bispecific T cell engager (BiTE), such as an inducible BiTE, a non-inducible BiTE, or a constitutively expressed BiTE, comprising an antigen-binding protein or a variant of a fragment thereof, as described in any one of claims 1 to 15.

28. The cell according to any one of claims 24 to 27, wherein the cell is an immune cell, selected from the group including, for example, T cells, macrophages, monocytes, and NK cells.

29. The cell according to any one of claims 24 to 28, wherein the immune cell is a T cell, particularly a CAR T cell.

30. The cell according to any one of claims 24 to 27, wherein the cell is a stem cell, selected from the group including, for example, mesenchymal stem cells, neural stem cells, and pluripotent stem cells such as induced pluripotent stem cells (iPSCs).

31. A composition comprising an antigen-binding protein according to any one of claims 1 to 16, a variant or fragment thereof, and / or a cell according to any one of claims 24 to 30.

32. An antigen-binding protein according to any one of claims 1 to 16, a variant or fragment thereof, a cell according to any one of claims 24 to 30, or a composition according to claim 31, for use in the treatment of a disease.

33. A method for treating a disease in a subject requiring such treatment, comprising administering to the subject an antigen-binding protein according to any one of claims 1 to 16, a variant or fragment thereof, a cell according to any one of claims 24 to 30, or a composition according to claim 31.

34. Use of an antigen-binding protein according to any one of claims 1 to 16, a variant or fragment thereof, a cell according to any one of claims 24 to 30, or a composition according to claim 31 in the manufacture of a pharmaceutical product for the prevention and / or treatment of a disease.

35. The method, use, antigen-binding protein, variant or fragment thereof, cell or composition for use, according to any one of claims 32 to 34, wherein the disease is a proliferative disorder such as a tumor or cancer.

36. A method for diagnosing / determining the prognosis or presence of an epithelial solid tumor, comprising the step of detecting high EpCAM expression on a solid tumor using an antigen-binding protein, a variant or fragment thereof, as described in any one of claims 1 to 16, or the composition described in claim 31.

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