HER2 chimeric antigen receptor secretion

Abstract

Modified cells are provided that express multispecific antigen-binding proteins, variants, or binding fragments that bind to one or more targets, including (a) a chimeric antigen receptor targeting HER2, and (b) a first antigen-binding protein, variant thereof, or binding fragment that binds to EpCAM (epithelial cell adhesion molecule), and a second antigen-binding protein, variant thereof, or binding fragment that binds to an immune cell marker, wherein the first antigen-binding protein, variant thereof, or binding fragment that binds to EpCAM includes a heavy chain variable region and / or a light chain variable region containing the sequence disclosed herein. Methods for producing / generating the cells disclosed herein and methods for employing the cells disclosed herein are also disclosed.

Description

[Technical Field]

[0001] This disclosure broadly relates to antigen-binding proteins, chimeric antigen receptors, and modified cells specific to epithelial cell adhesion molecules (EpCAM). In particular, this disclosure relates to cells expressing chimeric antigen receptors that target tumor antigens, as well as to multispecific antigen-binding proteins, variants, or binding fragments that bind to one or more targets, including a first antigen-binding protein, its variants, or binding fragments that bind to EpCAM (epithelial cell adhesion molecules), and a second antigen-binding protein, its variants, or binding fragments that bind to cell markers. [Background technology]

[0002] background Global cancer cases are projected to reach 28.4 million by 2040. Approximately 90% of all adult cancer cases are solid tumors. Solid tumors, such as liver cancer, lung cancer, stomach cancer, and female breast cancer, are among the leading causes of cancer-related death. Because epithelial tissue is the most abundant tissue in the body, malignant tumors of epithelial tissue, also known as "carcinomas," account for 80-90% of all solid tumor cases. For example, gastric adenocarcinoma accounts for 90-95% of stomach cancers. Female breast cancer is another example, where ductal carcinoma and lobular carcinoma, originating from epithelial tissue, are the most common primary breast cancer types, accounting for 90% of all breast cancer cases combined. Other examples include tumors found in the skin, ovaries (>90%), kidneys (>85%), lungs (>85%), liver (~85%), pancreas (>90%), head and neck (>90%), and prostate (>95%), the vast majority of which are carcinomas. While carcinoma in situ has a high chance of being cured, the five-year survival rate for most invasive or metastatic cancers is approximately 20-30%, even with the availability of multiple treatment options such as surgery, chemotherapy, radiation therapy, and targeted therapy.

[0003] Compared to conventional treatment strategies, antibody and cell-based therapies are gaining increasing attention and investment in their development as potentially more promising standard treatments for cancer. Cell-based therapies such as chimeric antigen receptor (CAR) T cells have shown great success in hematological malignancies, but have demonstrated limited efficacy in treating solid tumors. Among the complex challenges each approach has faced, one essential problem is the heterogeneous distribution of tumor markers in solid tumor cell populations and antigen escape during treatment. To overcome these challenges, there is an urgent medical need to explore next-generation CAR T cell therapies to prevent cancer recurrence.

[0004] Immune escape of target antigen-negative cells emerges as a major mechanism of cancer recurrence. Consequently, the unsatisfactory efficacy of single-antigen-targeting CAR-T cells has been attributed to resistance to CAR T-cell therapy. Because tumor cells express a diverse repertoire of surface antigens, a potential approach to overcome this challenge is to design CAR T cells with dual or multiple targeting, exemplified by bispecific anti-CD19 / CD20 CAR T-cell therapy, which has shown early promise in treating relapsed or refractory B-cell lymphoma to combat tumor escape. However, finding multiple tumor-specific targets in solid tumors is extremely difficult. In fact, most solid tumor-associated antigens (TAAs) are also expressed at low levels in normal tissues, and therefore, the highly potent properties of CARs can induce unbearable on-target off-tumor toxicity. Therefore, there is a need to provide alternative methods to prevent cancer recurrence.

[0005] There is a need to provide cell-based alternative therapies. There is an urgent need to provide alternative chimeric antigen receptor cells. [Overview of the Initiative]

[0006] overview EpCAM is a well-known tumor-associated antigen that is frequently overexpressed in almost all solid tumors originating from epithelium. However, because EpCAM is also widely expressed, albeit at low levels, in normal epithelium, both CAR T cell-targeted EpCAM and systemically administered BiTE have not been successful to date due to their high systemic toxicity. Secretion of anti-EpCAM BiTE by CAR-T cells not only localizes anti-EpCAM BiTE to the tumor site or its vicinity, reducing tumor escape but also significantly improving safety.

[0007] This disclosure demonstrates that HER2-targeted CAR T cells secreting anti-EpCAM BiTE (named "HE CAR-BiTE T") exhibit superior efficacy in killing human tumors originating from gastric carcinoma in vitro. In summary, our approach utilizes the following factors: 1) tumor-directed T cell proliferation, and 2) tumor-specific anti-EpCAM BiTE secretion, the latter of which cannot be achieved by conventional systemic treatment approaches. This disclosure also offers the potential to extend anti-EpCAM BiTE-secreting CAR T cell therapy to other cancer types in order to establish a broader range of next-generation CAR T cell therapies.

[0008] In one embodiment, modified cells expressing the following are provided. (a) Chimeric antigen receptors targeting HER2, and (b) A multispecific antigen-binding protein that binds to one or more targets, including a first antigen-binding protein that binds to EpCAM (epithelial cell adhesion molecule), a variant thereof or binding fragment, and a second antigen-binding protein that binds to an immune cell marker, a variant thereof or binding fragment, Here, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10) CDR-H1 includes the following: · GSIFSGND (Sequence IDs: 25-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·GSSERFTS (Sequence ID: 29-1B8) CDR-H2 includes the following: · ITSGGST (Sequence IDs: 26-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·ITNGGST (Sequence ID: 30-1B8), and CDR-H3 includes the following: ·TNGRWSGDTYYAHH (Sequence IDs: 27-2C4, hu2C4, 1A5, 2B7 and 2D10), ·MAGTS (Sequence ID: 31-1B8), or ·TNGRWSGDTYYAHL (Sequence number: 33-2B7) (ii) Heavy chain variable regions including the following: (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) (iii) Heavy chain variable region including the following: (1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7) (iv) Light chain variable regions including the following: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6) CDR-L1 includes the following: • QSLLHSNGYNY (Sequence IDs: 9-1B6, 1C1, 1C11, and 1H6) · QSLLHSNRYNY (Sequence ID: 17-1D4), or • QSISDF (Sequence ID: 19-1E4) CDR-L2 includes the following: ·LGS (Accession numbers: 10-1B6, 1C1, 1C11, 1D4 and 1H6), or ·AAS (Accession number: 20-1E4), and CDR-L3 containing the following: ·MQALQTPYT (Accession numbers: 11-1B6, 1C1 and 1D4), ·MQGLQSPWT (Accession number: 15-1C11), ·QQSYIMPDT (Accession number: 21-1E4), or ·MQGLQTPYT (Accession number: 23-1H6); and Or fragments or variants or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto.

[0009] In some examples, the first antigen-binding protein, its variant or binding fragment that binds to EpCAM comprises a heavy-chain variable region comprising: (2C4, hu2C4, 1A5, 1B8, 2B7 and 2D10) CDR-H1 containing the following: ·GSIFSGND (Accession numbers: 25-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·GSSERFTS (Accession number: 29-1B8) CDR-H2 containing the following: ·ITSGGST (Accession numbers: 26-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·ITNGGST (Accession number: 30-1B8); and CDR-H3 containing the following: ·TNGRWSGDTYYAHH (Accession numbers: 27-2C4, hu2C4, 1A5, 2B7 and 2D10), ·MAGTS (Accession number: 31-1B8), or ·TNGLWSGDTYYAHL (Accession number: 33-2B7) Or fragments or variants or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto.

[0010] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (2C4-VHH, hu2C4-VHH, 1A5-VHH, and 2D10-VHH) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID: 27) (ii) Heavy chain variable region including the following: (1B8-VHH) CDR-H1 containing GSSERFTS (Sequence ID: 29), CDR-H2 containing ITNGGST (Sequence ID: 30), and CDR-H3 containing MAGTS (SEQ ID NO: 31); and (iii) Heavy chain variable region including the following: (2B7-VHH) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0011] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (2C4, hu2C4, 1A5, and 2D10) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID: 27) (ii) Heavy chain variable region including the following: (1B6, 1C1, 1C11, 1D4, 1H6) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3) (iii) Heavy chain variable region including the following: (1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7) (iv) Light chain variable region including the following: (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) (v) Light chain variable region including the following: (1C11) CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (Sequence ID: 15) (vi) Light chain variable region including the following: (1D4) CDR-L1 containing QSLLHSNRYNY (Sequence ID: 17) CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11) (vii) Light chain variable region including the following: (1E4) CDR-L1 containing QSISDF (Sequence ID: 19), CDR-L2 containing AAS (Sequence ID: 20), and CDR-L3 containing QQSYIMPDT (Sequence ID: 21) (viii) Light chain variable region including the following: (1H6) CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9) CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (Sequence ID: 23) (ix) Heavy chain variable region including the following: (1B8-VHH) CDR-H1 containing GSSERFTS (Sequence ID: 29), CDR-H2 containing ITNGGST (Sequence ID: 30), and CDR-H3 containing MAGTS (SEQ ID NO: 31); and Heavy chain variable region including (x) below: (2B7-VHH) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0012] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of: (i)QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domain containing WGQGTL (SEQ ID NO: 37-hu2C4-VHH) (ii) QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHHHeavy chain variable domain containing WGQGTQ (SEQ ID NO: 35-2C4-VHH) (iii)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY Heavy chain variable domains including WGQGTL (SEQ ID NOs: 4-1B6, 1C1, 1C11, 1D4, and 1H6) (iv)QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI Heavy chain variable domains including WGQGTM (SEQ ID NO: 8-1E4), (v)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domains containing FGQGTK (SEQ ID NOs: 12-1B6 and 1C1) (vi)EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT Light chain variable domain containing FGQGTK (SEQ ID NO: 16-1C11) (vii)DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 18-1D4) (viii)DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT Light chain variable domain containing FGQGTK (SEQ ID NO: 22-1E4) (ix)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 24-1H6) (x)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domain containing WGQGTQ (SEQ ID NO: 28-1A5-VHH) (xi)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS Heavy chain variable domain containing WGQGTQ (SEQ ID NO: 32-1B8-VHH) (xii)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL Heavy chain variable domain containing WGQGTQ (SEQ ID NO: 34-2B7-VHH) (xiii)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domains including WGQGTQ (SEQ ID NO: 36-2D10-VHH); and Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0013] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a single-domain heavy chain variable domain having the following sequence: (i)QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID: 35-clone2C4-VHH), or (ii) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH WGQGTL (Sequence ID: 37-hu2C4-VHH), or (iii)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID: 28-Clone 1A5-VHH), or (iv)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ (Sequence ID: 32-clone1B8-VHH), or (v)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL WGQGTQ (Sequence ID: 34-Clone 2B7-VHH), or (vi)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID: 36-clone2D10-VHH), Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0014] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (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) (ii) Heavy chain variable region including the following: (1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7); and The light chain includes the following variable regions: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6) CDR-L1 includes the following: • QSLLHSNGYNY (Sequence IDs: 9-1B6, 1C1, 1C11, and 1H6) · QSLLHSNRYNY (Sequence ID: 17-1D4), or • QSISDF (Sequence ID: 19-1E4) CDR-L2 includes the following: · LGS (Sequence IDs: 10-1B6, 1C1, 1C11, 1D4 and 1H6), or · AAS (Sequence ID: 20-1E4), and CDR-L3 includes the following: • MQALQTPYT (Sequence IDs: 11-1B6, 1C1 and 1D4) • MQGLQSPWT (Sequence ID: 15-1C11) · QQSYIMPDT (Sequence ID: 21-1E4), or • MQGLQTPYT (Sequence ID: 23-1H6) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0015] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes 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: (1B6 and 1C1) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3); and The light chain variable region includes the following: CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11) (ii) Heavy chain variable region including the following: (1C11) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3); and The light chain variable region includes the following: CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (Sequence ID: 15) (iii) Heavy chain variable region including the following: (1D4) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3); and The light chain variable region includes the following: CDR-L1 containing QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11) (iv) Heavy chain variable region including the following: (1H6) CDR-H1 containing GGTFSSYA (Sequence ID: 1) CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3); and The light chain variable region includes the following: CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23); and (v) Heavy chain variable region including the following: (1E4) CDR-H1 (SEQ ID NO: 5) containing GDSISSNSVA, CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7); and The following is included in the light chain variable region: (1E4) CDR-L1 containing QSISDF (Sequence ID: 19), CDR-L2 containing AAS (Sequence ID: 20), and CDR-L3 containing QQSYIMPDT (Sequence ID: 21) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0016] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes 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: (1B6 and 1C1) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 12) (ii) Heavy chain variable domains including the following: (1C11) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWTLight chain variable domain containing FGQGTK (SEQ ID NO: 16) (iii) Heavy chain variable domains including the following: (1D4) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 18) (iv) Heavy chain variable domains including the following: (1E4) QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQGTM (SEQ ID NO: 8), and DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT A light chain variable domain containing FGQGTK (SEQ ID NO: 22), or (v) Heavy chain variable domains including the following: (1H6) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and Light chain variable domain DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT FGQGTK (Sequence ID: 24), or Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0017] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a light chain constant domain having the following sequence: (i)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (Sequence ID: 13-Clone 1B6-Light Chain Constant Domain), or (ii)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (Sequence ID: 14 ​​Clone 1C1-Light Chain Constant Domain), Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0018] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain encoded by a nucleotide sequence comprising: (i)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 72 - Clone 2C4), or (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (SEQ ID NO: 74 - Clone hu2C4 - VHH); or (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGGAGATTCGCTAC TGGGGCCAGGGAACCCTG (SEQ ID NO: 41 - for Clone 1B6, 1C1, 1C11, 1D4, 1H6), or (iv)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (SEQ ID NO: 45 - Clone 1E4); or (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 65 - Clone 1A5), or (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), or (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (Sequence ID: 71-Clone 2B7), or (viii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 73-Clone 2D10), and / or The aforementioned light chain variable domain is encoded by a nucleotide sequence including the following: (i)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 49-Clone 1B6 and 1C1), or (ii)GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID: 53-Clone 1C11), or (iii)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT 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), Or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have 10-20 nucleic acid substitutions.

[0019] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes heavy and light chain variable domains encoded by a nucleotide sequence selected from the following group: (i) Heavy chain variable domains encoded by nucleotide sequences including the following: (1B6 and 1C1) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 49) (ii) Heavy chain variable domain encoded by a nucleotide sequence including the following: (1C11) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and Light chain variable domain encoded by the following nucleotide sequence: GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID: 53) (iii) Heavy chain variable domain encoded by a nucleotide sequence including the following: (1D4) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 55) (iv) Heavy chain variable domain encoded by the following nucleotide sequence: (1H6) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG(Sequence ID: 61); and (v) Heavy chain variable domain encoded by a nucleotide sequence including the following: (1E4). CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (Sequence ID: 45), and Light chain variable domain encoded by the following nucleotide sequence: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence number: 59) Or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have 10-20 nucleic acid substitutions.

[0020] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain encoded by a nucleotide sequence comprising: (i)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 has 10-20 nucleic acid substitutions, (ii) 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 10-20 nucleic acid substitutions; or (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 has 10-20 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 has 10-20 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 has 10-20 nucleic acid substitutions, (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 73), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or have 10-20 nucleic acid substitutions, 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 has 10-20 nucleic acid substitutions, (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TGGGGCCAAGGGACAATG (Sequence ID: 45), and / or has 10-20 nucleic acid substitutions.

[0021] In some cases, the multispecific antigen-binding protein, its variant, or fragment is a bispecific antibody.

[0022] In some cases, the multispecific antigen-binding protein, its variants, or fragments are immune cell engagers selected from the group including T cell engagers, NK cell engagers, monocyte engagers, and macrophage engagers.

[0023] In some cases, the multispecific antigen-binding protein, its variants, or fragments are bispecific T cell engagers (BiTEs), such as inducible BiTEs, non-inducible BiTEs, or constitutively expressed BiTEs.

[0024] In some cases, the second antigen-binding protein of the immune cell engager, its variant, or binding fragment binds to an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0025] In some cases, 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).

[0026] In some cases, the cells are immune cells selected from a group consisting of, for example, T cells, macrophages, monocytes, and NK cells.

[0027] In some cases, the cells are T cells, optionally CAR T cells.

[0028] In some cases, these cells bind to HER2 and secrete inducible bispecific T cell engagers (HE CAR-BiTE T) that target EpCAM and CD3.

[0029] In another embodiment, polynucleotides encoding the cells described herein are provided.

[0030] In yet another embodiment, a vector expressing the polynucleotide described herein is provided.

[0031] In yet another embodiment, a host cell containing the vector described herein is provided.

[0032] In yet another embodiment, a method for producing / generating the cells described herein is provided, comprising introducing the polynucleotides described herein into the cells.

[0033] In yet another embodiment, a composition comprising the cells described herein is provided.

[0034] In yet another embodiment, a method is provided for treating a disease in a subject requiring such treatment, the method comprising administering the cells or compositions described herein to a subject, wherein the disease is optionally a proliferative disease and optionally cancer.

[0035] 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.

[0036] As used herein, the term “antibody” refers to a whole (i.e., full-length) antibody (i.e., comprising two heavy chains and two light chain elements) and a functionally active fragment thereof (i.e., a molecule containing an antigen-binding domain that specifically binds to an antigen, also referred to as an antibody fragment or antigen-binding fragment). The characteristics described herein with respect to antibodies also apply to antibody fragments unless the context indicates otherwise. The term “antibody” includes monovalent antibodies, i.e., antibodies containing only one antigen-binding domain (e.g., a one-armed antibody containing an interconnected full-length heavy chain and a full-length light chain, also referred to as a “half-antibody”), and polyvalent antibodies, i.e., antibodies containing more than one antigen-binding domain, e.g., bivalent antibodies.

[0037] The term “antigen-binding fragment” as used herein 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.

[0038] As used herein, “binding fragment” refers to a fragment capable of binding to a target peptide or antigen with sufficient affinity to characterize the fragment as specific to the target peptide or antigen.

[0039] The term "monoclonal antibody" (or "mAb") refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, each monoclonal antibody preparation is identical except for trace amounts of possible mutations (e.g., naturally occurring mutations). Nevertheless, certain differences in protein sequence related to post-translational modifications (e.g., cleavage of the heavy chain C-terminal lysine, deamidation of asparagine residues, and / or isomerization of aspartic acid residues) may exist among the various different antibody molecules present in the composition. In contrast to polyclonal antibody preparations, each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant of the antigen.

[0040] As used herein, the term “diabody” refers to a set of 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 linked to the VL of the second Fv, and the VL of the first Fv is linked to the VH of the second Fv.

[0041] As used herein, the term “tribody” (also referred to as Fab(scFv)2) refers to a Fab fragment comprising 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, comprising four Fvs and four inter-Fv linkers.

[0042] The term "multivalent antibody" refers to an antibody that contains more than one antigen-binding domain, such as a bivalent antibody.

[0043] The term "Fv" refers to a pair of two variable domains in a full-length antibody, such as a cognate pair or affinity maturation variable domain, i.e., a VH and VL pair. The term "scFv" refers to a single-chain variable fragment, which is a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin, linked by a short linker peptide of 10 to approximately 25 amino acids. The term "bis-scFv" as used herein refers to a bispecific scFv.

[0044] The terms “dsscFv” or “disulfide-stabilized single-chain variable fragment” as used herein refer to a single-chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains and also contains an interdomain disulfide bond between VH and VL.

[0045] The term "DVD-Ig" (also known as dual V-domain IgG) refers to a full-length antibody with four additional variable domains, one at the N-terminus of each heavy chain and one at the N-terminus of each light chain.

[0046] As used herein, the term “Fab” refers to a light chain fragment containing a VL (variable light chain) domain and a constant domain (CL) of the light chain, and an antibody fragment containing a VH (variable heavy chain) domain and a first constant domain (CHI) of the heavy chain. The dimers of Fab' described herein produce F(ab')2, for example, in which dimerization can be performed through a hinge. The term “F(ab')” refers to a monovalent fragment of a single light chain homodimer, which is obtained by pepsin digestion of IgG followed by reduction of the light chain disulfide bond. As used herein, the term “F(ab')2” refers to a fragment of IgG prepared by pepsin digestion of IgG. The F(ab')2 fragment is a disulfide-bonded homodimer of two light chain dimers and therefore retains a divalent epitope bond like whole IgG, but is smaller in size than whole IgG because it lacks a heavy chain. The F(ab')2 and F(ab') fragments do not bind to immunoglobulin receptors on cells and may be useful in achieving specific staining of the primary antibody target.

[0047] The terms “constant domains” or “constant regions” are used interchangeably herein and refer to the domains of an antibody located outside the variable region. Constant domains are identical in all antibodies of the same isotype, but differ from isotype to isotype. Typically, the constant region of a heavy chain is formed from the N-terminus to the C-terminus by CH1-hinge-CH2-CH3-optionally CH4 and contains three or four constant domains.

[0048] As used herein, the term “DiFab” refers to two Fab molecules linked via their C-terminuses in a heavy chain, or two Fab' molecules linked via one or more disulfide bonds in their hinge region.

[0049] The term "antigen-binding variant" refers to a polypeptide, for example, an antibody comprising VH and / or VL that has the desired properties described herein and has 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 with respect to the antibodies described herein, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. Optionally, a variant antibody may have up to one conserved amino acid substitution compared to the antibody sequence provided herein, or up to about two, three, four, five, six, seven, eight, nine, or ten conserved amino acid substitutions compared to the antibody sequence provided herein.

[0050] The term “immune cells” refers to a type of specialized cell that plays a vital role in the body’s defense against infections and foreign substances. They 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 or cancerous cells. As used herein, “immune cells” refers to any cell in 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.

[0051] In the context of antibodies, the term “specifically” as used herein is intended to refer to an antibody that recognizes only a specific antigen, or an antibody that has a significantly higher binding affinity to a specific antigen compared to its binding to a nonspecific antigen, for example, at least 5, 6, 7, 8, 9, or 10 times higher.

[0052] In the context of antibodies, the term “epitope” or “binding site” refers to the site (or portion) of an antigen to which an antibody paratope binds or recognizes. Epitopes can be formed from consecutive amino acids (often called “linear epitopes”) or from discontinuous amino acids formed by the tertiary folding of proteins (often called “conformal epitopes”). Epitopes formed from consecutive amino acids are usually retained even upon exposure to denaturing solvents, while epitopes formed by folding are usually lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, and more commonly at least 5-10, amino acids in a unique spatial conformation. Epitopes typically consist of chemically active surface groups of molecules such as amino acids and sugar side chains, and usually have specific three-dimensional structural and charge properties.

[0053] The "class" of an antibody refers to the type of constant domain or constant region present in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as 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.

[0054] 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 (variable region) or part thereof is modified, replaced, or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or modified class, effector function, and / or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new characteristics to the chimeric antibody; or (b) the variable region or part thereof is modified, replaced, or exchanged to 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. Due to the substitution with the human constant region, the chimeric antibody may retain its specificity in antigen recognition while having reduced antigenicity in humans compared to the original mouse antibody.

[0055] The term "chimeric antigen receptor" refers to a receptor protein designed to give T cells a new ability to target specific antigens. The receptor is a chimera, combining both antigen-binding and T-cell activation functions into a single receptor. CAR T-cell therapy uses T cells designed with CARs 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 originate from the patient's own blood (autologous) or from T cells of another healthy donor (allogeneic). Once isolated from a person, these T cells are genetically engineered to express a specific CAR, which directs them to target antigens present on the surface of tumors. For safety, CAR T cells are specifically designed to express antigens that are expressed in tumors but not on healthy cells. CAR T cells destroy cells by increasing their toxicity (cytotoxicity) to other living cells through widespread stimulated cell proliferation and by causing increased secretion of factors that can affect other cells, such as cytokines, interleukins, and growth factors. The surface of CAR T cells can carry either one of two co-receptors, CD4 or CD8, which have different cytotoxic effects and interacting cytotoxic effects, respectively.

[0056] The terms “human antibody” or “humanized antibody” (or its antigen-binding fragment) are used herein to include antibodies (and their antigen-binding fragments) having a variable region in which both the framework and CDR region are derived from human sequences. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies or immunoglobulins are classified into the following classes: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2. Thus, 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 may be used, in particular. Alternatively, when an antibody molecule is intended for therapeutic purposes and antibody effector function is not required, the IgG2 and IgG4 isotypes may be used. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a human sequence. Humanized antibodies (or their antigen-binding fragments) retain the reactivity of non-human antibodies while exhibiting lower immunogenicity in humans. This can be achieved, for example, by retaining non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., framework portions of the constant and variable regions). Modifications to additional framework regions can be made within human framework sequences and within CDR sequences derived from germline cells of another mammalian species. Humanized antibodies of this disclosure may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-directed mutations in vitro, or by somatic mutations in vivo, or conservative substitutions to enhance stability or production). This definition of humanized antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art (such as phage display libraries, or administering antigens to transgenic animals modified to produce such antibodies in response to antigen challenges, but whose endogenous loci have been deactivated (e.g., xenomouses immunized via human B-cell hybridoma technology)).

[0057] 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 (e.g., transfectomas), and antibodies prepared, expressed, produced, or isolated by any other means including splicing all or part of the sequence of a human immunoglobulin gene with another DNA sequence.

[0058] The term "isolated" throughout this specification means that an antibody, or possibly a polynucleotide, exists in a physical environment different from the physical environment in which it could naturally occur. The term "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or has been synthetically produced. Isolated nucleic acids include, for example, synthetic DNA, cDNA, genomic DNA, or any combination thereof produced by chemical processing. An isolated antibody refers to an antibody that is substantially free of other cellular material and / or chemicals.

[0059] 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 ("Chothia" numbering scheme); ImMunoGenTics (IMGT) numbering ("IMGT" numbering scheme); etc. 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 (H1, H2, H3) and three in the VL (L1, L2, L3).

[0060] As used herein, the term “sequence identity” refers to the percentage of sequence identity determined by the sequence of the antibody maximally aligned according to the Kabat numbering rules. After alignment, when the antibody region of interest (e.g., the entire maturation variable region of the heavy or light chain) is compared to the same region of the reference antibody, the percentage of sequence identity between the antibody region of interest and the reference antibody region is obtained by dividing the number of positions occupied by the same amino acids in both the antibody region of interest 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. In some examples, the antigen-binding proteins disclosed herein may contain sequences that are at least 60% identical to any one of the sequences disclosed herein.For example, the antigen-binding protein is 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%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% of any of the sequences disclosed herein. , at least about 97%, at least about 98%, at least about 99%, or 100% identical (for example, 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 The sequences may include sequences that are approximately 77%, or approximately 78%, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, approximately 99%, or approximately 100% sequence identity. In some examples, antigen-binding proteins include sequences or amino acid regions that differ from the sequences disclosed herein by approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10 or more amino acids or nucleic acid bases, or are encoded by such different nucleotide regions.In some examples, 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 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 cleavages.

[0061] In some examples, the amino acid mutation is an amino acid substitution and may include conserved and / or non-conserved substitutions.

[0062] "Conservative substitutions" can be made, for example, based on the similarity of the amino acid residues involved in terms of polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. The 20 naturally occurring amino acids can be classified into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, lIe; (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.

[0063] As used herein, “conservative substitution” is defined as the exchange of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the polypeptide thus modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0064] As used herein, “non-conservative substitution” is defined as the exchange of an amino acid by another amino acid listed in the six standard amino acid groups (1) through (6) shown above.

[0065] In some examples, the substitutions may also include non-classical amino acids. Examples of non-classical amino acids include, but are not limited to, selenocysteine, pyrrolicin, 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, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylamino acids, C-α-methylamino acids, N-α-methylamino acids, and amino acid analogs in general.

[0066] In some cases, the amino acid mutation may be located in the CDR region of the antigen-binding protein (e.g., the CDR1, CDR2, or CDR3 region). In other cases, the amino acid modification may be located in the framework region (FR) of the antigen-binding protein (e.g., the FR1, FR2, FR3, or FR4 region).

[0067] In some cases, the mutation does not substantially reduce the ability of the antigen-binding protein to specifically bind to the target. In some cases, the mutation does not substantially reduce the ability of the antigen-binding protein to specifically bind to the target and does not functionally modulate (e.g., partially or completely neutralize) the target.

[0068] Modification of amino acid sequences can be achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis.

[0069] The term "polynucleotide" refers to a linear polymer composed of many nucleotide units and forming part of a nucleic acid molecule. Polynucleotides are made up of long chains of nucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0070] The term "affinity" refers to the strength of all non-covalent interactions between an antibody and a target protein. Unless otherwise indicated, the term "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 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 represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0071] As used herein, the term "K D " refers to the ratio of K a to K d (i.e., K d / K a ), and refers to the dissociation constant 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 for an antibody can be determined using well-established methods in the art. As used herein, the term "low affinity" refers to a KD of 100 nM or more.

[0072] As used herein, the term "moderate affinity" refers to a K D ranging from 10 nM to 100 nM.

[0073] As used herein, the term "high affinity" refers to a K D ranging from 1 to 10 nM.

[0074] As used herein, the term "very high affinity" refers to a K D of 1 nM or less.

[0075] The term "EC50" as used herein refers to the antibody concentration or antigen-binding protein / part thereof that elicits a response that is 50% of the maximum response (i.e., midway between the maximum response and the baseline) in either an in vivo or in vitro assay.

[0076] As used herein, the term “multispecific antigen-binding protein” refers to a multispecific antigen-binding protein capable of binding to two or more host cell target antigens. Therefore, as used herein, the terms “multispecific” or “multispecific antibody” refer to an antibody described herein having at least two binding domains, i.e., two or more binding domains, for example, two or three binding domains, where at least two binding domains independently bind to two different antigens or two different epitopes of 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.

[0077] As used herein, the terms “bispecific” or “bispecific antibody” refer to an antibody that possesses two antigen specificities, or an antibody that has the ability to simultaneously bind to two target antigens / sites.

[0078] As used herein, “bispecific T cell engagers (BiTEs)” refers to a class of artificially created bispecific monoclonal antibodies that direct the host immune system, such as by inducing cytotoxic activity of T cells against target cells (e.g., cancer cells). A BiTE is a fusion protein on a single peptide chain of approximately 55 kDa, consisting of two single-chain variable fragments (scFv) of different antibodies, or amino acid sequences from four different genes. One scFv binds to an immune cell (e.g., T cells via the CD3 receptor), and the other binds to a target of interest (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs form a binding between the immune cell (e.g., T cell) and the target cell (e.g., tumor cell). This causes the immune cell (e.g., T cell) to exert cytotoxic activity against the tumor cell. For example, if the immune cell is a T cell, the T cell exerts cytotoxic activity by entering the tumor cell and producing proteins such as perforin and granzyme that initiate cellular apoptosis.

[0079] In some cases, BiTE may refer to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In some cases, BiTE may also refer to a bispecific T-cell engager platform known in the art, which is a recombinant protein that has the ability to simultaneously bind to two different antigens and engage immune cells (such as T cells).

[0080] As used herein, the term “nanobody” refers to a single-domain antibody (sdAb) consisting of an antibody fragment comprising a single monomeric variable antibody domain. In some examples, bispecific T-cell engagers (BiTEs) are nanobodies consisting of only heavy chains (VHH). As used herein, “nanobody consisting of only heavy chains” refers to a nanobody-based heavy chain antibody. A heavy chain antibody is an antibody consisting of two heavy chains and lacking the two light chains typically found in antibodies.

[0081] As used herein, “vector” is any molecule or composition having the ability to deliver a nucleic acid sequence into a suitable host cell where, for example, the synthesis of the encoded polypeptide may take place. Typically and preferably, a vector is a nucleic acid designed 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 contains one or more of the following components (if they are not already provided by the nucleic acid molecule): a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing a splice donor site and a splice acceptor site, 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.

[0082] Vectors are typically selected to be functional in the host cell in which they are used (the vectors are adapted to 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.

[0083] As used herein, the term “host cell” 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 the cell of a particular subject but also to the offspring of such a cell. Because certain modifications may occur in the progeny due to either mutation or environmental influences, such offspring may not actually be identical to the parent cell, but they still fall within the scope of the term “host cell” as used herein.

[0084] The terms “treating,” “treat,” and “therapy,” and their synonyms, refer to both therapeutic and prophylactic or preventative measures, the purpose of which is to prevent or delay (reduce) a medical condition, including but not limited to diseases, symptoms, and disorders. A medical condition also includes the body’s response to a disease or disorder, such as inflammation. Those who require such treatment include those who already have a medical condition, those prone to developing a medical condition, or those for whom a medical condition should be prevented.

[0085] 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, while “non-patient” refers to an individual who does not have and is not likely to have a medical condition. “Non-patient” includes healthy individuals, disease-free individuals and / or individuals without a medical condition. The term “subject” includes humans and animals. Animals may include, but are not limited to, mammals (e.g., non-human primates, dogs, murines, etc.). “Murrine” refers to any mammal of the Rodentia and / or Leporidae families, such as mice, rats, and rabbits.

[0086] 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, inhibits exacerbation, inhibits further exacerbation, and / or improves at least one sign or symptom of the disease.

[0087] 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 clear support for both meanings or either one of them.

[0088] Furthermore, in this description, the term “substantially” is understood to include, but not be limited to, “entirely” or “completely” whenever it is used. In addition, terms such as “comprising” and “comprise” are intended to be non-restrictive descriptive language in that, whenever they are used, they broadly include elements / components listed after such terms, in addition to other components that are not explicitly enumerated. 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” may be considered a subset of terms such as “comprising” and “comprise” in appropriate contexts. Thus, in embodiments disclosed herein using terms such as “comprising” and “comprise,” these embodiments are understood to provide teachings corresponding to embodiments using terms such as “consisting” and “consist.” Furthermore, whenever terms such as “approximately” or “about” are used, they generally mean a reasonable variation, such as 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.

[0089] Furthermore, in this description, certain values ​​may be disclosed as ranges. Values ​​indicating the endpoints of a range are intended to describe a preferred range. Whenever a range is given, it is intended to encompass and teach all possible partial ranges and the individual numerical values ​​within those ranges. That is, the endpoints of a range should not be interpreted as immutable limitations. For example, a description of a range from 1% to 5% is intended to have specific partial ranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, and individually have values ​​such as 1%, 2%, 3%, 4%, and 5% within those ranges. Individual numerical values ​​within a range should also be understood to include integers, fractions, and decimals. Furthermore, whenever a range is given, it is also intended to encompass and teach values ​​with up to two additional decimal places or significant figures (where appropriate) from the endpoints of the indicated numerical values. For example, a description of a range from 1% to 5% is intended to specifically disclose the range from 1.00% to 5.00% and also the range from 1.0% to 5.0%, as well as all the intermediate values ​​across the said range (1.01%, 1.02%...4.98%, 4.99%, 5.00%, and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The intent of the specific disclosure described above can be applied to any depth / breadth of the range.

[0090] 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 over its entire length, such as 96%, 97%, 98%, or 99% identical. A software program may be used to calculate the identity percentage.

[0091] Furthermore, in describing certain embodiments, this disclosure may disclose methods and / or processes as a specific set of steps. However, unless otherwise required, it should be understood that such methods or processes should not be limited to the specific set of steps disclosed. Other sets of steps may be possible. The specific order of the steps disclosed herein should not be construed as an unreasonable limitation. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the order written. The set of steps may be modified and still remain within the scope of this disclosure.

[0092] Furthermore, while the Disclosure provides embodiments having one or more features / characteristics discussed herein, it is understood that one or more of these features / characteristics may also be waived in other alternative embodiments, and the Disclosure provides support for such waivers and these related alternative embodiments. [Modes for carrying out the invention]

[0093] Description of the Embodiment This disclosure provides designed cells that express the following: (a) A chimeric antigen receptor that targets the first target antigen, and (b) A multispecific antigen-binding protein that binds to EpCAM (an epithelial cell adhesion molecule).

[0094] While we do not wish to be bound by any particular theory, designing cells to secrete EpCAM BiTE at target sites minimizes toxicity, reduces side effects, and / or reduces nonspecific cytotoxicity.

[0095] In some cases, the first target antigen is a disease-related antigen.

[0096] For example, the target antigen is a molecule associated with a disease. For example, the molecule may be an extracellular molecule, an intracellular molecule, and / or a transmembrane molecule. In some examples, the molecule may be a polypeptide, a polynucleotide, a carbohydrate, etc. In some examples, the first target antigen may be a diseased cell, etc.

[0097] In some examples, the diseased cells may include, but are not limited to, cells of proliferative disorders (tumors / cancer, inflammatory diseases, etc.). In some examples, the diseased cells may be cancer / tumor cells. In some examples, the cancer cells may be solid tumors. In some examples, the solid tumors may include epithelial tumors such as carcinomas. In some examples, carcinomas may include, but are not limited to, liver cancer, lung cancer, stomach cancer (gastric adenocarcinoma, etc.), breast cancer, skin cancer, ovarian cancer, kidney cancer, pancreatic cancer, head and neck cancer, prostate cancer, esophageal cancer, bladder cancer, colon cancer, etc. In some examples, the tumor cells may include benign tumors, precancerous tumors, malignant tumors, etc. In some examples, the tumor cells may include stem cells, progenitor cells, etc. In some examples, the tumor cells may include, but are not limited to, tumor cell lines such as gastric adenocarcinoma cell lines (AGS, etc.), breast cancer cell lines (MDA-MB468, MCF7, SKBR3, BT474, MDA-MB231, MDA-MB436, etc.), and glioblastoma (A-172).

[0098] In some cases, the target antigen is an epithelial marker. In some cases, the epithelial marker includes, but is not limited to, receptor tyrosine protein kinase erbB-2 (HER2), glypican 3 (GPC3), claudin 18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), delta-like canonical Notch ligand 3 (DLL3), carcinoembryonic antigen (CEA), mucin 1 (MUC1), mucin 16 (MUC16), CEA cell adhesion molecule 7 (CEACAM7), prominin-1 (CD133), differentiation cluster 147 (CD147), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), mesoserine (MSLN), mesenchymal-epithelial conversion factor (c-Met), and folate receptor alpha (FRα). In some cases, the first antigen is HER2. Therefore, in some embodiments, the chimeric antigen receptor binds to HER2.

[0099] In some cases, HER2-expressing cancers may include, but are not limited to, gastric / esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, bladder cancer, lung cancer, and colorectal cancer.

[0100] In some cases, the aforementioned CAR T cells target HER2 alone (HE CAR-T).

[0101] In some cases, the cells express multispecific antigen-binding proteins that bind to one or more markers. For example, the multispecific antigen-binding proteins may bind to EpCAM and immunomarkers.

[0102] While we do not wish to be bound by theory, EpCAM is widely expressed in almost all cancers, but also at low levels in normal epithelium. Anti-EpCAM CAR T cells have been demonstrated to be highly toxic to normal tissues, and anti-EpCAM BiTEs (such as solitomab and catumaxomab) have shown dose-limiting toxicity and failed to obtain FDA approval.

[0103] EpCAM (epidermal cell adhesion molecule) represents another category of biomarkers that are widely expressed in almost all cancers. Cancer therapies targeting EpCAM have undergone more than a decade of clinical development. However, because EpCAM is also expressed at low levels in normal epithelium, anti-EpCAM CAR T cells have proven to be highly toxic to normal tissue. For the same reason, both solitomab and catumaxomab (anti-EpCAM BiTE) exhibited dose-limiting toxicity and failed to obtain FDA approval.

[0104] HER2 amplification or overexpression is found in approximately 10–30% of gastric / esophageal cancers and 15–30% of breast cancers. In addition, HER2 overexpression has also been reported in other cancers such as ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer. Immunotherapy, such as HER2-targeted CAR T-cell therapy, has been clinically proven to be somewhat effective and generally safe, provided that antibody clones with very high antigen-binding affinity are used. However, only a very limited number of patients have achieved complete remission (CR). Disease metastasis and recurrence following treatment targeting these markers can largely be attributed to the fact that not all tumor cells express HER2, even though it is found at high levels in these cancers at diagnosis. Reappearing tumor masses often originate from the proliferation of antigen-negative cancer cells, or from escaped cancer cells that have downregulated or even lost antigen expression following CAR T-cell therapy. Despite reports of very high CR (complete remission), CAR T cell therapies using FDA-approved anti-CD19 targeted "Kymriah" or "Yescarta" for the treatment of various B-cell origin leukemias and lymphomas also faced the same problem of antigen escape. To overcome this challenge, the idea of ​​dual-target CAR T cells has often been applied, and bispecific anti-CD19 / CD20 CAR T cell therapy has shown early promise for treating relapsed or refractory B-cell lymphomas. However, since most tumor markers are also expressed, albeit at relatively low levels, on normal tissues, finding two or more targetable tumor-specific antigens that are highly specific to tumors is extremely difficult in solid tumors. Because CAR T cells have the inherent property of being highly potent in killing cancer cells, dual-CAR designs that multi-target two or more antigens are considered to be extremely dangerous for patients with solid tumors, as they can induce unbearable on-target off-tumor toxicity.

[0105] Interestingly, as a broad pan-marker for epithelial cells, EpCAM has been found to co-express with HER2 in many cancers. Instead of using a dual-CAR setup, the present invention has developed treatment of HER2-targeted CAR T cells that secrete an anti-EpCAM bispecific T cell engager (BiTE), thereby limiting the presence of anti-EpCAM BiTE to the tumor site, contributing to maximum tumor eradication and prevention of tumor escape, while simultaneously avoiding systemic toxicity. These CAR T cells were named "HE CAR-BiTE T" (HER2-targeted CAR T cells that secrete anti-EpCAM BiTE).

[0106] CAR-BiTE T cells that secrete anti-EpCAM BiTE initially penetrate and retain the tumor site via CAR recognition of specific tumor antigens expressed on tumor cells. Binding to the CAR target activates the CAR T cells, causing them to proliferate and simultaneously secrete anti-EpCAM BiTE. While CAR T cells can directly kill target cells, anti-EpCAM BiTE exerts its cytotoxicity by recruiting nearby T cells. This approach physically directs bystander T cells near the tumor, simultaneously activating them and subsequently assisting in the removal of tumor cells. Because HER2 overexpression is tumor-specific, the proliferation of anti-EpCAM BiTE is likely limited to the tumor site or its vicinity. Furthermore, since the secretion of recombinant anti-EpCAM BiTE antibodies by anti-HER2 CAR T cells is not highly efficient, further diffusion into normal tissue, coupled with the requirement for just-in-time T cell presence, is likely to diminish on-target, off-tissue toxicity. Additionally, EpCAM has also been found to be expressed on cancer progenitor cells and cancer stem cells, and is therefore defined as a cancer stem cell marker. The secretion of anti-EpCAM BiTE by CAR T cells simultaneously contributes to preventing cancer recurrence and relapse by eliminating cancer stem cells and cancer progenitor cells.

[0107] In experimental data presented herein, EpCAM was shown to have the highest expression levels compared to other epithelial tumor markers in multiple stained HER2-positive tumor cells. Therefore, EpCAM was selected as a BiTE target due to its broad coverage of epithelial-derived cancer cells. The much higher expression of EpCAM in cancerous cells compared to normal epithelial cells provides a broader therapeutic area compared to other targets. Furthermore, anti-EpCAM BiTE can be injected into the tumor site for targeted treatment. The anti-EpCAM BiTE of this disclosure, secreted only locally to the tumor site by CAR T cells (and not administered systemically), limits its potential toxicity. This transforms an undrug-unavailable (or difficult-to-drug) target into a drug-available target.

[0108] Therefore, in one embodiment, modified cells expressing the following are provided. (a) Chimeric antigen receptors targeting HER2, and (b) A multispecific antigen-binding protein that binds to one or more targets, including a first antigen-binding protein that binds to EpCAM (epithelial cell adhesion molecule), a variant thereof or binding fragment, and a second antigen-binding protein that binds to an immune cell marker, a variant thereof or binding fragment, Here, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10) CDR-H1 includes the following: · GSIFSGND (Sequence IDs: 25-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·GSSERFTS (Sequence ID: 29-1B8) CDR-H2 includes the following: · ITSGGST (Sequence IDs: 26-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·ITNGGST (Sequence ID: 30-1B8), and CDR-H3 includes the following: ·TNGRWSGDTYYAHH (Sequence IDs: 27-2C4, hu2C4, 1A5 and 2D10), ·MAGTS (Sequence ID: 31-1B8), or ·TNGRWSGDTYYAHL (Sequence number: 33-2B7) (ii) Heavy chain variable regions including the following: (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) (iii) Heavy chain variable region including the following: (1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7) (iv) Light chain variable regions including the following: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6) CDR-L1 includes the following: • QSLLHSNGYNY (Sequence IDs: 9-1B6, 1C1, 1C11, and 1H6) · QSLLHSNRYNY (Sequence ID: 17-1D4), or • QSISDF (Sequence ID: 19-1E4) CDR-L2 includes the following: · LGS (Sequence IDs: 10-1B6, 1C1, 1C11, 1D4 and 1H6), or · AAS (Sequence ID: 20-1E4), and CDR-L3 includes the following: • MQALQTPYT (Sequence IDs: 11-1B6, 1C1 and 1D4) • MQGLQSPWT (Sequence ID: 15-1C11) · QQSYIMPDT (Sequence ID: 21-1E4), or ·MQGLQTPYT(Sequence ID: 23-1H6); and Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0109] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region comprising: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10) CDR-H1 includes the following: · GSIFSGND (Sequence IDs: 25-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·GSSERFTS (Sequence ID: 29-1B8) CDR-H2 includes the following: · ITSGGST (Sequence IDs: 26-2C4, hu2C4, 1A5, 2B7 and 2D10), or ·ITNGGST(Sequence ID: 30-1B8); and CDR-H3 includes the following: ·TNGRWSGDTYYAHH (Sequence IDs: 27-2C4, hu2C4, 1A5 and 2D10), ·MAGTS (Sequence ID: 31-1B8), or ·TNGRWSGDTYYAHL (Sequence number: 33-2B7). Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0110] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (2C4-VHH, hu2C4-VHH, 1A5-VHH, and 2D10-VHH) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID: 27) (ii) Heavy chain variable region including the following: (1B8-VHH) CDR-H1 containing GSSERFTS (Sequence ID: 29), CDR-H2 containing ITNGGST (Sequence ID: 30), and CDR-H3 containing MAGTS (SEQ ID NO: 31); and (iii) Heavy chain variable region including the following: (2B7-VHH) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID: 33) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0111] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (2C4, hu2C4, 1A5, and 2D10) CDR-H1 containing GSIFSGND (Sequence ID: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID: 27) (ii) Heavy chain variable region including the following: (1B6, 1C1, 1C11, 1D4, 1H6) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID: 3) (iii) Heavy chain variable region including the following: (1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7) (iv) Light chain variable region including the following: (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) (v) Light chain variable region including the following: (1C11) CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (Sequence ID: 15) (vi) Light chain variable region including the following: (1D4) CDR-L1 containing QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11) (vii) Light chain variable region including the following: (1E4) CDR-L1 containing QSISDF (Sequence ID: 19), CDR-L2 containing AAS (Sequence ID: 20), and CDR-L3 containing QQSYIMPDT (Sequence ID: 21) (viii) Light chain variable region including the following: (1H6) CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (Sequence ID: 23) (ix) Heavy chain variable region including the following: (1B8-VHH) CDR-H1 containing GSSERFTS (Sequence ID: 29), CDR-H2 containing ITNGGST (Sequence ID: 30), and A CDR-H3 comprising MAGTS (SEQ ID NO: 31); and (x) a heavy chain variable region comprising: (2B7-VHH) A CDR-H1 comprising GSIFSGND (SEQ ID NO: 25), A CDR-H2 comprising ITSGGST (SEQ ID NO: 26), and A CDR-H3 comprising TNGRWSGDTYYAHL (SEQ ID NO: 33) Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto.

[0112] In some examples, the first antigen-binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of: (i) QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH A heavy chain variable domain comprising WGQGTQ (SEQ ID NO: 35 - 2C4-VHH) (ii) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH A heavy chain variable domain comprising WGQGTL (SEQ ID NO: 37 - hu2C4-VHH) (iii) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY A heavy chain variable domain comprising WGQGTL (SEQ ID NO: 4 - 1B6, 1C1, 1C11, 1D4 and 1H6) (iv) QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVAWNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI Heavy chain variable domains including WGQGTM (SEQ ID NO: 8-1E4), (v)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domains containing FGQGTK (SEQ ID NOs: 12-1B6 and 1C1) (vi)EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT Light chain variable domain containing FGQGTK (SEQ ID NO: 16-1C11) (vii)DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 18-1D4) (viii)DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT Light chain variable domain containing FGQGTK (SEQ ID NO: 22-1E4) (ix)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 24-1H6) (x)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGNDMAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domain containing WGQGTQ (SEQ ID NO: 28-1A5-VHH) (xi)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS Heavy chain variable domain containing WGQGTQ (SEQ ID NO: 32-1B8-VHH) (xii)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL Heavy chain variable domain containing WGQGTQ (SEQ ID NO: 34-2B7-VHH) (xiii)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domains including WGQGTQ (SEQ ID NO: 36-2D10-VHH); and Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0113] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain selected from the following group: (i)QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHHHeavy chain variable domain containing WGQGTQ (Accession No.: 35-2C4-VHH) (ii) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domain containing WGQGTL (Accession No.: 37-hu2C4-VHH) (iii) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY Heavy chain variable domain containing WGQGTL (Accession No.: 4-1B6, 1C1, 1C11, 1D4 and 1H6) (iv) QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI Heavy chain variable domain containing WGQGTM (Accession No.: 8-1E4), (v) QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domain containing WGQGTQ (Accession No.: 28-1A5-VHH) (vi) QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS Heavy chain variable domain containing WGQGTQ (Accession No.: 32-1B8-VHH) (vii) QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL Heavy chain variable domain containing WGQGTQ (SEQ ID NO: 34-2B7-VHH) (viii)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH Heavy chain variable domains including WGQGTQ (SEQ ID NO: 36-2D10-VHH); and Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0114] In some cases, the first-stage antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain a single-domain heavy chain variable domain having the following sequence: (i)QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID: 35-clone2C4-VHH), or QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH WGQGTL (Sequence ID: 37-hu2C4-VHH), or (ii) QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID: 28-Clone 1A5-VHH), or (iii)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ (Sequence ID: 32-clone1B8-VHH), or (iv)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL WGQGTQ (Sequence ID: 34-Clone 2B7-VHH), or (v)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID: 36-clone2D10-VHH), Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0115] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable region selected from the group consisting of: (i) Heavy chain variable regions including the following: (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) (ii) Heavy chain variable region including the following: (1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7); and / or Includes light chain variable regions including: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6) CDR-L1 includes the following: • QSLLHSNGYNY (Sequence IDs: 9-1B6, 1C1, 1C11, and 1H6) · QSLLHSNRYNY (Sequence ID: 17-1D4), or • QSISDF (Sequence ID: 19-1E4) CDR-L2 includes the following: · LGS (Sequence IDs: 10-1B6, 1C1, 1C11, 1D4 and 1H6), or · AAS (Sequence ID: 20-1E4), and CDR-L3 includes the following: • MQALQTPYT (Sequence IDs: 11-1B6, 1C1 and 1D4) • MQGLQSPWT (Sequence ID: 15-1C11) · QQSYIMPDT (Sequence ID: 21-1E4), or • MQGLQTPYT (Sequence ID: 23-1H6) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0116] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM 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: (1B6 and 1C1) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3); and / or The light chain variable region includes the following: CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11) (ii) Heavy chain variable region including the following: (1C11) CDR-H1 containing GGTFSSYA (Sequence ID: 1) CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3); and / or The light chain variable region includes the following: CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQSPWT (Sequence ID: 15) (iii) Heavy chain variable region including the following: (1D4) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3); and / or The light chain variable region includes the following: CDR-L1 containing QSLLHSNRYNY (Sequence ID: 17), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQALQTPYT (Sequence ID: 11) (iv) Heavy chain variable region including the following: (1H6) CDR-H1 containing GGTFSSYA (Sequence ID: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3); and / or The light chain variable region includes the following: CDR-L1 containing QSLLHSNGYNY (Sequence ID: 9), CDR-L2 containing LGS (Sequence ID: 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23); and (v) Heavy chain variable region including the following: (1E4) CDR-H1 containing GDSISSNSVA (Sequence ID: 5), CDR-H2 containing TYYRSKWYS (Sequence ID: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID: 7); and / or The following is included in the light chain variable region: (1E4) CDR-L1 containing QSISDF (Sequence ID: 19), CDR-L2 containing AAS (Sequence ID: 20), and CDR-L3 containing QQSYIMPDT (Sequence ID: 21) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0117] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of: (vi) Heavy chain variable domains including the following: (1B6 and 1C1) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and / or DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 12) (vii) Heavy chain variable domains including the following: (1C11) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and / or EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT Light chain variable domain containing FGQGTK (SEQ ID NO: 16) (viii) Heavy chain variable domains including the following: (1D4) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and / or DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT Light chain variable domain containing FGQGTK (SEQ ID NO: 18) (ix) Heavy chain variable domains including the following: (1E4) QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQGTM (Sequence ID: 8), and / or DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT A light chain variable domain containing FGQGTK (SEQ ID NO: 22), or (x) Heavy chain variable domain including the following: (1H6) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence ID: 4), and / or Light chain variable domain DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT FGQGTK (Sequence ID: 24), or Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0118] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a light chain constant domain having the following sequence: (iii)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (Sequence ID: 13-Clone 1B6-Light Chain Constant Domain), or (iv)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (Sequence ID: 14 ​​Clone 1C1-Light Chain Constant Domain), Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have two or three amino acid substitutions.

[0119] In one embodiment, the antigen-binding protein, its variant, or fragment comprises a heavy chain variable region and / or a light chain variable region encoded by a nucleic acid sequence selected from the group consisting of: (i) Heavy chain variable region including the following: (1B6 and 1C1) CDR-H1 (Sequence ID: 38) containing GGAGGCACCTTCAGCAGCTATGCT, CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (Sequence ID: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID: 40); and / or The light chain variable region includes the following: CDR-L1 containing CAGAGCCTCCTGCATAGTAATGGATACAACTAT (Sequence ID: 46), CDR-L2 containing TTGGGTTCT (Sequence ID: 47), and CDR-L3 containing ATGCAAGCTCTACAAACTCCGTACACT (Sequence ID: 48) (ii) Heavy chain variable region including the following: (1C11) CDR-H1 containing GGAGGCACCTTCAGCAGCTATGCT (Sequence ID: 38), CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (Sequence ID: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID: 40); and / or The light chain variable region includes the following: CDR-L1 (Sequence ID: 46) containing CAGAGCCTCCTGCATAGTAATGGATACAACTAT, CDR-L2 containing TTGGGTTCT (Sequence ID: 47), and CDR-L3 containing ATGCAAGGTCTACAAAGTCCCTGGACG (Sequence ID: 52) (iii) Heavy chain variable region including the following: (1D4) CDR-H1 containing GGAGGCACCTTCAGCAGCTATGCT (Sequence ID: 38), CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (Sequence ID: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID: 40); and / or The light chain variable region includes the following: CDR-L1 containing CAGAGCCTCCTGCATAGTAATAGATACAACTAT (Sequence ID: 54), CDR-L2 containing TTGGGTTCT (Sequence ID: 47), and CDR-L3 containing ATGCAAGCTCTACAAACTCCGTACACT (Sequence ID: 48) (iv) Heavy chain variable region including the following: (1H6) CDR-H1 containing GGAGGCACCTTCAGCAGCTATGCT (Sequence ID: 38), CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (Sequence ID: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID: 40); and / or The light chain variable region includes the following: CDR-L1 containing CAGAGCCTCCTGCATAGTAATGGATACAACTAT (Sequence ID: 46), CDR-L2 containing TTGGGTTCT (Sequence ID: 47), and CDR-L3 containing ATGCAAGGTCTACAGACTCCGTACACT (SEQ ID NO: 60); and (v) Heavy chain variable region including the following: (1E4) CDR-H1 containing GGGGACAGTATCTCTAGTAACAGTGTTGCT (Sequence ID: 42), CDR-H2 containing ACATACTACAGGTCCAAGTGGTACAGT (Sequence ID: 43), and CDR-H3 containing GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC (Sequence ID: 44); and / or The following is included in the light chain variable region: (1E4) CDR-L1 containing CAGAGTATTAGCGACTTT (Sequence ID: 56), CDR-L2 containing GCTGCATCG (Sequence ID: 57), and CDR-L3 containing TTACATTATGCCCGACACT (Sequence ID: 58) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0120] In one embodiment, the antigen-binding protein, its variant, or fragment comprises a heavy chain variable region encoded by a nucleic acid sequence selected from the group consisting of: (i) Heavy chain variable regions including the following: (2C4-VHH, 1A5-VHH, 2D10-VHH, and hu2C4-VHH) CDR-H1 containing GGAAGCATCTTCAGTGGCAATGAC (Sequence ID: 62), CDR-H2 containing ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC (Sequence ID: 64) (ii) Heavy chain variable region including the following: (1B8-VHH) CDR-H1 containing GGAAGCTCCGAAAGATTCACATCA (Sequence ID: 66), CDR-H2 containing ATTACTAATGGTGGTAGCACA (Sequence ID: 67), and CDR-H3 containing ATGGCGGGTACGTCC (Sequence ID: 68); and (iii) Heavy chain variable region including the following: (2B7-VHH) CDR-H1 containing GGAAGCATCTTCAGTGGCAATGAC (Sequence ID: 62), CDR-H2 containing ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC (Sequence ID: 70) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

[0121] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain encoded by a nucleotide sequence comprising: (i)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 72-Clone 2C4), (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (SEQ ID NO: 74 - Clone hu2C4 - VHH); (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (SEQ ID NO: 41 - for Clones 1B6, 1C1, 1C11, 1D4, 1H6), or (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (SEQ ID NO: 45 - Clone 1E4); (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 65 - Clone 1A5), (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), (vii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), (viii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 73-Clone 2D10), or and / or The light chain variable domain is encoded by a nucleotide sequence that includes the following: (vi)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 49-Clone 1B6 and 1C1), or (vii)GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID: 53-Clone 1C11), or (viii)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (SEQ ID NO: 55 - Clone 1D4), or (ix)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (SEQ ID NO: 59 - Clone 1E4), or (x)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (SEQ ID NO: 61 - Clone 1H6), Or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have 10-20 nucleic acid substitutions.

[0122] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain encoded by a nucleotide sequence comprising: (i)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 72-Clone 2C4), or (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG(Sequence ID:74-clonehu2C4-VHH); or (iii)GAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41 - Clone 1B6, 1C1, 1C11, 1D4, 1H6), or (iv)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (Sequence ID: 45-Clone1E4); or (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 65-Clone 1A5), or (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), or (vii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), or (viii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 73-Clone 2D10), 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-Clone 1B6 and 1C1), or (ii)GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID: 53-Clone 1C11), or (iii)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT 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), Or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have 10-20 nucleic acid substitutions.

[0123] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes heavy and / or light chain variable domains encoded by a nucleotide sequence selected from the group consisting of: (i) Heavy chain variable domains encoded by nucleotide sequences including the following: (1B6 and 1C1) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and / or Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 49) (ii) A heavy chain variable domain encoded by a nucleotide sequence including the following: (1C11). GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and / or Light chain variable domain encoded by the following nucleotide sequence: GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID: 53) (iii) Heavy chain variable domain encoded by a nucleotide sequence including the following: (1D4) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and / or Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID: 55) (iv) Heavy chain variable domain encoded by the following nucleotide sequence: (1H6) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID: 41), and / or Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG(Sequence ID: 61); and (v) Heavy chain variable domain encoded by a nucleotide sequence including the following: (1E4) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (Sequence ID: 45), and / or Light chain variable domain encoded by the following nucleotide sequence: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence number: 59) Or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them, and / or have 10-20 nucleic acid substitutions.

[0124] In one embodiment, the antigen-binding protein, its variant, or fragment comprises a light chain constant domain encoded by a nucleotide sequence consisting of the following: CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (Sequence ID: 50-Clone 1B6-Light Chain Constant Domain), or CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT (Sequence ID: 51-Clone 1C1-Light Chain Constant Domain), or Sequences that are at least 60% identical to them and / or have 10-20 nucleic acid substitutions.

[0125] In some examples, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM includes a heavy chain variable domain encoded by a nucleotide sequence comprising: (i)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 has 10-20 nucleic acid substitutions, (ii) 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 10-20 nucleic acid substitutions; or (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 has 10-20 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 has 10-20 nucleic acid substitutions, (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC 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 has 10-20 nucleic acid substitutions, (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID: 73), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or have 10-20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (Sequence ID: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10-20 nucleic acid substitutions, (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC A sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10-20 nucleic acid substitutions.

[0126] In some examples, the multispecific antigen-binding protein may comprise 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, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and cleavages. In some examples, the amino acid mutations are amino acid substitutions and may include conserved and / or non-conserved substitutions. In some examples, the mutations do not substantially reduce the ability of the antigen-binding protein to specifically bind to a target. In some examples, the mutations do not substantially reduce the ability of the antigen-binding protein to specifically bind to a target and do not functionally modulate (e.g., partially or completely neutralize) the target.

[0127] In some examples, the multispecific antigen-binding protein may be a bispecific, triplicate, or quadruplicate antigen-binding protein. In some examples, the multispecific antigen-binding protein is a bispecific antigen-binding protein. In some examples, the multispecific antigen-binding protein, its variants, or fragments are bispecific antibodies. In some examples, the multispecific antigen-binding protein may be provided as a nanobody. In some examples, the multispecific antigen-binding protein may be provided as an Fc region. 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). In some embodiments, the bispecific T cell engager (BiTE) is a nanobody with only a heavy chain (VHH).

[0128] In some cases, the multispecific antigen-binding protein, its variants, or fragments are inducible bispecific immune cell engagers. In some cases, the multispecific antigen-binding protein is secreted by cells, optionally immune cells. In some cases, the multispecific antigen-binding protein, its variants, or fragments are bispecific T cell engagers (BiTEs).

[0129] As used herein, bispecific T cell engagers (BiTEs) refer to a class of artificial bispecific monoclonal antibodies that direct the host immune system, such as by inducing cytotoxic activity of T cells against target cells (e.g., cancer cells). A BiTE is a fusion protein on a single peptide chain of approximately 55 kDa, consisting of two single-chain variable fragments (scFv) of different antibodies, or amino acid sequences from four different genes. One scFv binds to T cells via the CD3 receptor, and the other binds to a target of interest (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs form a binding between T cells and target cells (e.g., tumor cells). This causes T cells to exert cytotoxic activity on target cells (e.g., tumor cells) by producing proteins such as perforin and granzyme that enter tumor cells and initiate cellular apoptosis.

[0130] In some cases, BiTE may refer to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In some cases, BiTE may also refer to a bispecific T-cell engager platform known in the art, which is a recombinant protein that has the ability to simultaneously bind to two different antigens and engage immune cells (such as T cells).

[0131] In some cases, the bispecific T cell engager (BiTE) binds to two antigens; where the first antigen is EpCAM; and where the second antigen is an immune cell marker. In some cases, the second antigen-binding protein, its variant, or binding fragment binds to an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, CD64, etc.

[0132] In some cases, 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).

[0133] In some cases, the bispecific T cell engager (BiTE) binds to two antigens; where the first antigen is an epithelial cell adhesion molecule (EpCAM); and where the second antigen is CD3. In some cases, the CD3 may be CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0134] In some examples, the modified / designed immune cells may be, 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, etc. In some examples, the modified / designed immune cells are T cells, NK cells, or macrophages. In some examples, the modified / designed immune cells are T cells.

[0135] In some cases, these immune cells bind to HER2 and secrete inducible bispecific T cell engagers (HE CAR-BiTE T) that target EpCAM and CD3.

[0136] While we do not wish to be constrained by theory, the CAR cells directly kill target cells, while the anti-EpCAM BiTE exerts its cytotoxicity by recruiting nearby immune cells (such as T cells). This approach physically directs bystander T cells near the tumor, simultaneously activating them to help eliminate tumor cells. By applying local secretion of anti-EpCAM BiTE by CAR T cells, it transforms a non-drug-unavailable target into a drug-available target by mitigating the on-target off-tumor toxicity that can result from systemic delivery. Since EpCAM is also defined as a cancer stem cell marker expressed on cancer progenitor cells and cancer stem cells, secretion of anti-EpCAM BiTE by CAR cells simultaneously contributes to preventing cancer relapse and recurrence by eliminating cancer stem cells and cancer progenitor cells.

[0137] In addition, as described in the experimental data of this disclosure, HER2-targeted CAR T cells secreting anti-EpCAM BiTE (HE CAR-BiTE T) are demonstrated to be superior in vitro to HE CAR-T alone in killing human tumors originating from gastric or breast cancer (Figures 1D and 1E). Both clones 4D5 and F5 of HE CAR-BiTE T were superior to HE CAR-T alone in killing AGS cells (HER2 High , EpCAM High ) and MDA-MB468 cells (HER2 VeryLow , EpCAM High ) shows a higher cytolysis %. MDA-MB468 (HER2 VeryLow , EpCAM HighIn (Figure 1E), clones F5 and 4D5 showed minimal or reduced cytolysis of MDA-MB468 compared to their respective HE CAR-BiTE T cells. This suggests that the primary cytotoxic function in MDA-MB468 cells is contributed by anti-EpCAM BiTE. These results further highlight that the HE CAR-BiTE T cells of the present invention target host cells only when EpCAM expression levels are high, and avoid targeting cells with low EpCAM expression. Clones 4D5 of the HE CAR-T cells were unable to initiate efficient killing of AGS cells (Figure 1D) with low levels of interferon-γ secretion (Figure 1F), suggesting that the cytotoxic function of the HE CAR-BiTE T cells of clone 4D5 was primarily contributed by secreted anti-EpCAM BiTE. This disclosure also shows that the modified cells disclosed herein achieved more efficient and prolonged tumor control than treatment with anti-HER2 CAR T cells alone (see Figure 2).

[0138] In some examples, the cells disclosed herein 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 others, which can engage immune cells in a target environment. In some examples, the immune cells are T cells or macrophages or monocytes or NK cells. In some examples, the immune cells are T cells.

[0139] Therefore, in some cases, the cells bind to HER2 and secrete multispecific antigen-binding proteins that bind to both EpCAM and CD3 (e.g., HE CAR-BiTE T).

[0140] Polypeptides comprising multispecific antigen-binding proteins are also disclosed. In some examples, the multispecific antigen-binding protein is a bispecific antibody. In some examples, the polypeptide comprises multispecific antigen-binding proteins that bind to EpCAM (epithelial cell adhesion molecule) and immune cells.

[0141] In some cases, the multispecific antigen-binding protein is a bispecific cell engager capable of engaging both antigens and immune cells.

[0142] 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).

[0143] In some examples, the polypeptide includes an anti-immune cell antigen-binding protein. In some examples, the polypeptide includes 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 can be CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0144] 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.

[0145] In some cases, the immune cells are T cells, NK cells, macrophages, or monocytes. In some cases, the immune cells are T cells.

[0146] 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), such as an inducible BiTE, a non-inducible BiTE, or a constitutively expressed 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 be, but is not limited to, CD3, NKG2D, CD28, CD16, or CD64. In some examples, the polypeptide comprises a BiTE that bispecifically binds to EpCAM and T cells. In some examples, the polypeptide comprises a BiTE that bispecifically binds to EpCAM and CD3. In some examples, the EpCAM targeted by the BiTE is modified with anti-EpCAM VHH paired with anti-CD3 scFv.

[0147] In some cases, the anti-EpCAM VHH is paired with an anti-CD3 scFv which may contain clone Okt3(Nb01-013A).

[0148] In another embodiment, polynucleotides encoding cells and / or polypeptides and / or multispecific antigen-binding proteins described herein are provided.

[0149] Furthermore, polynucleotides comprising sequences encoding an immune cell engager and a chimeric antigen receptor (CAR) are disclosed, wherein the immune cell engager is capable of bispecifically binding to EpCAM (epithelial cell adhesion molecule) and immune cells, and wherein the CAR is capable of binding to a first antigen.

[0150] In some examples, polynucleotides are provided that include a sequence encoding a chimeric antigen receptor capable of recognizing, binding to, and engaging with HER2-positive cells. In some examples, the first antigen is HER2. Thus, in some examples, the CAR is capable of binding to HER2.

[0151] In some examples, the polynucleotide further comprises sequences encoding one or more costimulatory domains, signal peptides, hinges, and / or signal transduction domains.

[0152] In some examples, the aforementioned co-stimulatory domains may include, but are not limited to, 4-1BB, CD28, CD27, and OX-40.

[0153] In some examples, the signal peptide may be, but is not limited to, an IgH signal peptide, an IgK signal peptide, or a CD8 signal peptide.

[0154] In some examples, the hinge may be, but is not limited to, an IgH hinge, a hinge of an immunoglobulin-like protein (such as IgA, IgD, IgE, IgG, IgM), or a transmembrane domain, CD28, CD8, 4-1 BB, etc.

[0155] In some examples, the sequence encoding the CAR encodes an antigen-binding protein (or anti-HER2 antigen-binding protein) capable of binding to HER2, or a fragment thereof, or a variant thereof. In some examples, the sequence encoding the CAR encodes a single-chain variable fragment. In some examples, the sequence encoding the CAR encodes an anti-HER2 scFv.

[0156] In some cases, the sequence encoding the CAR further encodes an immune cell signaling domain. In some cases, the signaling domain may include, but is not limited to, the intracellular domains of CD3, TCFζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3ε, CD3η, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some cases, the intracellular domain of CD3 may be one or more such as CD3ζ, CD3ε, CD3γ, and CD3δ. In some cases, the sequence encoding the CAR further encodes an intracellular domain of CD3 (signaling domain).

[0157] In some examples, the sequence encoding the CAR encodes the anti-HER2 scFv, CD28 costimulatory domain, 4-1BB costimulatory domain, IgH signaling peptide, IgH hinge, and CD3 signaling domain.

[0158] 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), a fragment thereof, a variant thereof, and an anti-immune cell antigen-binding protein that can bind to EpCAM.

[0159] In some examples, the sequence encoding the immune cell engager encodes a single-chain variable fragment (scFv) or a single-chain variable domain located in the heavy chain (VHH).

[0160] In some examples, the sequence encoding the immune cell engager encodes anti-EpCAM scFv or anti-EpCAM VHH.

[0161] In some examples, 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 examples, the immune cell activation marker may include, but is not limited to, CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0162] In some cases, the immune activation marker may be of the VHH type or scFv type. In some cases, the sequence encoding the immune cell engager encodes an antigen-binding protein (anti-CD3 antigen-binding protein), a fragment thereof, or a variant thereof, that can bind to CD3. In some cases, the sequence encoding the immune cell engager encodes a single-chain variable fragment (scFv) or a VHH type. In some cases, the sequence encoding the immune cell engager encodes anti-CD3 scFv or anti-CD3 single-domain VHH.

[0163] In some cases, the immune cell engager may include a His tag.

[0164] In some examples, 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 examples, the linker is a cleavable linker, and examples of such cleavable linkers include, but are not limited to, P2A, T2A, and F2A.

[0165] In some cases, the immune cells are T cells, NK cells, macrophages, or monocytes. In some cases, the immune cells are T cells.

[0166] In some examples, the polynucleotide comprising the sequence encoding an immune cell engager is a sequence encoding a bispecific T cell engager (BiTE), such as an inducible BiTE, a non-inducible BiTE, or a constitutively expressed BiTE. In some examples, the polynucleotide comprises the sequence encoding a BiTE that bispecifically binds to EpCAM and T cells. In some examples, the polynucleotide comprises a sequence encoding an anti-HER2 scFv CAR with an intracellular CD3 domain, as well as a sequence encoding a BiTE that binds to EpCAM and T cells.

[0167] In yet another embodiment, vectors expressing polynucleotides or polypeptides described herein are provided. In some examples, 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 virus vectors, DNA virus vectors, and vectors derived from combinations of plasmids and phage DNA, further optionally wherein the polynucleotide is operably linked to an expression control sequence(s) for directing peptide synthesis, and further optionally wherein the vector comprises one or more selectable marker genes for providing phenotypic traits for selection of transformed host cells.

[0168] In some cases, the vector is a lentiviral vector.

[0169] In some embodiments, the host cells include the above-mentioned cloning or expression vector and / or nucleic acid sequences encoding the above-mentioned antigen-binding protein, antibody and its binding fragment.

[0170] The host cell may be any type of cell that can be transformed or transfected with the nucleic acid or vector to produce the antigen-binding protein or its binding fragment / protein encoded by it. The host cell containing the nucleic acid or vector may be used to produce the antigen-binding protein or its binding fragment / protein, or a portion thereof (e.g., a heavy chain sequence or light chain sequence encoded by the nucleic acid or vector). After the introduction of 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, or antibody fragment, or a portion of the antibody may then be isolated from the cell.

[0171] The host cells may be prokaryotic host cells (such as Escherichia coli) or eukaryotic host cells (such as yeast cells, insect cells, or vertebrate cells). When cultured under appropriate conditions, the host cells express antibodies or their conjugated fragments, which can then be harvested from the culture medium (if the host cells secrete them into the medium) or directly from the host cells producing them (if they do not secrete them). The selection of appropriate host cells depends on various factors, including the desired expression level, the modification of the polypeptide desirable or required for activity, such as glycosylation or phosphorylation, and the ease of folding into a biologically active molecule. The selection of host cells also depends in part on whether the antibody or its conjugated fragment should be post-transcriptionally modified (e.g., glycosylation and / or phosphorylation). The host cells may include bacterial cells, yeast cells, animal cells, such as mammalian cells and / or plant cells.

[0172] Suitable mammalian host cells include CHO cells, myeloma cells, or hybridoma cells. Many are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include mammalian cells such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61), human embryonic kidney (HEK) 293 cells or 293T cells (ATCC No. CRL1573), 3T3 cells (ATCC No. CCL92), or PER.C6 cells. Other cell types used to express antibodies include lymphoid cell lines such as NSO myeloma cells and SP2 cells, and COS cells.

[0173] In one embodiment, the host cell expresses / secretes an antigen-binding protein, a variant thereof, or a fragment thereof as disclosed herein.

[0174] In one embodiment, cells that express / secrete EpCAM-specific immune cell engagers are provided.

[0175] 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.

[0176] In one embodiment, the cells are immune cells.

[0177] In one embodiment, the 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.

[0178] In one embodiment, the immune cells express / secrete EpCAM-specific bispecific T cell engagers (BiTEs).

[0179] In one embodiment, the immune cells express / secrete bispecific T cell engagers (BiTEs), such as inducible, non-inducible, or constitutively expressed BiTEs, which include the antigen-binding proteins, variants, or fragments thereof disclosed herein.

[0180] Surprisingly, the inventors have demonstrated that immune cells, such as CAR T cells, can secrete EpCAM BiTE. This was previously unknown. Advantageously, by designing immune cells that express the EpCAM immune engager, it is possible to enable the EpCAM immune engager to be secreted at target sites (e.g., solid tumor sites), thereby minimizing toxicity and / or side effects.

[0181] In some examples, the immune cells may 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, and monocytes. 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.

[0182] 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.

[0183] In some cases, the immune cells may bind to multiple host cell antigens. Therefore, in some cases, the 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.

[0184] In yet another embodiment, a method for producing / generating cells as described herein is provided, comprising introducing the polynucleotides described herein into the cells. In some examples, the cells are immune cells.

[0185] Furthermore, a method for producing / generating the cells described herein is disclosed, wherein one or more nucleic acids encoding the host cell target antigen are fused in a vector via a linker (such as a P2A cleavable linker); and wherein the vector is transformed or transfected into host cells. In some examples, the culture supernatant of the host cells transformed or transfected with the vector is collected.

[0186] In some examples, the method includes introducing the vector described herein into the cells. In some examples, the vector is introduced via viral transduction.

[0187] Furthermore, compositions comprising cells or cell supernatants described herein are disclosed.

[0188] Furthermore, pharmaceutical compositions comprising cells or cell supernatants described herein, and suitable pharmaceutical compositions thereof are disclosed.

[0189] In some examples, the composition is a prophylactic and / or therapeutic composition.

[0190] Pharmaceutically acceptable agents for use in this pharmaceutical composition include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings 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.

[0191] Furthermore, compositions described herein or pharmaceutical compositions for use in therapeutic / pharmaceutical / vaccine applications are disclosed, and such compositions may optionally further comprise excipients and / or stabilizers.

[0192] In another context, a method is provided for treating a disease in a subject that requires it, the method comprising administering the cells described herein to the subject, wherein the disease is optionally a proliferative disorder.

[0193] Furthermore, a method for treating a disease in a subject requiring such treatment is disclosed, the method comprising administering the cells or compositions designed herein to the subject.

[0194] Furthermore, methods are disclosed for preventing and / or reducing the severity of symptoms caused by a disease in subjects requiring such prevention, the methods comprising administering the designed cells or compositions described herein to the subjects.

[0195] In some cases, the cells or composition or pharmaceutical composition should be administered to the subject through one or more routes of administration, including but not limited to topical, intravascular, intravenous, oral, subcutaneous, intra-arterial, intrathecal, intraperitoneal, nasal, intradermal, and intramuscular.

[0196] Furthermore, the use of cells described herein in the manufacture of pharmaceuticals for preventing and / or treating diseases is disclosed.

[0197] Furthermore, polynucleotides, antibodies, bispecific T cell engagers, designed cells, designed immune cells, methods, compositions, or pharmaceutical compositions described herein are disclosed.

[0198] In some cases, the disease is a proliferative disorder. In some cases, the disease is a tumor or cancer. In some cases, the disease is a carcinoma, and may include, but is not limited to, liver cancer (hepatocellular carcinoma, etc.), lung cancer (squamous cell carcinoma of the lung, etc.), stomach cancer (adenocarcinoma of the stomach, etc.), breast cancer, skin cancer (malignant melanoma, etc.), ovarian cancer (clear cell carcinoma of the ovary, etc.), kidney cancer, pancreatic cancer, head and neck cancer, prostate cancer, esophageal cancer, bladder cancer, colon cancer, and childhood cancers (hepatoblastoma, nephroblastoma, yolk sac tumor, etc.).

[0199] Furthermore, in describing certain embodiments, this disclosure may disclose a method and / or process as a specific set of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific set of steps disclosed. Other sets of steps may be possible. The specific order of the steps disclosed herein should not be construed as an unreasonable limitation. Unless otherwise required, the method and / or process disclosed herein should not be limited to steps performed in the order written. The set of steps may be modified and still remain within the scope of this disclosure.

[0200] Furthermore, it is understood that while this disclosure provides embodiments having one or more features / characteristics discussed herein, one or more of these features / characteristics may also be omitted in other alternative embodiments, and that this disclosure provides support for such omissions and these related alternative embodiments.

[0201] array CDRs are shown in bold (CDR1); bold and italic (CDR2); or bold, italic and underlined (CDR3). Highlighted residues / bases indicate the differences between the 1B6 and 1C1 light chain constant domains.

[0202] 1. amino acid sequence

[0203] 1.1. Heavy chain variable domain of human IgG1 JPEG2026519827000002.jpg43145

[0204] 1.2. Light chain variable domain of human IgG1 JPEG2026519827000003.jpg160145

[0205] 1.3. Single-domain heavy chain variable domain of Lama VHH JPEG2026519827000004.jpg119145

[0206] 1.4. Single-domain heavy chain variable domain of humanized Lama VHH JPEG2026519827000005.jpg23145

[0207] 1.5. Anti-EpCAM Okt3 BiTE JPEG2026519827000006.jpg91145

[0208] 2. Nucleotide sequence

[0209] 2.1. Heavy chain variable domain of human IgG1 JPEG2026519827000007.jpg86145

[0210] 2.2. Light chain variable domain of human IgG1 JPEG2026519827000008.jpg157145 JPEG2026519827000009.jpg155145

[0211] 2.3. Single-domain heavy chain variable domain of Lama VHH JPEG2026519827000010.jpg128146 JPEG2026519827000011.jpg86145

[0212] 2.4. Single-domain heavy chain variable domain of humanized Lama VHH JPEG2026519827000012.jpg44145

[0213] Explanation of the diagram Examples of embodiments of this disclosure will be better understood and readily apparent to a person of ordinary skill in the art from the following discussion and, where applicable, in conjunction with the drawings. It should be understood that other modifications may be made without departing from the scope of the invention. The examples of embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new exemplary embodiments. The examples of embodiments should not be construed as limiting the scope of this disclosure. [Brief explanation of the drawing]

[0214] [Figure 1A-G]Figure 1A shows the construction and structural map of HE CAR-BiTE T cells (clones 4D5 or F5) using anti-EpCAM VHH BiTE (Nb01-013A). Figure 1B shows FACS analysis of tumor markers (HER2 and EpCAM) expressed in AGS cells and MDA-MB468 cells. Figure 1C shows the percentage of CAR expression in anti-HER2 CAR T (4D5 or F5) cells and HE CAR-BiTE T (4D5 or F5) cells detected by flow cytometry analysis. Figure 1D shows the percentage of cell lysis of AGS cells mediated by anti-HER2 CAR T (4D5 or 5F) cells and HE CAR-BiTE T (4D5 or F5) cells, with time-course measurements using xCelligence impedance assay (E:T ratio = 2:1). Figure 1E shows the percentage of cell lysis of MDA-MB468 cells mediated by anti-HER2 CAR T (4D5 or 5F) cells and HE CAR-BiTE T (4D5 or F5) cells, with time-course measurement using xCelligence impedance assay (E:T ratio = 1:1). Figure 1F shows bar graphs of ELISA measurements of interferon-r and IL-2 measured using culture supernatant collected 36 hours after co-culturing AGS cells and effector CAR T cells. Figure 1G shows bar graphs of ELISA measurements of interferon-r and IL-2 measured using culture supernatant collected 36 hours after co-culturing MDA-MB468 cells and effector CAR T cells. [Figure 2A-C]Figure 2 shows that human HE CAR-BiTE T cells using BiTE constructed with anti-EpCAM VHH demonstrated superior tumor killing in in vivo AGS xenografting. 1.3 million AGS(Her2High,EpCAMHigh) cells were subcutaneously injected into the right flank of female NSG mice (-9 days). On day 0, the mice were regrouped according to measurable tumor size, and 5 million CAR T(4D5) cells, CAR T(F5) cells, HE CAR-BiTE T(4D5) cells ("Nb01-013A" used as BiTE), HE CAR-BiTE T(F5) cells ("Nb01-013A" used as BiTE), or mock T cells were intravenously injected into these mice via the tail vein. Figure 2A shows that the tumor size of each mouse was measured and recorded every 3 to 7 days. Figure 2B shows that the tumor size of each individual mouse in each treatment group was measured and recorded every 3 to 7 days. Figure 2C shows that the body weight of each mouse was measured and recorded every 3 to 7 days. [Figure 3]Figure 3 shows the detection of the anti-EpCAM BiTE molecule Nb01-013A secreted from HE CAR-BiTE T cells by ELISA. Anti-HER2 CAR T (4D5 or F5) cells or anti-HER2 CAR T cells secreting anti-EpCAM BiTE (Nb01-013A) (named "HE CAR-BiTE T (4D5 or F5)") were cultured in T cell proliferation medium containing IL-7 (20 ng / ml) and IL-15 (5 ng / ml). The initial cell density was 1.5 million per ml, and the percentage of CAR expression ranged from 36.8% to 57.2%. Cell culture supernatants were collected at 24 and 48 hours for ELISA to detect the amount of secreted anti-EpCAM BiTE molecule Nb01-013A. In short, a human EpCAM-Fc tag protein was used to coat an ELISA plate overnight. After blocking with casein for two hours, the culture supernatant containing the anti-EpCAM BiTE molecule Nb01-013A was added to the plate. After one hour of incubation, the plate was washed, and the bound BiTE molecule was detected by an HRP-conjugated secondary antibody against the His tag. [Examples]

[0215] Experiment Section Construction of HE CAR-BiTE T constructs and evaluation of their functional properties in vitro. In this invention, the inventors constructed "HE CAR-BiTE T" by fusing the anti-EpCAM BiTE gene (the sequence "Nb01-013A" was used) to a lentiviral construct of an anti-HER2 CAR ("trastuzumab" clone 4D5, or the inventors' own anti-HER2 clone F5) via a P2A cleavable linker (Figure 1A). Following lentiviral transduction and proliferation (Figure 1C), AGS cells (HER2 High , EpCAM High ) and MDA-MB468 cells (HER2 VeryLow , EpCAM HighBoth (Figure 1B) were used to test the in vitro killing efficacy of CAR T cells. The HE CAR-BiTE T cells were shown to kill AGS cells at a much faster rate than the anti-HER2 CAR alone (Figure 1D), suggesting the involvement of BiTE in target cell killing. Surprisingly, anti-HER2 CAR T cells constructed with clone 4D5 were unable to initiate efficient killing of AGS cells with low levels of interferon-γ secretion (Figure 1F) (Figure 1D), suggesting that the cytotoxic function of HE CAR-BiTE T cells (clone 4D5) was primarily contributed by secreted anti-EpCAM BiTE. More interestingly, anti-HER2 CAR T cells (clone 4D5) were found to be MDA-MB468 (HER2 VeryLow , EpCAM High While these cells showed a similar level of killing, anti-HER2 CAR T cells constructed using our patented clone F5 demonstrated high specificity by preserving cells expressing HER2 at very low levels (Figures 1E and 1G).

[0216] In summary, to overcome the challenge of tumor heterogeneity, we developed a novel CAR T-cell therapy for targeting carcinomas (solid tumors originating from epithelial cells) by giving single-antigen-targeting CAR T cells additional targeting of EpCAM-positive cancer cells through localized secretion of anti-EpCAM BiTE. By using CAR T cells that secrete anti-EpCAM BiTE, we were also able to maximize the efficacy of CAR T-cell therapy by recruiting nearby bystander T cells (a population of T cells that do not express CAR) to contribute to tumor removal, while simultaneously avoiding the systemic toxicity that can be caused by other delivery methods of anti-EpCAM BiTE. We demonstrated the development of a prototype (HE CAR-BiTE) that also serves as proof of concept. While HE CAR-BiTE has been successfully demonstrated to work, our technology is not limited to HER2-targeting CARs and can be extended to many other tumor antigens that can be applied as CAR targets for various types of carcinomas occurring in multiple organs.

[0217] Human HE CAR-BiTE cells demonstrated superior killing efficacy in a mouse xenograft model. The anti-EpCAM BiTE "Nb01-013A" was constructed by pairing anti-EpCAM VHH clone 2C4 with the anti-human CD3 agonist clone Okt3. After testing the in vitro killing efficacy of HE CAR-BiTE T cells ("Nb01-013A" was used as the BiTE) using different CAR clones (4D5 or F5), we performed in vivo AGS(Her2 High , EpCAM High We then proceeded to test their tumor control efficacy using a xenograft model (Figure 2). Consistent with in vitro data, AGS appeared to be resistant to 4D5 CAR-T. While CAR T(4D5) failed to control tumor growth, treatment with CAR T(F5) cells or CAR-BiTE T cells from the other groups showed some in vivo tumor control efficacy without significant weight loss (Figure 2C) (Figure 2A). CAR T(F5) treatment showed effective control of tumor growth in all five mice. However, after 5-6 weeks of tumor reduction, the residual tumors began to grow again, with tumor size reaching 1000 mm². 3 It has exceeded Her2 Negative This suggested tumor escape from the cell population. 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 for up to 181 days after T cell injection (Figure 2B), and Her2 Negative EpCAM Positive This demonstrated extended tumor control through the removal of tumor cells.

[0218] Anti-EpCAM rama VHH BiTE, secreted by HE CAR-BiTE T cells, can be detected from cell cultures. The Nb01-013A BiTE molecule can also be detected from HE CAR-BiTE T(4D5) and HE CAR-BiTE T(F5) cells in in vitro culture. 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 an initial cell density of 1.5 million cells per ml. Cell culture supernatants were collected at 24 and 48 hours, followed by ELISA to detect the amount of secreted anti-EpCAM BiTE molecule Nb01-013A. In both HE CAR-BiTE T(4D5) and HE CAR-BiTE T(F5) cells, the amount of secreted Nb01-013A molecule increased over time, suggesting continuous secretion and accumulation of anti-EpCAM VHH BiTE from both cultures (Figure 3).

[0219] In summary, we demonstrated that the use of a BiTE molecule constructed from anti-EpCAM VHH(Nb01-013A) in HE CAR-BiTE T cells achieved more efficient and prolonged tumor control than treatment with anti-Her2 CAR T cells alone. [Industrial applicability]

[0220] application Embodiments of modified cells disclosed herein utilize the following factors: 1) tumor-directed T cell proliferation, and 2) tumor-limited anti-EpCAM BiTE secretion, the latter of which is not achievable by conventional systemic delivery approaches. The anti-EpCAM BiTE-secreting CAR T cells disclosed herein may be extended to other cancer types to establish a broader range of next-generation CAR T cell therapies.

[0221] The technology described in the current TDF may have the following applications: 1. Anti-HER2 CAR T cells that secrete anti-EpCAM BiTE can be used to treat HER2-expressing cancers. These anti-EpCAM BiTE-secreting CAR T cells can be used to target a variety of solid cancers, including liver cancer, breast cancer, head and neck cancer, gastric cancer, pancreatic cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, esophageal cancer, bladder cancer, and colon cancer. 2. CAR T cells that secrete anti-EpCAM BiTE may be configured to target other tumor-specific antigens, including but not limited to GPC3, claudin 18.2, ROR1, DLL3, CEA, MUC1, MUC16, CEACAM7, CD133, CD147, PSCA, PSMA, MSLN, c-Met, and FRα.

[0222] This disclosure provides an improvement in the efficacy of current single-target CAR T-cell treatments for solid tumors by advantageously overcoming tumor heterogeneity and treatment-related tumor escapes using anti-EpCAM BiTE. Furthermore, by applying local secretion of anti-EpCAM BiTE by CAR T cells, non-drug-capable targets are transformed into drug-capable targets to mitigate on-target off-tumor toxicity that may result from systemic delivery. In addition, targeting cancer progenitor cells and cancer stem cells that express EpCAM further contributes to complete tumor eradication and prevention of tumor recurrence.

[0223] It will be understood by those skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the broader disclosure. For example, features of different exemplary embodiments may be mixed, combined, replaced, incorporated, adopted, modified, included, or similarly incorporated across different exemplary embodiments in this description. These embodiments should therefore be considered in all respects to be exemplary and not limiting.

Claims

1. Modified cells that express the following: (a) Chimeric antigen receptors targeting HER2, and (b) A multispecific antigen-binding protein that binds to one or more targets, comprising a first antigen-binding protein that binds to EpCAM (epithelial cell adhesion molecule), a variant thereof or binding fragment, and a second antigen-binding protein that binds to an immune cell marker, a variant thereof or binding fragment, Here, the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM 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 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, - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSSERFTS (Sequence ID: 29), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2 includes the following: - ITSGST (Sequence ID: 26), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, - 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: - TNGRWSGDTYYAHH (Sequence ID: 27), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. - MAGTS (Sequence ID: 31), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, - Sequence TNGRWSGDTYYAHL (Sequence ID: 33), or sequences 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 (Sequence ID: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, 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). (iii) Heavy chain variable region including the following: CDR-H1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GDSISSNSVA (Sequence ID: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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). Light chain variable region including (iv) and below: CDR-L1 includes the following: - QSLLHSNGYNY (Sequence ID: 9), or sequences 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, - QSISDF (Sequence ID: 19), or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. 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 sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, 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 with it. - MQGLQSPWT (Sequence ID: 15), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. - QQSYIMPDT (Sequence ID: 21), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, - MQGLQTPYT (Sequence ID: 23), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it.

2. The cell according to claim 1, wherein the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM 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 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (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), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical 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 (Sequence ID: 1), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, 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). (iii) Heavy chain variable region including the following: CDR-H1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GDSISSNSVA (Sequence ID: 5), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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). Light chain variable region including (iv) and below: CDR-L1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity 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 (Sequence ID: 11), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. (v) Light chain variable region including the following: CDR-L1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity 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 (Sequence ID: 15), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. (vi) Light chain variable region including the following: CDR-L1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity 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 (Sequence ID: 11), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. (vii) Light chain variable region including the following: CDR-L1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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 (Sequence ID: 21). Light chain variable region including (viiii) 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 (Sequence ID: 23), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. Heavy chain variable region including (ix) and below: CDR-H1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSSERFTS (Sequence ID: 29), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITNGGST (Sequence ID: 30), and CDR-H3 containing MAGTS (Sequence ID: 31), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and Heavy chain variable region including (x) below: CDR-H1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (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 TNGRWSGDTYYAHL (Sequence ID: 33).

3. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant or binding fragment that binds to EpCAM 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 the sequence GGTFSSYA (Sequence ID: 1), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-H2 containing the sequence IIPIFGTA (Sequence ID: 2), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3), or a sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and / or Light chain variable region including the following: CDR-L1 containing the sequence QSLLHSNGYNY (Sequence ID: 9), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-L2 containing the sequence LGS (Sequence ID: 10), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 containing MQALQTPYT (Sequence ID: 11), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. (ii) Heavy chain variable region including the following: CDR-H1 containing the sequence GGTFSSYA (Sequence ID: 1), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-H2 containing the sequence IIPIFGTA (Sequence ID: 2), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3), or a sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and / or Light chain variable region including the following: CDR-L1 containing the sequence QSLLHSNGYNY (Sequence ID: 9), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-L2 containing the sequence LGS (Sequence ID: 10), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 containing MQGLQSPWT (Sequence ID: 15), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. (iii) Heavy chain variable region including the following: CDR-H1 containing the sequence GGTFSSYA (Sequence ID: 1), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-H2 containing the sequence IIPIFGTA (Sequence ID: 2), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3), or a sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and / or Light chain variable region including the following: CDR-L1 containing QSLLHSNRYNY (Sequence ID: 17), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-L2 containing the sequence LGS (Sequence ID: 10), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 containing MQALQTPYT (Sequence ID: 11), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. Heavy chain variable region including (iv) and below: CDR-H1 containing the sequence GGTFSSYA (Sequence ID: 1), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-H2 containing the sequence IIPIFGTA (Sequence ID: 2), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 containing ARSLGGRFRY (SEQ ID NO: 3), or a sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and / or Light chain variable region including the following: CDR-L1 containing the sequence QSLLHSNGYNY (Sequence ID: 9), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-L2 containing the sequence LGS (Sequence ID: 10), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 containing MQGLQTPYT (Sequence ID: 23), or a sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and (v) Heavy chain variable region including the following: CDR-H1 containing the sequence GDSISSNSVA (Sequence ID: 5), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-H2 containing the sequence TYYRSKWYS (Sequence ID: 6), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 containing the sequence AREVEGSSYDAFDI (SEQ ID NO: 7), or a sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and / or Light chain variable region including the following: QSISDF (Sequence ID: 19), or CDR-L1 containing the same sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. AAS (Sequence ID: 20), or CDR-L2 containing the same sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 containing QQSYIMPDT (Sequence ID: 21), or a sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

4. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a heavy chain variable region including: 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, - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSSERFTS (Sequence ID: 29), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2 includes the following: - ITSGST (Sequence ID: 26), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, - 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: - TNGRWSGDTYYAHH (Sequence ID: 27), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. - MAGTS (Sequence ID: 31-1B8), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, - TNGRWSGDTYYAHL (Sequence ID: 33), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to it.

5. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds to EpCAM 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 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (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), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. (ii) Heavy chain variable region including the following: CDR-H1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSSERFTS (Sequence ID: 29), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITNGGST (Sequence ID: 30), and CDR-H3 containing MAGTS (Sequence ID: 31), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and (iii) Heavy chain variable region including the following: CDR-H1 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (Sequence ID: 25), CDR-H2 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (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.

6. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant or binding fragment that binds to EpCAM comprises a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of: (i) A heavy chain variable domain comprising QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (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. (ii) A heavy chain variable domain comprising QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (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. (iii)EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID: 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 has two or three amino acid substitutions, comprising a heavy chain variable domain (iv) QVQLQQSGPGLVKPSQTLSLTCCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (Sequence ID: 8), or a heavy chain variable domain comprising 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 domain comprising a fragment, variant or sequence thereof having two or three amino acid substitutions, or being identical to (vvvmtqsplslpvtpgepasiscrssqsllhsngynyldwylqkpgqspqllliyligsnrasgvpdrfsgsgsgsgtdftlkiisrveaedvvvyycmqalqtpytytfgqgtk (Sequence ID: 12), or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. (vi) Light chain variable domain comprising EIVLTQSPLSLPVTPPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (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. (vii) Light chain variable domain comprising (vii) DVVMTQSPLSLPVTPPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLLIYLGSNRASGVPDRFSGSGSGTDFTTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (Sequence ID: 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. (viiii)DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAAPKLLIYAASSLQTGVPSRFGGGSGSGTEFFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (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 has two or three amino acid substitutions, comprising a light chain variable domain (ix) DVVMTQSPLSLPVTPPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (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 has two or three amino acid substitutions, comprising a light chain variable domain. (x) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (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 has two or three amino acid substitutions, comprising a heavy chain variable domain (xi)QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (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, comprising a heavy chain variable domain (xi)QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAAHLWGQGTQ (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 has two or three amino acid substitutions, comprising a heavy chain variable domain. (xiiii)QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (Sequence ID: 36), or a heavy chain variable domain comprising 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.

7. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant or binding fragment that binds to EpCAM comprises a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of: (i) EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID: 4), or a heavy chain variable domain comprising 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 / or Light chain variable domains comprising DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLLIYLGSNRASGVPDRFSGSGSGTDFTTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (Sequence ID: 12), or fragments, variants, or sequences thereof that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (ii) EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID: 4), or a heavy chain variable domain comprising 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 / or Light chain variable domains comprising EIVLTQSPLSLPPVTPPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLLIYLGSNRASGVPDRFSGSGSGTDFFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (Sequence ID: 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)EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID: 4), or a heavy chain variable domain comprising a fragment, variant or sequence having two or three amino acid substitutions, which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions therein, and / or Light chain variable domains comprising DVVMTQSPLSLPVTPPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (Sequence ID: 18), or fragments, variants, or sequences thereof that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. (iv) QVQLQQSGPGLVKPSQTLSLLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), or heavy chain variable domains comprising fragments, variants or sequences having two or three amino acid substitutions, and / or being at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAAPKLLIYAASSLQTGVPSRFGGGSGSGTEFLTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or a light chain variable domain comprising 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 (v) EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID: 4), or heavy chain variable domains comprising fragments, variants or sequences thereof that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions, and / or Light chain variable domain DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (Sequence ID: 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.

8. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a single-domain heavy chain variable domain having the following sequence: (i) QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (Sequence ID: 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 has two or three amino acid substitutions, or (ii) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (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 has two or three amino acid substitutions, or (iii) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (Sequence ID: 28), or any 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, or (iv) QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (Sequence ID: 32), or any 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, or (v) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (Sequence ID: 34), or any 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) QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (Sequence ID: 36), 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. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a heavy chain variable domain encoded by a nucleotide sequence including: (i) Heavy chain variable region including the following: CDR-H1 containing GGAAGCATCTTCAGTGGCAATATGAC (Sequence ID: 62), CDR-H2 containing ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATAC (Sequence ID: 64) (ii) Heavy chain variable region including the following: CDR-H1 containing GGAAGTCCCGAAAAGATTCACATCA (Sequence ID: 66), CDR-H2 containing ATTACTAATGGGGTAGCCACA (SEQ ID NO: 67), and CDR-H3 containing ATGGCGGGTAACGTCC (Sequence ID: 68); and (iii) Heavy chain variable region including the following: CDR-H1 containing GGAAGCATCTTCAGTGGCAATATGAC (Sequence ID: 62), CDR-H2 containing ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATTCC (Sequence ID: 70) Or fragments, variants, or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to them.

10. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a heavy chain variable domain encoded by a nucleotide sequence including: (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGG TACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATG CCCAGAAGACCGTATATCTGCCAAAACGAACGAACCTGGAAAACCGCCGTGTATATACTGCCAAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGCCCAGGGGACCCAG (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 10-20 nucleic acid substitutions, or (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTG GTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCaAGAACACCcTATATCTGCAAATGAACAGCCCTGAGAGCCTGAGGACACGGCCGTTATTACTGCCAAAACGGGAAAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGCCCAGGGGACCCTG (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 10-20 nucleic acid substitutions, (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAG CTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCG CAGACACCTCCATAAGCAACAGCCTACAATGGAGCTGAGCCAGCCTGAGAATCTGAGCACAGGCCCGTGTATATACTGTGCCGAGATCGTTGGGGGGAGATTTCGCTACTGGGGGCCAGGGCAGACCTG (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 10-20 nucleic acid substitutions, or (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAA CTGGATCAGGCAGTCCCCATCGAGAGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCCAACCCA GACACATCCCAAGAACCAGTTCTCCCCTGCCAGCTGAACTCTGTGACTCCCCGAGGACACGGCTGTGTTATTATTTGTGCCAAGAGAAGTTGAGGGCCAAGCAAGCTATGAATGCCTTTTTGAATCTGGGGGCCAAGGGAATG (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 10-20 nucleic acid substitutions, or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGG TACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATG CCCAGAAGACCGTATATCTGCAAACGAACGAACTTTGAAAACCTGACACGGCCGTGTATATGTGCACAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGCCCAGGGGACCACCAG (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 10-20 nucleic acid substitutions, or (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGT GGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCAT CTCCAGAGAACACGCCAAGAACACGGGTGTATCTGCAAATGAACAGCCCTGAAAAAGCTGAGGCCCGTCTATTATTTGTTATTGGCGGTACGTCCCTGGGGCCCAGGGGACCCAG (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 10-20 nucleic acid substitutions, or (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTG GTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAT GCCCAGAAGACCGTATATCTGCAAACGAACGAACCTGAAACCTGAGGACACGGCCGTGTATATACTGCACAAACGGGAAGATGGTCCAGGGCGAATACTATACTATGCCCCATCTCTGGGGGCCAGGGGACCACAG (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 10-20 nucleic acid substitutions, or (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTG GTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGC CCAGAAAGACCGTATATCTGCAAACGAACGAACCTGAAACACGGCCCGGTTATTACTGCCAACAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGCCCAGGGGACCCAG (Sequence ID: 73), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or sequences having 10-20 nucleic acid substitutions, and / or The aforementioned light chain variable domain is encoded by a nucleotide sequence including: (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGAT ACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAG TGGATCAGGCACAGATTTTTACACTGAAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGGTTTTATTATTACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGGCCAGGGGACCAAG (Sequence ID: 49), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10-20 nucleic acid substitutions, or (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGA TACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAG TGGATCAGGTACAGATTTTTACACTGAAAAATAGCAGAGTGGAGCTGAGGATGTGGGGTTTTATTATTAGCATGCCAAGGTTCTCTACAAAAGTTCCCCTGGACGTTCCGGCCAAGGGACCAAG (Sequence ID: 53), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10-20 nucleic acid substitutions, or (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAG ATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGCACAGATTTTTACACTGAAAAATCAGCAGAGGCCTGAGGATGTGGGGGTTTTTATACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGCCCAGGGGACCAAG (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 10-20 nucleic acid substitutions, or (iv) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTT AAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCT GGGACAGAATTCCAACTTCTCCAACCATAAGCAGTCTAACACACTGAAGATTTGGGAACTTATTTACTGTCAACAGAGTTTAACATTTAGCCCCGAACACTTTTTGGCCCAGGGGGAAAA (Sequence ID: 59), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has 10-20 nucleic acid substitutions, or (v) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGA TACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGCAACAGATTTTTACACTGCCAAAATCAGCCAGAGTGGGAGGCTGAGGATGCCTGGGGGTTTTTATACTGCATGCCAAGGTTCTACAGAACTCCCGTAACTTTTGGCCCAAGGGGGACCAAG (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 10-20 nucleic acid substitutions.

11. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a heavy chain and / or light chain variable domain encoded by a nucleotide sequence selected from the group consisting of: (i) Heavy chain variable domains encoded by nucleotide sequences including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGG TGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGAC ACCTCCATAAGCAACAGCCTACACATGGAGCTGAGCCAGCCTGAGAGTCTGAGGACACGGCCCGGTGTATATACTGTGCGAGAATCGTTGGGGGGGAGAATTTTCGCCTACTGGGGGCCAGGGGAACCCTG (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 10-20 nucleic acid substitutions, and / or Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAG TGGATCAGGCACAGATTTTTACACTGAAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGGTTTTATTATTACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGGCCAGGGGACCAAG (Sequence ID: 49), or a sequence having 10-20 nucleic acid substitutions that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. (ii) Heavy chain variable domains encoded by nucleotide sequences including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGG TGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGAC ACCTCCATAAGCAACAGCCTACACATGGAGCTGAGCCAGCCTGAGAGTCTGAGGACACGGCCCGGTGTATATACTGTGCGAGAATCGTTGGGGGGGAGAATTTTCGCCTACTGGGGGCCAGGGGAACCCTG (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 10-20 nucleic acid substitutions, and / or Light chain variable domain encoded by the following nucleotide sequence: GAAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAG TGGATCAGGTACAGATTTTTACACTGAAAAATAAGCAGAGTGGAGCTGAGGATGTGGGGTTTTATTATTAGCATGCCAAGGTTCTCTACAAAAGTTCCCCTGGAGACGTTCCGGCCAAGGGACCCAAG (Sequence ID: 53), or a sequence having 10-20 nucleic acid substitutions that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. (iii) Heavy chain variable domains encoded by nucleotide sequences including the following: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGG TGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGAC ACCTCCATAAGCAACAGCCTACACATGGAGCTGAGCCAGCCTGAGAGTCTGAGGACACGGCCCGGTGTATATACTGTGCGAGAATCGTTGGGGGGGAGAATTTTCGCCTACTGGGGGCCAGGGGAACCCTG (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 10-20 nucleic acid substitutions, and / or Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAG TGGATCAGGCACAGATTTTTACACTGAAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGGTTTTATTATTACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGGCCAGGGGACCAAG (Sequence ID: 55), or a sequence having 10-20 nucleic acid substitutions that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. Heavy chain variable domains encoded by nucleotide sequences including (iv) and below: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGG TGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGAC ACCTCCATAAGCAACAGCCTACACATGGAGCTGAGCCAGCCTGAGAGTCTGAGGACACGGCCCGGTGTATATACTGTGCGAGAATCGTTGGGGGGGAGAATTTTCGCCTACTGGGGGCCAGGGGAACCCTG (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 10-20 nucleic acid substitutions, and / or Light chain variable domain encoded by the following nucleotide sequence: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATAC AACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTG GATCAGGCACAGATTTTTACACTGCCAAAATCAGCCAGAGTGGAGCTGAGGATGCCTGGGGGTTTATATACTGCATGCCAAGGTTCTACAGAACTCCGTTAACACTTTTTGGGCCAAGGGGCACCAAG (Sequence ID: 61), or sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10-20 nucleic acid substitutions; and (v) Heavy chain variable domains encoded by nucleotide sequences including the following: CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGA TCAGGCAGTCCCCATCGAGAGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACAC ATCCAAGAACCAGTTCTCCCCTGCAGCTGAACTCTGTGAACTCCCCGAGGACACGGCTGTGTTATTATTTGTGCCAAG Light chain variable domain encoded by the following nucleotide sequence: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAA TTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTG GGACAGAAATTCCAACTTCTCCAACCATAAGCAGTCTAACACCTGAAGATTTGGGAACTTATTTACTGTCAACAGAGTTTAACATTTAGCCCCGAACACTTTTTGGCCCAGGGGGAAAA (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 10-20 nucleic acid substitutions.

12. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a heavy chain variable domain encoded by a nucleotide sequence including: (i) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGG TACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATT CCaAGAAAcACCcTATATCTGCAAATGAACAGCCCTGAGAGCCTGAGGACACGGCCGTGTATATTAACTGCCAACAAACGGGAAAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGCCCAGGGGACCCTG (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 10-20 nucleic acid substitutions; or (ii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTG GTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAT GCCCAGAAGACCGTATATCTGCAAACGAACGAACCTGAAACCTGAGGACACGGCCGTGTATATACTGCACAAACGGGAAGATGGTCCAGGGCGAATACTATACTATGCCCCATCAACTGGGGGCCAGGGGACCACAG (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 10-20 nucleic acid substitutions, or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTG GTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAT GCCCAGAAGACCGTATATCTGCAAACGAACGAACTTTGAAAACCTGAGCCCGGTTATTACTGCACAAACGGGAAGAGAGAGATAGGCGAATACTATACTATGCCCCATCAACTGGGGGCCAGGGGACCACAG (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 10-20 nucleic acid substitutions, or (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGT GGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCAT CTCCAGAGAACACGCCAAGAACACGGGTGTATCTGCAAATGAACAGCCCTGAAAAAGCTGAGGCCCGTCTATTATTTGTTATTGGCGGTACGTCCCTGGGGCCCAGGGGACCCAG (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 10-20 nucleic acid substitutions, or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGG TACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATG CCCAGAAGACCGTATATCTGCCAAAACGAACGAACCTGGAAAACCTGAGGCCCGGTATATACTGCCAAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCTCTGGGGCCCAGGGGACCCAG (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 10-20 nucleic acid substitutions, or (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCC TGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGA CAATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACCTGAAACAGGCCCGTGTATATACTGCCAAAACGGGAAGATGGGCCAAGAGACCCAG (Sequence ID: 73), or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having 10-20 nucleic acid substitutions GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAG CTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCG CAGACACCTCCATAAGCAACAGCCTACAATGGAGCTGAGCCAGCCTGAGAATCTGAGCACAGGCCCGTGTATATACTGTGCCGAGATCGTTGGGGGGAGATTTCGCTACTGGGGGCCAGGGCAGACCTG (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 10-20 nucleic acid substitutions, or (vii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGA ACTGGATCAGGCAGTCCCCATCGAGAGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCCAAACC CAGAACACATCCCAAGAACCACAGTTTCCCCTGCCAGCTGAACTCTGTGAACTCCCCGAGGACACGGCTGTGTTATTATTTGTGCCAAGAGAAGTTGAGGGCCAGCCAAGCTATGAATGCCTTTTTGAATCTGGGGGCCAAGGGAACAATG (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 10-20 nucleic acid substitutions.

13. A cell according to any one of the preceding claims, wherein the multispecific antigen-binding protein, a variant thereof, or a fragment thereof is a bispecific antibody.

14. The cell according to any one of the preceding claims, wherein the multispecific antigen-binding protein, its variant or fragment is an immune cell engager selected from the group including T cell engagers, NK cell engagers, monocyte engagers and macrophage engagers.

15. The cell according to any one of the preceding claims, wherein the multispecific antigen-binding protein, its variant or fragment is a bispecific T cell engager (BiTE), such as an inducible BiTE, a non-inducible BiTE, or a constitutively expressed BiTE.

16. The cell according to any one of the preceding claims, wherein the second antigen-binding protein, a variant thereof, or a binding fragment binds to an immunomarker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

17. The cell according to any one of the preceding claims, wherein the multispecific antigen-binding protein is an inducible bispecific T cell engager comprising a heavy-chain antibody variable region (i.e., VHH) or a single-chain variable fragment (scFv).

18. The cell according to any one of the preceding claims, wherein the cell is an immune cell selected from the group consisting of, for example, T cells, macrophages, monocytes, and NK cells.

19. The cell according to any one of the preceding claims, wherein the cell is a T cell, optionally a CAR T cell.

20. The cells according to any one of the preceding claims, wherein the cells bind to HER2 and secrete an inducible immune cell engager that targets EpCAM and CD3 (HECAR-BiTE T).

21. A polynucleotide encoding a cell according to any one of the preceding claims.

22. A vector expressing the polynucleotide of claim 21.

23. A host cell containing the vector of claim 22.

24. A method for producing / generating a cell according to any one of claims 1 to 20, comprising introducing the polynucleotide of claim 21 into a cell.

25. A composition comprising the cells according to any one of claims 1 to 20.

26. A method for treating a disease in a subject requiring such treatment, the method comprising administering to the subject cells of any one of claims 1 to 20 or the composition of claim 25, wherein the disease is optionally a proliferative disorder and optionally cancer.

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