HER2 chimeric antigen receptor secretion

Abstract

Modified cells are provided that express (a) a chimeric antigen receptor targeting HER2, and (b) a multispecific antigen binding protein, variant or binding fragment thereof that binds to one or more targets, the present invention relates to antigen binding proteins comprising a first antigen binding protein, a variant or a binding fragment thereof, that binds to EpCAM (epithelial cell adhesion molecule) and a second antigen binding protein, a variant or a binding fragment thereof, that binds to an immune cell marker, wherein a first antigen binding protein, a variant or a binding fragment thereof binding to EpCAM comprises a heavy chain variable region and / or a light chain variable region comprising a sequence as disclosed herein. Also disclosed are methods of producing / producing the cells as disclosed herein and methods of treatment employing the cells as disclosed herein.

Description

Technical Field

[0001] This disclosure broadly relates to antigen-binding proteins, chimeric antigen receptors, and modified cells that are specific to epithelial cell adhesion molecule (EpCAM). Specifically, this disclosure relates to cells expressing chimeric antigen receptors that target tumor antigens and multispecific antigen-binding proteins, variants, or binding fragments that bind to one or more targets, said multispecific antigen-binding proteins, variants, or binding fragments comprising a first antigen-binding protein, variant, or binding fragment that binds to EpCAM (epithelial cell adhesion molecule) and a second antigen-binding protein, variant, or binding fragment that binds to cellular markers.

[0002] background

[0003] The global cancer incidence rate is projected to rise to 28.4 million by 2040. Of all adult cancer cases, approximately 90% are solid tumors. Solid tumors, such as liver cancer, lung cancer, stomach cancer, and breast cancer in women, are leading causes of cancer-related deaths. Due to the abundant presence of epithelial 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. Breast cancer in women is another example, where ductal carcinoma and lobular carcinoma originating from epithelial tissue are the most common types of primary breast cancer, accounting for a total of 90% of all breast cancer cases. Other examples include tumors found in the skin, ovaries (>90%), kidneys (>85%), lungs (>85%), liver (approximately 85%), pancreas (>90%), head and neck (>90%), and prostate (>95%), most of which are carcinomas. Although the chances of curing carcinoma in situ are high, even with a variety of treatment options available, such as surgery, chemotherapy, radiation therapy, and targeted therapy, the five-year survival rate for most invasive or metastatic cancers is only about 20-30%.

[0004] Compared to conventional treatment strategies, antibody- and cell-based therapies are gaining increasing attention and investment as potentially more promising standards of cancer care. Cell-based therapies such as chimeric antigen receptor (CAR) T cells have achieved great success in hematologic malignancies but have shown limited efficacy in treating solid tumors. Among the complex challenges encountered by each approach, an inherent problem lies in the heterogeneous distribution of tumor markers within the solid tumor cell population 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.

[0005] Immune escape from target antigen-negative cells has become a major mechanism of cancer recurrence. Therefore, the unsatisfactory efficacy of CAR-T cells targeting a single antigen is attributed to resistance to CAR T-cell therapy. Given the diverse repertoire of surface antigens expressed by tumor cells, a potential approach to overcome this challenge is to engineer CAR T cells with dual or multiple targets to combat tumor escape, as exemplified by the promising early treatment of relapsed or refractory B-cell lymphomas using bispecific anti-CD19 / CD20 CAR T-cell therapy. However, identifying 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, so the high potency of CARs may induce intolerable on-target off-tumor toxicity. Therefore, alternative methods for preventing cancer recurrence are needed.

[0006] There is a need to provide alternative cell-based therapies. There is an urgent need to provide alternative chimeric antigen receptor cells. Summary of the Invention

[0007] EpCAM is a well-known tumor-associated antigen that is frequently overexpressed in almost all epithelial solid tumors. However, because EpCAM is also widely expressed (albeit at low levels) in normal epithelial cells, both EpCAM-targeting CAR-T cells and systemically administered BiTE have failed due to their high systemic toxicity. Localizing anti-EpCAM BiTE to the tumor site or its vicinity via secretion by CAR-T cells not only reduces tumor escape but also significantly improves safety.

[0008] This disclosure demonstrates the superior efficacy of HER2-targeting CAR T cells secreting anti-EpCAM BiTE (named "HE CAR-BiTET") in killing human tumors originating from gastric cancer in vitro. In summary, the inventors' method utilizes 1) tumor-directed T cell expansion and 2) tumor-restricted anti-EpCAM BiTE secretion, the latter of which cannot be achieved through conventional systemic therapies. This disclosure also provides the possibility of extending anti-EpCAM BiTE-secreting CAR T cell therapy to other cancer types to establish a broader range of next-generation CAR T cell therapies.

[0009] On the one hand, it provides modified cells that express...

[0010] (a) Chimeric antigen receptors targeting HER2, and

[0011] (b) A multispecific antigen-binding protein, its variants, or binding fragments that bind to one or more targets, comprising a first antigen-binding protein, its variants, or binding fragments that bind to EpCAM (epithelial cell adhesion molecule) and a second antigen-binding protein, its variants, or binding fragments that bind to immune cell markers.

[0012] The first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy chain variable regions and / or light chain variable regions selected from the following groups:

[0013] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5, 1B8, 2B7 and 2D10)

[0014] CDR-H1 includes:

[0015] GSIFSGND (SEQ ID NO:25 - 2C4, hu2C4, 1A5, 2B7 and 2D10) or

[0016] GSSERFTS (SEQ ID NO:29 – 1B8)

[0017] CDR-H2 contains:

[0018] ITSGGST (SEQ ID NO:26–2C4, hu2C4, 1A5, 2B7 and 2D10) or

[0019] ITNGGST (SEQ ID NO:30 – 1B8) and

[0020] CDR-H3 contains:

[0021] TNGRWSGDTYYAHH (SEQ ID NO:27–2C4, hu2C4, 1A5, 2B7 and 2D10)

[0022] MAGTS (SEQ ID NO:31 – 1B8) or

[0023] TNGRWSGDTYYAHL (SEQ ID NO:33 – 2B7),

[0024] (ii) Heavy chain variable regions, which include: (1B6, 1C1, 1C11, 1D4, and 1H6)

[0025] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0026] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0027] CDR-H3 contains ARSLGGRFRY (SEQ ID NO:3)

[0028] (iii) Heavy chain variable region, which contains: (1E4)

[0029] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5)

[0030] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0031] CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO:7)

[0032] (iv) Light chain variable regions, comprising: (1B6, 1C1, 1C11, 1D4, 1E4 and 1H6)

[0033] CDR-L1 includes:

[0034] QSLLHSNGYNY (SEQ ID NO:9 - 1B6, 1C1, 1C11 and 1H6)

[0035] QSLLHSNRYNY (SEQ ID NO:17 - 1D4) or

[0036] QSISDF (SEQ ID NO:19 – 1E4)

[0037] CDR-L2 includes:

[0038] LGS (SEQ ID NO:10 - 1B6, 1C1, 1C11, 1D4 and 1H6) or

[0039] AAS (SEQ ID NO:20 - 1E4), and

[0040] CDR-L3 includes:

[0041] MQALQTPYT (SEQ ID NO:11 - 1B6, 1C1 and 1D4)

[0042] MQGLQSPWT (SEQ ID NO:15 - 1C11),

[0043] QQSYIMPDT (SEQ ID NO:21 – 1E4) or

[0044] MQGLQTPYT (SEQ ID NO:23 – 1H6); and

[0045] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0046] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain heavy chain variable regions, including: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10).

[0047] CDR-H1 includes:

[0048] GSIFSGND (SEQ ID NO:25 - 2C4, hu2C4, 1A5, 2B7 and 2D10) or

[0049] GSSERFTS (SEQ ID NO:29 – 1B8)

[0050] CDR-H2 contains:

[0051] ITSGGST (SEQ ID NO:26–2C4, hu2C4, 1A5, 2B7 and 2D10) or

[0052] ITNGGST (SEQ ID NO:30 – 1B8); and

[0053] CDR-H3 contains:

[0054] TNGRWSGDTYYAHH (SEQ ID NO:27–2C4, hu2C4, 1A5, 2B7 and 2D10)

[0055] MAGTS (SEQ ID NO:31 – 1B8) or

[0056] TNGRWSGDTYYAHL (SEQ ID NO:33 – 2B7)

[0057] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0058] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or the binding fragment contains a heavy chain variable region selected from the group consisting of:

[0059] (i) Heavy chain variable regions, which include: (2C4-VHH, hu2C4-VHH, 1A5-VHH and 2D10-VHH)

[0060] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0061] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0062] CDR-H3 contains TNGRWSGDTYYAHH (SEQ ID NO:27)

[0063] (ii) Heavy chain variable region, which contains: (1B8-VHH)

[0064] CDR-H1 contains GSSERFTS (SEQ ID NO:29).

[0065] CDR-H2, which contains ITNGGST (SEQ ID NO:30), and

[0066] CDR-H3, which contains MAGTS (SEQ ID NO:31); and

[0067] (iii) Heavy chain variable region, which contains: (2B7-VHH)

[0068] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0069] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0070] CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO:33)

[0071] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0072] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy chain variable regions and / or light chain variable regions selected from the group consisting of:

[0073] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5 and 2D10)

[0074] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0075] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0076] CDR-H3 contains TNGRWSGDTYYAHH (SEQ ID NO:27)

[0077] (ii) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1H6)

[0078] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0079] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0080] CDR-H3 contains ARSLGGRFRY (SEQ ID NO:3)

[0081] (iii) Heavy chain variable region, which contains: (1E4)

[0082] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5).

[0083] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0084] CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO:7)

[0085] (iv) Light chain variable region, which includes: (1B6 and 1C1)

[0086] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0087] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0088] CDR-L3 contains MQALQTPYT (SEQ ID NO:11)

[0089] (v) Light chain variable region, containing: (1C11)

[0090] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0091] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0092] CDR-L3 contains MQGLQSPWT (SEQ ID NO:15)

[0093] (vi) Light chain variable region, which contains: (1D4)

[0094] CDR-L1 contains QSLLHSNRYNY (SEQ ID NO:17).

[0095] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0096] CDR-L3 contains MQALQTPYT (SEQ ID NO:11)

[0097] (vii) Light chain variable region, which includes: (1E4)

[0098] CDR-L1, which contains QSISDF (SEQ ID NO:19),

[0099] CDR-L2, which contains AAS (SEQ ID NO:20), and

[0100] CDR-L3 contains QQSYIMPDT (SEQ ID NO:21)

[0101] (viii) Light chain variable region, which contains: (1H6)

[0102] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0103] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0104] CDR-L3 contains MQGLQTPYT (SEQ ID NO:23)

[0105] (ix) Heavy chain variable region, which contains: (1B8-VHH)

[0106] CDR-H1 contains GSSERFTS (SEQ ID NO:29).

[0107] CDR-H2, which contains ITNGGST (SEQ ID NO:30), and

[0108] CDR-H3, which contains MAGTS (SEQ ID NO:31); and

[0109] (x) Heavy chain variable region, which contains: (2B7-VHH)

[0110] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0111] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0112] CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO:33)

[0113] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0114] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of:

[0115] (i) Heavy-chain variable structural domains, which contain

[0116] QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO:37- hu2C4-VHH)

[0117] (ii) Heavy-chain variable structural domains, which contain

[0118] QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:35 - 2C4-VHH)

[0119] (iii) Heavy-chain variable structural domains, which contain

[0120] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4 - 1B6, 1C1, 1C11, 1D4 and 1H6)

[0121] (iv) Heavy-chain variable structural domains, which contain

[0122] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO:8 - 1E4),

[0123] (v) Light chain variable structural domain, which contains

[0124] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:12 - 1B6 and 1C1)

[0125] (vi) Light chain variable structural domain, which contains

[0126] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO:16 - 1C11)

[0127] (vii) Light chain variable structural domain, which contains

[0128] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:18 - 1D4)

[0129] (viii) Light chain variable structural domain, which contains

[0130] DIQLTQSPSSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO:22 - 1E4)

[0131] (ix) Light chain variable structural domain, which contains

[0132] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO:24- 1H6)

[0133] (x) Heavy-chain variable structural domain, which contains

[0134] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:28- 1A5-VHH)

[0135] (xi) Heavy-chain variable structural domain, which contains

[0136] QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO:32 - 1B8-VHH)

[0137] (xii) Heavy-chain variable structural domain, which contains

[0138] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO:34 - 2B7-VHH)

[0139] (xiii) Heavy-chain variable structural domains, which contain

[0140] and

[0141] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0142] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain a single-domain heavy-chain variable domain having the following sequence:

[0143] (i) QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:35 - clone 2C4-VHH), or

[0144] (ii) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO:37 - hu2C4-VHH), or

[0145] Or

[0146] (iv) QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO:32 - clone 1B8-VHH), or

[0147] Or

[0148] (vi)QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:36 - clone 2D10-VHH),

[0149] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0150] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or the binding fragment contains a heavy chain variable region selected from the group consisting of:

[0151] (i) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4 and 1H6)

[0152] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0153] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0154] CDR-H3 contains ARSLGGRFRY (SEQ ID NO:3).

[0155] (ii) Heavy chain variable region, which contains: (1E4)

[0156] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5)

[0157] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0158] CDR-H3, which contains AREVEGSSYDAFDI (SEQ ID NO:7); and

[0159] It contains a light chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6).

[0160] CDR-L1 includes:

[0161] QSLLHSNGYNY (SEQ ID NO:9 - 1B6, 1C1, 1C11 and 1H6),

[0162] QSLLHSNRYNY (SEQ ID NO:17 - 1D4), or

[0163] QSISDF (SEQ ID NO:19 – 1E4),

[0164] CDR-L2 includes:

[0165] LGS (SEQ ID NO:10 - 1B6, 1C1, 1C11, 1D4 and 1H6) or

[0166] AAS (SEQ ID NO:20 - 1E4), and

[0167] CDR-L3 includes:

[0168] MQALQTPYT (SEQ ID NO:11 - 1B6, 1C1 and 1D4),

[0169] MQGLQSPWT (SEQ ID NO:15 - 1C11),

[0170] QQSYIMPDT (SEQ ID NO:21 – 1E4), or

[0171] MQGLQTPYT (SEQ ID NO:23 – 1H6),

[0172] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0173] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy chain variable regions and light chain variable regions selected from the group consisting of:

[0174] (i) Heavy chain variable region, which contains: (1B6 and 1C1)

[0175] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0176] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0177] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and

[0178] The light chain variable region, which includes:

[0179] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0180] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0181] CDR-L3 contains MQALQTPYT (SEQ ID NO:11);

[0182] (ii) Heavy chain variable region, which contains: (1C11)

[0183] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0184] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0185] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and

[0186] The light chain variable region, which includes:

[0187] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0188] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0189] CDR-L3 contains MQGLQSPWT (SEQ ID NO:15);

[0190] (iii) Heavy chain variable region, which contains: (1D4)

[0191] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0192] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0193] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and

[0194] The light chain variable region, which includes:

[0195] CDR-L1 contains QSLLHSNRYNY (SEQ ID NO:17).

[0196] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0197] CDR-L3 contains MQALQTPYT (SEQ ID NO:11);

[0198] (iv) Heavy chain variable region, which contains: (1H6)

[0199] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0200] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0201] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and

[0202] The light chain variable region, which includes:

[0203] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0204] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0205] CDR-L3, which contains MQGLQTPYT (SEQ ID NO:23); and

[0206] (v) Heavy chain variable region, which contains: (1E4)

[0207] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5).

[0208] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0209] CDR-H3, which contains AREVEGSSYDAFDI (SEQ ID NO:7); and

[0210] The light chain variable region contains: (1E4)

[0211] CDR-L1, which contains QSISDF (SEQ ID NO:19),

[0212] CDR-L2, which contains AAS (SEQ ID NO:20), and

[0213] CDR-L3 contains QQSYIMPDT (SEQ ID NO:21)

[0214] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0215] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain a heavy chain variable domain and a light chain variable domain selected from the group consisting of:

[0216] (i) Heavy chain variable structural domain, which contains: (1B6 and 1C1)

[0217] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and

[0218] Light chain variable structural domain, which includes:

[0219] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:12);

[0220] (ii) Heavy chain variable structural domain, which contains: (1C11)

[0221] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and

[0222] Light chain variable structural domain, which includes:

[0223] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO:16);

[0224] (iii) Heavy chain variable structural domain, which contains: (1D4)

[0225] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and

[0226] Light chain variable structural domain, which includes:

[0227] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:18);

[0228] (iv) Heavy chain variable structural domain, which contains: (1E4)

[0229] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO:8), and

[0230] Light chain variable structural domain, which includes:

[0231] OR

[0232] (v) Heavy chain variable structural domain, which contains: (1H6)

[0233] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and

[0234] Light chain variable structural domain, which includes:

[0235] OR

[0236] It has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with and / or has 2 or 3 amino acid substitutions.

[0237] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain a light chain constant domain having the following sequence:

[0238] (i)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:13 - clone 1B6-light chain constant domain), or

[0239] (ii)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (SEQ ID NO:14 clone 1C1 - light chain constant domain),

[0240] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0241] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0242] (i)CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:72 - clone 2C4), or

[0243] (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO:74 - Clone hu2C4-VHH); or

[0244] (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41 - For clones 1B6, 1C1, 1C11, 1D4, 1H6), or

[0245] (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45 - Clone 1E4); or

[0246] (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:65 - Clone 1A5), or

[0247] (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO:69 - Clone 1B8), or

[0248] (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO:71 - Clone 2B7), or

[0249] (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:73 - Clone 2D10), and / or

[0250] The light chain variable domain is encoded by the following nucleotide sequence, which comprises:

[0251] (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:49 - Clone 1B6 and 1C1), or

[0252] (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO:53 - Clone 1C11), or

[0253] (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:55 - Clone 1D4), or

[0254] (iv)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGC ATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO:59 - clone 1E4), or

[0255] (v)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCT ATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:61 - clone 1H6),

[0256] Or a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or has 10-20 nucleic acid substitutions.

[0257] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy and light chain variable domains encoded by nucleotide sequences selected from the group consisting of:

[0258] (i) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1B6 and 1C1)

[0259] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and

[0260] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0261] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:49);

[0262] (ii) a heavy chain variable domain encoded by a nucleotide sequence comprising: (1C11)

[0263] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and

[0264] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0265] GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO:53)

[0266] (iii) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1D4)

[0267] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and

[0268] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0269] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:55)

[0270] (iv) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1H6)

[0271] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and

[0272] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0273] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:61); and

[0274] (v) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1E4)

[0275] CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGT ACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45), and

[0276] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0277] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCAT CGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO:59);

[0278] Or a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or has 10-20 nucleic acid substitutions.

[0279] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0280] (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCAGGGACCCAG (SEQ ID NO:72) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0281] (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO:74) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions; or

[0282] (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:65) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0283] (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGACCCAG (SEQ ID NO:69) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0284] (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGACCCAG (SEQ ID NO:71) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0285] (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTAC ACACTATGCAGACTCCGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:73) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with and / or having 10-20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGAGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0286] (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions.

[0287] In some instances, the multispecific antigen-binding protein, its variants, or fragments are bispecific antibodies.

[0288] In some embodiments, the multispecific antigen-binding protein, its variants, or fragments are immune cell adjuvants selected from the group consisting of T-cell adjuvants, NK-cell adjuvants, monocyte adjuvants, and macrophage adjuvants.

[0289] In some instances, the multispecific antigen-binding protein, its variants, or fragments are bispecific T-cell adaptors (BiTEs), such as inducible BiTEs, non-inducible BiTEs, or constitutively expressed BiTEs.

[0290] In some instances, the second antigen-binding protein of the immune cell adjuvant, its variants, or binding fragments can bind to immune markers selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0291] In some instances, the multispecific antigen-binding protein is an inducible bispecific T-cell adaptor containing a heavy chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv).

[0292] In some instances, the cells are immune cells selected from, for example, a group consisting of T cells, macrophages, monocytes, and NK cells.

[0293] In some instances, the cells are T cells, optionally CAR T cells.

[0294] In some instances, the cells bind to HER2 and secrete an inducible bispecific T cell adaptor (HECAR-BiTE T) that targets EpCAM and CD3.

[0295] On the other hand, it provides polynucleotides encoding the cells described in this article.

[0296] On the other hand, vectors for expressing the polynucleotides described herein are provided.

[0297] On the other hand, a host cell containing the vector described herein is provided.

[0298] In yet another aspect, a method for producing / generating the cells described herein is provided, which includes introducing the polynucleotides described herein into the cells.

[0299] In yet another aspect, compositions comprising the cells described herein are provided.

[0300] In yet another aspect, a method for treating a disease in a subject in need is provided, the method comprising administering to the subject the cells or compositions described herein, optionally the disease being a proliferative disease, optionally cancer.

[0301] definition

[0302] The term “antigen-binding protein” is used in the broadest sense herein to include various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies, as long as they exhibit the desired antigen-binding activity.

[0303] As used herein, the term "antibody" refers to both full-length (i.e., a component consisting of two heavy chains and two light chains) antibodies and their functionally active fragments (i.e., molecules containing an antigen-binding domain that specifically binds to an antigen, also referred to as antibody fragments or antigen-binding fragments). Unless the context otherwise requires, the characteristics of antibodies described herein also apply to antibody fragments. The term "antibody" encompasses both monovalent antibodies, which contain only one antigen-binding domain (e.g., a single-arm antibody consisting of an interconnected full-length heavy chain and a full-length light chain, also referred to as a "half-antibody"), and multivalent antibodies, which contain more than one antigen-binding domain, such as bivalent antibodies.

[0304] The term “antigen-binding fragment” as used in this article 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, biscFv, biantibody, triantibody, tetraantibody, and epitope-binding fragments of any of the above antibodies.

[0305] The term "binding fragment" as used in this article refers to a fragment that can bind to a target peptide or antigen with sufficient affinity to characterize the fragment as specific to that peptide or antigen.

[0306] The term "monoclonal antibody" (or "mAb") refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that each individual monoclonal antibody formulation is identical except for a small number of possible mutations (e.g., naturally occurring mutations). However, some differences in protein sequence related to post-translational modifications (e.g., cleavage of heavy chain C-terminal lysine residues, deamidation of asparagine residues, and / or isomerization of aspartic acid residues) may exist between the various antibody molecules present in the composition. Unlike polyclonal antibody formulations, each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0307] The term "double antibody" as used in this article refers to two Fv pairs, a first VH / VL pair and another VH / VL pair, which have two Fv intermolecular connectors 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.

[0308] The term "triantibody" (also known as Fab(scFv)2) used in this paper refers to a Fab fragment with a first scFv appended to the C-terminus of the light chain and a second scFv appended to the C-terminus of the heavy chain. The term "tetraantibody" used in this paper refers to a form similar to a biantibody containing four Fvs and four Fv intermolecular heads.

[0309] The term "multivalent antibody" refers to an antibody that contains more than one antigen-binding domain (e.g., a bivalent antibody).

[0310] The term "Fv" refers to the two variable domains of a full-length antibody, such as a co-variable domain, or a homology pair or affinity-maturated variable domain, i.e., the 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 "bi-scFv" as used herein refers to a bispecific scFv.

[0311] The term “dsscFv” or “disulfide-stable single-chain variable fragment” used in this article refers to a single-chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains and also includes the interdomain disulfide bond between the VH and VL domains.

[0312] The term "DVD-Ig" (also known as double-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.

[0313] As used herein, the term "Fab" refers to an antibody fragment comprising a light chain segment containing a VL (variable light) domain and a constant domain of a light chain (CL) and a VH (variable heavy) domain and a first constant domain (CHI) of a heavy chain. The dimer of Fab' according to this disclosure generates F(ab')2, where dimerization can, for example, occur via a hinge. The term "F(ab')" refers to a monovalent fragment of a single light chain homodimer obtained by digesting IgG with pepsin and then reducing the light chain disulfide bonds. The term "F(ab')2" as used herein refers to an IgG fragment prepared by digesting IgG with pepsin. The F(ab')2 fragment is a homodimer of two light chain dimers linked by disulfide bonds, thus retaining divalent epitope binding like intact IgG, but being smaller in size compared to intact IgG due to the lack of a heavy chain. Both F(ab')2 and the F(ab') fragment do not bind to immunoglobulin receptors on cells, which may be useful for achieving specific staining of an antibody target.

[0314] The terms "constant domain" or "constant region" used herein are used interchangeably and refer to the domain of an antibody outside the variable region. The constant domain is identical in all antibodies of the same isotype, but differs between isotypes. Typically, the constant region of the heavy chain is formed from the N to C-terminus by a CH1-hinge-CH2-CH3-optionally CH4, containing three or four constant domains.

[0315] The term “DiFab” as used in this article refers to two Fab molecules linked by the C-terminus of their heavy chains, or two Fab' molecules linked by one or more disulfide bonds in their hinge regions.

[0316] The term "antigen-binding variant" refers to a polypeptide, for example, an antibody having the desired characteristics described herein and comprising a VH and / or VL with at least about 80% amino acid sequence identity to a reference antibody's VH and / or VL. 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, the antibody variant has at least about 80% amino acid sequence identity to the antibody described herein, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. Optionally, the variant antibody will have no more than one conserved amino acid substitution compared to the antibody sequence provided herein, or no more than about 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions compared to the antibody sequence provided herein.

[0317] The term "immune cells" refers to a type of specialized cell that plays a vital role in the body's defense against infection 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 (such as basophils, eosinophils, and neutrophils), mast cells, monocytes, and macrophages.

[0318] The term “specifically” as used in the context of antibodies in this article is intended to refer only to antibodies that recognize antigens that are specific to them, or antibodies that have a significantly higher binding affinity (e.g., up to at least 5, 6, 7, 8, 9, or 10 times higher) for antigens that are specific to them compared to their binding affinity for antigens that are nonspecific to them.

[0319] In the context of antibodies, the term "epitope" or "binding site" refers to the site (or portion) on an antigen that is bound to or recognized by the complementary site of an antibody. Epitopes can be formed from consecutive amino acids (often called "linear epitopes") or from discontinuous amino acids formed through the tertiary folding of proteins (often called "conformatory epitopes"). Epitopes formed from consecutive amino acids are generally preserved upon exposure to denaturing solvents, while epitopes formed through folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least three, and more often at least five to ten, amino acids in a unique spatial conformation. Epitopes are typically composed of chemically active surface groups of a molecule, such as amino acids or sugar side chains, and usually possess specific 3D structural and charge characteristics.

[0320] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called a, d, e, g, and m.

[0321] The term "chimeric antibody" (or its antigen-binding fragment) is an antibody molecule (or its antigen-binding fragment) in which (a) a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged by a variable region with a different or altered antigen specificity. For example, mouse antibodies can be modified by replacing their constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, chimeric antibodies can maintain their antigen recognition specificity while exhibiting reduced antigenicity in humans compared to the original mouse antibody.

[0322] The term "chimeric antigen receptor" refers to receptor proteins that have been engineered to give T cells a new ability to target specific antigens. Receptors are chimeric because they combine antigen binding and T cell activation functions into a single receptor. CAR T-cell therapy uses CAR-modified T cells to treat cancer. The T cells in CAR T immunotherapy are modified to recognize cancer cells so that they can be targeted and destroyed more effectively. CAR T cells can be derived from T cells in the patient's own blood (autologous) or from T cells in another healthy donor (allogeneic). Once isolated from the body, these T cells are genetically engineered to express a specific CAR, which programs them to target antigens present on the surface of tumors. For safety reasons, CAR T cells are designed to be specific to antigens expressed on tumors but not on healthy cells. CAR T cells destroy cells by broadly stimulating cell proliferation, increasing their cytotoxicity to other living cells, and by inducing increased secretion of factors that can affect other cells, such as cytokines, interleukins, and growth factors. CAR T cells can carry two types of co-receptors, CD4 and CD8, on their surface, each with different and interacting cytotoxic effects.

[0323] As used herein, the term "human antibody" or "humanized antibody" (or its antigen-binding fragment) is intended to include antibodies (and their antigen-binding fragments) having variable regions in which both the framework region and the CDR region are derived from human sequences. Antibodies or immunoglobulins are classified into the following types based on the amino acid sequence of their heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (subtypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Therefore, when antibody molecules are intended for therapeutic use and require antibody effector function, the human IgG constant region domain, particularly the constant region domains of the IgG1 and IgG3 isotypes, can be used. Alternatively, when antibody molecules are intended for therapeutic purposes and do not require antibody effector function, the IgG2 and IgG4 isotypes can be used. Furthermore, if the antibody contains a constant region, that 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 the human body. For example, this can be achieved by retaining the non-human CDR region and replacing the remainder of the antibody with its human counterpart (i.e., the framework portion of the constant region and the variable region). Additional framework region modifications can be made in the human framework sequence as well as in the CDR sequences of lineages derived from 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 mutagenesis in vitro or somatic mutations in vivo, or conserved substitutions that promote stability or production). This definition of humanized antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using various techniques known in the art, including phage display libraries, administration of antigens to transgenic animals modified to produce such antibodies in response to antigen stimulation but whose endogenous loci have been inactivated, such as xenogeneic mice immunized via human B-cell hybridoma technology.

[0324] As used herein, the term "recombinant humanized antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant methods, such as antibodies isolated from host cells transformed to express the humanized antibody (e.g., from transfected tumors), and antibodies prepared, expressed, generated, or isolated by any other method involving splicing all or part of the human immunoglobulin gene sequence into other DNA sequences.

[0325] Throughout this specification, the term "isolated" refers to an antibody or polynucleotide (as the case may be) existing in a physical environment different from that in which it might exist in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or has been synthesized. Isolated nucleic acids may include synthetic DNA (e.g., synthetic DNA produced through chemical processing), cDNA, genomic DNA, or any combination thereof. Isolated antibody refers to an antibody that is substantially free of other cellular material and / or chemicals.

[0326] The term "complementarity-determining region" ("CDR") refers to an amino acid sequence having defined boundaries using any of a variety of known schemes, including those described by Kabat (i.e., the "Kabat" numbering scheme); Al-Lazikani ("Chothia" numbering scheme); ImMunoGenTics ("IMGT" numbering scheme); and so on. The term "complementarity-determining region" ("CDR") refers to a hypervariable region containing a binding domain that interacts with the antigen. Antibodies typically contain six CDRs: three in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3).

[0327] As used herein, the term "sequence identity" refers to the percentage of sequence identity determined by maximally aligning antibody sequences using the Kabat numbering rules. After alignment, if a region of the test antibody (e.g., a fully matured variable region of the heavy or light chain) is compared to an identical region of a reference antibody, the percentage of sequence identity between the test antibody and reference antibody regions is calculated by dividing the number of positions in both regions occupied by the same amino acid by the total number of aligned positions in both regions (excluding gaps), and then multiplying by 100 to convert it to a percentage. In some instances, the antigen-binding proteins disclosed herein may contain sequences with at least 60% identity to any of the sequences disclosed herein. For example, the antigen-binding protein may comprise any sequence disclosed herein having 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%, and at least about 97%. A sequence having at least about 98%, at least about 99%, or 100% identity (e.g., having about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, about 99%, or about 100% sequence identity with any of the sequences disclosed herein). In some instances, the antigen-binding protein comprises a sequence or amino acid region, or is encoded by a nucleotide region differing from the sequences disclosed herein by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more amino acids or nucleobases. In some instances, the antigen-binding protein comprises an amino acid sequence having one or more amino acid mutations relative to any of the sequences disclosed herein.In some instances, 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 relative to any of the sequences disclosed herein. In some instances, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0328] In some instances, the amino acid mutation is an amino acid substitution, and may include conserved and / or non-conserved substitutions.

[0329] “Conservative substitution” can be based, for example, on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the amino acid residues involved. The 20 naturally occurring amino acids can be divided 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.

[0330] As used herein, “conservative substitution” is defined as the replacement of an amino acid by another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the exchange of Glu for Asp retains a negative charge in such a modified polypeptide. Furthermore, glycine and proline can substitute for each other based on their ability to disrupt the α-helix.

[0331] As used herein, “non-conservative substitution” is defined as the replacement of an amino acid with another amino acid listed in one of the different groups of the six standard amino acid groups (1) to (6) shown above.

[0332] In some instances, substitutions may also include non-classical amino acids. Exemplary non-classical amino acids include, but are not limited to, selenocysteine, pyrrolidone, N-formylmethionine, β-alanine, GABA and δ-aminovaleric 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, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, engineered amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and general amino acid analogs.

[0333] In some instances, amino acid mutations may be present in the CDR (e.g., CDR1, CDR2, or CDR3 region) of an antigen-binding protein. In another instance, amino acid alterations may be present in the framework region (FR) (e.g., FR1, FR2, FR3, or FR4 region) of an antigen-binding protein.

[0334] In some instances, the mutation does not significantly reduce the ability of the antigen-binding protein to specifically bind to its target, and does not functionally modulate (e.g., partially or completely neutralize) the target.

[0335] The modification of the amino acid sequence can be achieved using any technique known in the art (e.g., site-directed mutagenesis or PCR-based mutagenesis).

[0336] The term "polynucleotide" refers to a linear polymer whose molecule is composed of many nucleotide units, forming part of a nucleic acid molecule. Polynucleotides are composed of long-chain nucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0337] The term "affinity" refers to the strength of all non-covalent interactions between an antibody and its target protein. Unless otherwise stated, as used herein, the term "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule for its binding partner is typically expressed as a dissociation constant (KD). Affinity can be measured using methods commonly known in the art, including those described herein.

[0338] The term "K" used in this article D "Refers to K" d With K a The ratio (i.e., K) d / K a The dissociation constant obtained from K is expressed as molar concentration (M). d K and K represent the dissociation rate and binding rate of a specific antigen-antibody interaction, respectively. The K of an antibody... D The value can be determined using methods already established in the art. As used herein, the term "low affinity" refers to a K0 value of 100 nM or higher. D .

[0339] As used in this article, the term "moderate affinity" refers to K+ in the range of 10 nM to 100 nM. D .

[0340] As used in this article, the term "high affinity" refers to K+ with a K+ of 1 to 10 nM. D .

[0341] As used in this article, the term "very high affinity" refers to a K0.05 value of 1 nM or lower. D .

[0342] As used herein, the term “EC50” refers to the concentration of an antibody or its antigen-binding protein / fraction that induces a response of 50% of the maximum response (i.e., the midpoint between the maximum response and the baseline) in an in vivo or in vitro assay.

[0343] 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, the terms "multispecific" or "multispecific antibody" as used herein refer to the antibody described herein, which has at least two binding domains, i.e., two or more binding domains, such as two or three binding domains, wherein at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. Multispecific antibodies are typically monovalent for each specificity (antigen). The multispecific antibodies described herein encompass both monovalent and multivalent (e.g., bivalent, trivalent, and tetravalent) multispecific antibodies.

[0344] The term “bispecific” or “bispecific antibody” as used in this article refers to an antibody that is specific to two antigens or has the ability to bind to two target antigens / sites simultaneously.

[0345] As used herein, “bispecific T-cell adaptors (BiTEs)” refer to a class of artificial bispecific monoclonal antibodies that direct the host’s immune system, such as T cells, to exert cytotoxic activity against target cells, such as cancer cells. BiTEs are fusion proteins composed of two single-chain variable fragments (scFvs) of different antibodies or amino acid sequences from four different genes on a single peptide chain of approximately 55 kDa. One scFv binds to an immune cell (e.g., T cells via the CD3 receptor), and the other binds to a target (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs establish a link between immune cells (e.g., T cells) and target cells (e.g., tumor cells). This results in the immune cell (e.g., T cell) exerting cytotoxic activity against the tumor cell. For example, if the immune cell is a T cell, the T cell will exert cytotoxic activity by producing proteins such as perforin and granzymes that enter the tumor cell and initiate apoptosis.

[0346] In some instances, BiTE can refer to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In other instances, BiTE can also refer to a bispecific T-cell adjuvant platform known in the art, which refers to a recombinant protein that simultaneously binds to two different antigens and has the ability to adjuvant immune cells (such as T cells).

[0347] As used herein, the term "nanobody" refers to a single-domain antibody (sdAb) whose antibody fragment consists of a single monomeric variable antibody domain. In some instances, bispecific T-cell adaptors (BiTEs) are nanobodies that consist only of the heavy chain (VHH). As used herein, "nanobody with only heavy chain" refers to a heavy-chain antibody based on a nanobody. Heavy-chain antibodies consist of two heavy chains and lack the two light chains typically found in antibodies.

[0348] As described herein, a “vector” is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell, in which, for example, the synthesis of a polypeptide can occur. Typically, and preferably, a vector is a nucleic acid that has been engineered using recombinant DNA techniques known in the art to incorporate a desired nucleic acid sequence (e.g., the nucleic acid of this disclosure). Expression vectors typically contain one or more of the following components (if they are not already provided by a nucleic acid molecule): a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a secretion leader sequence, a ribosome binding site, a polyadenylation sequence, a multi-connector region for inserting a nucleic acid encoding the polypeptide to be expressed, and a selection marker element.

[0349] Vectors are typically selected to be functional in the host cell where they will be used (the vector is compatible with host cell mechanisms, enabling gene amplification and / or gene expression to occur). The vectors described herein can be expression vectors and / or cloning vectors.

[0350] As used herein, the term "host cell" refers to a cell into which the expression vector has been introduced. It should be understood that this term refers not only to the specific test cell but also to its progeny. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may actually differ from the parent cells but are still included within the scope of the term "host cell" as used herein.

[0351] The terms “treating,” “treatment,” and “therapy,” and their synonyms, refer to therapeutic procedures and preventative or protective measures aimed at preventing or alleviating (reducing) medical conditions, including but not limited to diseases, symptoms, and symptoms. Medical conditions also include the body’s response to a disease or symptom (such as inflammation). People who need such treatment include those who already have a medical condition, those who are susceptible to it, or those who need to prevent it.

[0352] The term "subject" as used in this document includes both patients and non-patients. The term "patient" refers to an individual who has or may have a medical condition, while "non-patient" refers to an individual who does not have or may not have the medical condition. "Non-patient" includes healthy individuals, individuals who are not ill, and / or individuals without a medical condition. The term "subject" includes both humans and animals. Animals may include, but are not limited to, mammals (e.g., non-human primates, canines, rodents, etc.). "Rodents" refers to any mammal from the Muridae and / or Leporidae families, such as mice, rats, rabbits, etc.

[0353] As used in this article, the terms “prevention” and / or “reducing the severity of symptoms” refer to the process of delaying the onset of disease, reducing the severity of symptoms, reducing and / or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and / or improving at least one sign or symptom of the disease.

[0354] The term “and / or”, such as “X and / or Y”, is understood to mean “X and Y” or “X or Y”, and should be used to provide clear support for both meanings or either meaning.

[0355] Furthermore, in the description herein, the word “substantially” is understood to include, but is not limited to, “completely” or “thoroughly” whenever used. Additionally, terms such as “comprising” and “comprise” are intended to be non-restrictive descriptive language whenever used, as they broadly include elements / components stated after these terms, except for other components not explicitly stated. 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” can be considered, in the appropriate context, as a subset of terms such as “comprising” and “comprise”. Therefore, in the embodiments disclosed herein that use terms such as “comprising” and “comprise”, it should be understood that these embodiments provide instruction for corresponding embodiments using terms such as “consisting” and “consist”. In addition, whenever terms such as “about” or “approximately” are used, they usually refer to reasonable variations, 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.

[0356] Furthermore, in this description, certain values ​​may be disclosed within a range. The values ​​shown at the endpoints of a range are intended to illustrate preferred ranges. Whenever a range is described, it means that the range covers and teaches all possible subranges as well as individual values ​​within that range. That is, the endpoints of a range should not be interpreted as immutable limitations. For example, the description of a range of 1% to 5% is intended to have specifically disclosed subranges of 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., and individual values ​​within that range, such as 1%, 2%, 3%, 4%, and 5%. It should be understood that individual values ​​within this range also include integers, fractions, and decimals. Furthermore, whenever a range is described, it also means that the range covers and teaches values ​​with up to two additional decimal places or significant digits (if appropriate) starting from the endpoint of the indicated value. For example, the description of a range of 1% to 5% is intended to specifically disclose ranges of 1.00% to 5.00% and 1.0% to 5.0%, as well as all their intermediate values ​​across that range (such as 1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The above specific disclosure is intended to apply to ranges of any depth / width.

[0357] The term "at least 95% identity" as used in this article refers to an amino acid sequence that is 95% or higher identical (such as 96%, 97%, 98%, or 99%) to the reference sequence in its entirety. Software programs can be used to calculate the percentage of identity.

[0358] Furthermore, when describing some embodiments, this disclosure may have disclosed methods and / or processes in a specific order of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific order of steps disclosed. Other orders of steps are also possible. The specific order of steps disclosed herein should not be construed as an undue limitation. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the order they are written. The order of steps may be changed, and still remain within the scope of this disclosure.

[0359] Furthermore, it should be understood that while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may be omitted in other alternative embodiments, and this disclosure provides support for such omissions and these related alternative embodiments.

[0360] Description of the implementation plan

[0361] This disclosure provides engineered cells that express...

[0362] (a) Chimeric antigen receptors targeting the first target antigen, and

[0363] (b) A multispecific antigen-binding protein that binds to EpCAM (epithelial cell adhesion molecule).

[0364] Without being bound by any theory, we engineer cells to enable EpCAM to be secreted at the target site, thereby minimizing toxicity, reducing side effects, and / or reducing nonspecific cytotoxicity.

[0365] In some instances, the primary target antigen is a disease-associated antigen.

[0366] For example, the target antigen is a molecule associated with a disease. This molecule can be an extracellular molecule, an intracellular molecule, and / or a transmembrane molecule. In some instances, the molecule can be a polypeptide, a polynucleotide, a carbohydrate, etc. In some instances, the primary target antigen is a diseased cell, etc.

[0367] In some instances, diseased cells may include, but are not limited to, cells from proliferative diseases (such as tumors / cancer, inflammatory diseases). In some instances, diseased cells may be cells from cancer / tumors. In some instances, cancer cells are solid tumors. In some instances, solid tumors may include tumors of epithelial origin, such as carcinoma. In some instances, carcinomas may include, but are not limited to, liver cancer, lung cancer, gastric cancer (such as gastric adenocarcinoma), 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 instances, tumor cells may include benign, precancerous, malignant tumors, etc. In some instances, tumor cells may include stem cells, progenitor cells, etc. In some instances, tumor cells may include tumor cell lines, such as, but not limited to, gastric adenocarcinoma cell lines (such as AGS), breast cancer cell lines (such as MDA-MB468, MCF7, SKBR3, BT474, MDA-MB231, MDA-MB436), glioblastoma (A-172), etc.

[0368] In some instances, the target antigen is an epithelial marker. In some instances, epithelial markers may include, but are not limited to, receptor tyrosine protein kinase erbB-2 (HER2), phosphatidylinositol proteoglycan 3 (GPC3), closure protein 18.2 (Claudin 18.2), receptor tyrosine kinase-like orphan receptor 1 (ROR1), delta-like canonical nick 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), mesothelin (MSLN), mesenchymal-epithelial transition factor (c-Met), folate receptor α (FRα), etc. In some instances, the primary antigen is HER2. Therefore, in some embodiments, the chimeric antigen receptor binds to HER2.

[0369] In some cases, cancers that express HER2 may include, but are not limited to, stomach / esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, endometrial cancer, bladder cancer, lung cancer, and colon cancer.

[0370] In some instances, CAR T cells target only HER2 (HE CAR-T).

[0371] In some instances, the cells express multispecific antigen-binding proteins that bind to one or more markers. For example, the multispecific antigen-binding protein may bind to EpCAM and immunomarkers.

[0372] Not wanting 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 shown to be highly toxic to normal tissues, and anti-EpCAM BiTEs (such as soritoxamab and caputoxamab) have exhibited dose-limiting toxicity and have failed to obtain FDA approval.

[0373] EpCAM (epithelial cell adhesion molecule) represents another class of biomarkers widely expressed in almost all cancers. Cancer therapies targeting EpCAM have undergone more than 10 years of clinical development. However, because EpCAM is also expressed at low levels in normal epithelial cells, anti-EpCAM CAR T cells have been shown to be highly toxic to normal tissues. For the same reason, soritomab and caputomab (anti-EpCAM BiTE) have both exhibited dose-limiting toxicities and have failed to obtain FDA approval.

[0374] HER2 amplification or overexpression has been found in approximately 10-30% of gastric / gastroesophageal cancers and 15-30% of breast cancers. Furthermore, HER2 overexpression has 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-targeting CAR T-cell therapy, has been clinically proven to be effective and is generally safe unless antibody clones with very high antigen-binding affinity are used. However, only a very limited number of patients achieve complete remission (CR). Metastasis and recurrence of the disease after treatment targeting these markers are largely attributed to the fact that not all tumor cells express HER2, despite high levels of HER2 expression found at diagnosis in these cancers. Re-emerging tumor masses often originate from the growth of antigen-negative cancer cells or escaped cancer cells that are subsequently downregulated or even lose antigen expression after CAR T-cell therapy. Despite the reported high complete response (CR) rates, CAR-T therapies using FDA-approved anti-CD19-targeted "Kymriah" or "Yescarta" to treat various B-cell-derived leukemias and lymphomas face the same antigen escape problem. To overcome this challenge, the idea of ​​dual-targeting CAR T cells has been frequently employed, and bispecific anti-CD19 / CD20 CAR T-cell therapy has shown early promise for treating relapsed or refractory B-cell lymphomas. However, in solid tumors, it is extremely difficult to find two or more highly tumor-specific, targetable tumor-specific antigens, as most tumor markers are also expressed in normal tissues, albeit at relatively low levels. Because CAR-T cells possess the inherently high efficiency in killing cancer cells, designing multi-targeting dual CARs against two or more antigens could induce intolerable non-tumor-targeting toxicities and is therefore considered extremely dangerous for patients with solid tumors.

[0375] Interestingly, EpCAM, a broad-spectrum pan marker for epithelial cells, has been found to be co-expressed with HER2 in many cancers. Instead of using a dual CAR setup, this invention develops a HER2-targeting CAR-T cell therapy that secretes an anti-EpCAM bispecific T-cell adaptor (BiTE). This restricts the presence of anti-EpCAM BiTE to the tumor site, maximizing tumor eradication and preventing tumor escape while avoiding systemic toxicity. These CAR T cells are named "HE CAR-BiTE T" (HER2-targeting CAR T cells with anti-EpCAM BiTE secretion).

[0376] CAR-BiTE T cells secreting anti-EpCAM BiTE first infiltrate tumor cells by recognizing specific tumor antigens expressed on them via CAR and remain at the tumor site. When the CAR target binds, the CAR T cells are activated and begin to proliferate, simultaneously secreting anti-EpCAM BiTE. While CAR T cells can directly kill target cells, anti-EpCAM BiTE exerts its cytotoxicity by recruiting nearby T cells. In this way, bystander T cells are physically guided to the vicinity of the tumor, activated, and subsequently help clear tumor cells. Because HER2 overexpression is tumor-specific, the spread of anti-EpCAM BiTE may be limited to or near the tumor site. Furthermore, the inefficient secretion of recombinant anti-EpCAM BiTE antibodies by anti-HER2 CAR T cells, coupled with the need for immediate T cell presence, may reduce their extra-tissue toxicity. Additionally, EpCAM is also defined as a cancer stem cell marker because it has been found to be expressed on cancer progenitors and cancer stem cells. CAR T cell secretion of anti-EpCAM will help prevent cancer recurrence and relapse by clearing cancer stem cells and progenitors.

[0377] The experimental data disclosed herein demonstrate that EpCAM exhibits the highest expression level in multiple stained HER2-positive tumor cells compared to other epithelial tumor markers. Therefore, EpCAM was chosen as a BiTE target due to its broad coverage of epithelial-derived cancer cells. The significantly higher expression of EpCAM in cancer cells compared to normal epithelial cells provides a wider therapeutic window than other targets. Furthermore, anti-EpCAM BiTE can be injected directly into the tumor site for targeted therapy. The fact that the anti-EpCAM BiTE of this disclosure is secreted locally at the tumor site (rather than systemically administered) by CAR T cells limits its potential toxicity. This transforms a previously unmanageable (or difficult) target into a manageable one.

[0378] Therefore, in one respect, modified cells are provided that express...

[0379] (a) Chimeric antigen receptors targeting HER2, and

[0380] (b) A multispecific antigen-binding protein, its variants, or binding fragments that bind to one or more targets, comprising a first antigen-binding protein, its variants, or binding fragments that bind to EpCAM (epithelial cell adhesion molecule) and a second antigen-binding protein, its variants, or binding fragments that bind to immune cell markers.

[0381] The first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy chain variable regions and / or light chain variable regions selected from the following groups:

[0382] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5, 1B8, 2B7 and 2D10)

[0383] CDR-H1 includes:

[0384] GSIFSGND (SEQ ID NO:25-2C4, hu2C4, 1A5, 2B7 and 2D10), or

[0385] GSSERFTS (SEQ ID NO:29 – 1B8)

[0386] CDR-H2 contains:

[0387] ITSGGST (SEQ ID NO:26–2C4, hu2C4, 1A5, 2B7 and 2D10) or

[0388] ITNGGST (SEQ ID NO:30 – 1B8) and

[0389] CDR-H3 contains:

[0390] TNGRWSGDTYYAHH (SEQ ID NO:27–2C4, hu2C4, 1A5, and 2D10),

[0391] MAGTS (SEQ ID NO:31 – 1B8) or

[0392] TNGRWSGDTYYAHL (SEQ ID NO:33 – 2B7)

[0393] (ii) Heavy chain variable regions, which include: (1B6, 1C1, 1C11, 1D4, and 1H6)

[0394] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0395] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0396] CDR-H3 contains ARSLGGRFRY (SEQ ID NO:3)

[0397] (iii) Heavy chain variable region, which contains: (1E4)

[0398] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5)

[0399] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0400] CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO: 7)

[0401] (iv) Light chain variable regions, comprising: (1B6, 1C1, 1C11, 1D4, 1E4 and 1H6)

[0402] CDR-L1 includes:

[0403] QSLLHSNGYNY (SEQ ID NO:9 - 1B6, 1C1, 1C11 and 1H6)

[0404] QSLLHSNRYNY (SEQ ID NO:17 - 1D4) or

[0405] QSISDF (SEQ ID NO:19 – 1E4)

[0406] CDR-L2 includes:

[0407] LGS (SEQ ID NO:10 - 1B6, 1C1, 1C11, 1D4 and 1H6), or

[0408] AAS (SEQ ID NO:20 - 1E4), and

[0409] CDR-L3 includes:

[0410] MQALQTPYT (SEQ ID NO:11 - 1B6, 1C1 and 1D4)

[0411] MQGLQSPWT (SEQ ID NO:15 - 1C11),

[0412] QQSYIMPDT (SEQ ID NO:21 – 1E4) or

[0413] MQGLQTPYT (SEQ ID NO:23 – 1H6); and

[0414] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0415] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain heavy chain variable regions, including: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10).

[0416] CDR-H1 includes:

[0417] GSIFSGND (SEQ ID NO:25 - 2C4, hu2C4, 1A5, 2B7 and 2D10) or

[0418] GSSERFTS (SEQ ID NO:29 – 1B8)

[0419] CDR-H2 contains:

[0420] ITSGGST (SEQ ID NO:26–2C4, hu2C4, 1A5, 2B7 and 2D10) or

[0421] ITNGGST (SEQ ID NO:30 – 1B8); and

[0422] CDR-H3 contains:

[0423] TNGRWSGDTYYAHH (SEQ ID NO:27–2C4, hu2C4, 1A5 and 2D10)

[0424] MAGTS (SEQ ID NO:31 – 1B8) or

[0425] TNGRWSGDTYYAHL (SEQ ID NO:33 – 2B7)

[0426] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0427] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or the binding fragment contains a heavy chain variable region selected from the group consisting of:

[0428] (i) Heavy chain variable regions, which include: (2C4-VHH, hu2C4-VHH, 1A5-VHH and 2D10-VHH)

[0429] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0430] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0431] CDR-H3 contains TNGRWSGDTYYAHH (SEQ ID NO:27);

[0432] (ii) Heavy chain variable region, which contains: (1B8-VHH)

[0433] CDR-H1 contains GSSERFTS (SEQ ID NO:29).

[0434] CDR-H2, which contains ITNGGST (SEQ ID NO:30), and

[0435] CDR-H3, which contains MAGTS (SEQ ID NO:31); and

[0436] (iii) Heavy chain variable region, which contains: (2B7-VHH)

[0437] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0438] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0439] CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO:33);

[0440] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0441] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy chain variable regions and / or light chain variable regions selected from the group consisting of:

[0442] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5 and 2D10)

[0443] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0444] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0445] CDR-H3 contains TNGRWSGDTYYAHH (SEQ ID NO:27);

[0446] (ii) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1H6)

[0447] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0448] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0449] CDR-H3 contains ARSLGGRFRY (SEQ ID NO:3);

[0450] (iii) Heavy chain variable region, which contains: (1E4)

[0451] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5).

[0452] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0453] CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO:7);

[0454] (iv) Light chain variable region, which includes: (1B6 and 1C1)

[0455] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0456] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0457] CDR-L3 contains MQALQTPYT (SEQ ID NO:11);

[0458] (v) Light chain variable region, which includes: (1C11)

[0459] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0460] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0461] CDR-L3 contains MQGLQSPWT (SEQ ID NO:15);

[0462] (vi) Light chain variable region, which contains: (1D4)

[0463] CDR-L1 contains QSLLHSNRYNY (SEQ ID NO:17).

[0464] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0465] CDR-L3 contains MQALQTPYT (SEQ ID NO:11);

[0466] (vii) Light chain variable region, which includes: (1E4)

[0467] CDR-L1, which contains QSISDF (SEQ ID NO:19),

[0468] CDR-L2, which contains AAS (SEQ ID NO:20), and

[0469] CDR-L3 contains QQSYIMPDT (SEQ ID NO:21);

[0470] (viii) Light chain variable region, which contains: (1H6)

[0471] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0472] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0473] CDR-L3 contains MQGLQTPYT (SEQ ID NO:23);

[0474] (ix) Heavy chain variable region, which contains: (1B8-VHH)

[0475] CDR-H1 contains GSSERFTS (SEQ ID NO:29).

[0476] CDR-H2, which contains ITNGGST (SEQ ID NO:30), and

[0477] CDR-H3, which contains MAGTS (SEQ ID NO:31); and

[0478] (x) Heavy chain variable region, which contains: (2B7-VHH)

[0479] CDR-H1, which contains GSIFSGND (SEQ ID NO:25),

[0480] CDR-H2, which contains ITSGGST (SEQ ID NO:26), and

[0481] CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO:33);

[0482] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0483] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of:

[0484] (i) Heavy-chain variable structural domains, which contain

[0485] QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:35 - 2C4-VHH)

[0486] (ii) Heavy-chain variable structural domains, which contain

[0487] QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO:37- hu2C4-VHH)

[0488] (iii) Heavy-chain variable structural domains, which contain

[0489] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4 - 1B6, 1C1, 1C11, 1D4 and 1H6)

[0490] (iv) Heavy-chain variable structural domains, which contain

[0491] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO:8 - 1E4),

[0492] (v) Light chain variable structural domain, which contains

[0493] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:12 - 1B6 and 1C1)

[0494] (vi) Light chain variable structural domain, which contains

[0495] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO:16 - 1C11)

[0496] (vii) Light chain variable structural domain, which contains

[0497] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:18 - 1D4)

[0498] (viii) Light chain variable structural domain, which contains

[0499] DIQLTQSPSSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO:22 - 1E4)

[0500] (ix) Light chain variable structural domain, which contains

[0501] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO:24- 1H6)

[0502] (x) Heavy-chain variable structural domain, which contains

[0503] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:28- 1A5-VHH)

[0504] (xi) Heavy-chain variable structural domain, which contains

[0505] QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO:32 - 1B8-VHH)

[0506] (xii) Heavy-chain variable structural domain, which contains

[0507] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO:34 - 2B7-VHH)

[0508] (xiii) Heavy-chain variable structural domains, which contain

[0509] and

[0510] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0511] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or the binding fragment contains a heavy-chain variable domain selected from the group consisting of:

[0512] (i) Heavy-chain variable structural domains, which contain

[0513] QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:35 - 2C4-VHH)

[0514] (ii) Heavy-chain variable structural domains, which contain

[0515] QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO:37- hu2C4-VHH)

[0516] (iii) Heavy-chain variable structural domains, which contain

[0517] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4 - 1B6, 1C1, 1C11, 1D4 and 1H6)

[0518] (iv) Heavy-chain variable structural domains, which contain

[0519] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO:8 - 1E4),

[0520] (v) Heavy-chain variable structural domains, which contain

[0521] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:28- 1A5-VHH)

[0522] (vi) Heavy-chain variable structural domains, which contain

[0523] QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO:32 - 1B8-VHH)

[0524] (vii) Heavy-chain variable structural domains, which contain

[0525] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO:34 - 2B7-VHH)

[0526] (viii) Heavy-chain variable structural domains, which contain

[0527] and

[0528] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0529] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain a single-domain heavy-chain variable domain having the following sequence:

[0530] (i) QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:35 - clone 2C4-VHH), or

[0531] OR

[0532] (ii) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:28 - clone 1A5-VHH), or

[0533] (iii) QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO:32 - clone 1B8-VHH), or

[0534] Or

[0535] (v)QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO:36 - clone 2D10-VHH),

[0536] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0537] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or the binding fragment contains a heavy chain variable region selected from the group consisting of:

[0538] (i) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4 and 1H6)

[0539] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0540] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0541] CDR-H3 contains ARSLGGRFRY (SEQ ID NO:3)

[0542] (ii) Heavy chain variable region, which contains: (1E4)

[0543] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5)

[0544] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0545] CDR-H3, which contains AREVEGSSYDAFDI (SEQ ID NO:7); and / or

[0546] It contains a light chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6).

[0547] CDR-L1 includes:

[0548] QSLLHSNGYNY (SEQ ID NO:9 - 1B6, 1C1, 1C11 and 1H6)

[0549] QSLLHSNRYNY (SEQ ID NO:17 - 1D4) or

[0550] QSISDF (SEQ ID NO:19 – 1E4)

[0551] CDR-L2 includes:

[0552] LGS (SEQ ID NO:10 - 1B6, 1C1, 1C11, 1D4 and 1H6) or

[0553] AAS (SEQ ID NO:20 - 1E4), and

[0554] CDR-L3 includes:

[0555] MQALQTPYT (SEQ ID NO:11 - 1B6, 1C1 and 1D4)

[0556] MQGLQSPWT (SEQ ID NO:15 - 1C11),

[0557] QQSYIMPDT (SEQ ID NO:21 – 1E4) or

[0558] MQGLQTPYT (SEQ ID NO:23 – 1H6)

[0559] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0560] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy chain variable regions and / or light chain variable regions selected from the group consisting of:

[0561] (i) Heavy chain variable region, which contains: (1B6 and 1C1)

[0562] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0563] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0564] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and / or

[0565] The light chain variable region, which includes:

[0566] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0567] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0568] CDR-L3 contains MQALQTPYT (SEQ ID NO:11)

[0569] (ii) Heavy chain variable region, which contains: (1C11)

[0570] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0571] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0572] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and / or

[0573] The light chain variable region, which includes:

[0574] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0575] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0576] CDR-L3 contains MQGLQSPWT (SEQ ID NO:15)

[0577] (iii) Heavy chain variable region, which contains: (1D4)

[0578] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0579] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0580] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and / or

[0581] The light chain variable region, which includes:

[0582] CDR-L1 contains QSLLHSNRYNY (SEQ ID NO:17).

[0583] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0584] CDR-L3 contains MQALQTPYT (SEQ ID NO:11)

[0585] (iv) Heavy chain variable region, which contains: (1H6)

[0586] CDR-H1 contains GGTFSSYA (SEQ ID NO:1).

[0587] CDR-H2, which contains IIPIFGTA (SEQ ID NO:2), and

[0588] CDR-H3, which contains ARSLGGRFRY (SEQ ID NO:3); and / or

[0589] The light chain variable region, which includes:

[0590] CDR-L1, which contains QSLLHSNGYNY (SEQ ID NO:9),

[0591] CDR-L2, which includes LGS (SEQ ID NO:10), and

[0592] CDR-L3, which contains MQGLQTPYT (SEQ ID NO:23); and

[0593] (v) Heavy chain variable region, which contains: (1E4)

[0594] CDR-H1 contains GDSISSNSVA (SEQ ID NO:5).

[0595] CDR-H2, which contains TYYRSKWYS (SEQ ID NO:6), and

[0596] CDR-H3, which contains AREVEGSSYDAFDI (SEQ ID NO:7); and / or

[0597] The light chain variable region contains: (1E4)

[0598] CDR-L1, which contains QSISDF (SEQ ID NO:19),

[0599] CDR-L2, which contains AAS (SEQ ID NO:20), and

[0600] CDR-L3 contains QQSYIMPDT (SEQ ID NO:21)

[0601] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0602] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of:

[0603] (vi) Heavy chain variable structural domain, which contains: (1B6 and 1C1)

[0604] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and / or

[0605] Light chain variable structural domain, which includes:

[0606] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:12)

[0607] (vii) Heavy chain variable structural domain, which contains: (1C11)

[0608] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and / or

[0609] Light chain variable structural domain, which includes:

[0610] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO:16)

[0611] (viii) Heavy-chain variable structural domains, which contain: (1D4)

[0612] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and / or

[0613] Light chain variable structural domain, which includes:

[0614] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO:18)

[0615] (ix) Heavy chain variable structural domain, which contains: (1E4)

[0616] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO:8), and / or

[0617] Light chain variable structural domain, which includes:

[0618] DIQLTQSPSSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO:22), or

[0619] (x) A heavy-chain variable structural domain containing: (1H6)

[0620] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO:4), and / or

[0621] Light chain variable structural domain, which includes:

[0622] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO:24), or

[0623] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0624] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain a light chain constant domain having the following sequence:

[0625] (iii)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:13 - clone 1B6 - light chain constant domain), or

[0626] (iv)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (SEQ ID NO:14 clone 1C1 - light chain constant domain),

[0627] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical with it and / or has 2 or 3 amino acid substitutions.

[0628] In one embodiment, the antigen-binding protein, its variants, or fragments comprise a heavy chain variable region and / or a light chain variable region encoded by a nucleic acid sequence selected from the group consisting of:

[0629] (i) Heavy chain variable region, which contains: (1B6 and 1C1)

[0630] CDR-H1 contains ggaggcaccttcagcagctatgct (SEQ ID NO:38).

[0631] CDR-H2, which contains atcatccctatctttggtacagca (SEQ ID NO:39), and

[0632] CDR-H3, which contains gcgagatcgttgggtgggagatttcgctac (SEQ ID NO:40); and / or

[0633] The light chain variable region, which includes:

[0634] CDR-L1 contains cagagcctcctgcatagtaatggatacaactat (SEQ ID NO:46).

[0635] CDR-L2, which contains ttgggttct (SEQ ID NO:47), and

[0636] CDR-L3 contains atgcaagctctacaaactccgtacact (SEQ ID NO:48).

[0637] (ii) Heavy chain variable region, which contains: (1C11)

[0638] CDR-H1 contains ggaggcaccttcagcagctatgct (SEQ ID NO:38).

[0639] CDR-H2, which contains atcatccctatctttggtacagca (SEQ ID NO:39), and

[0640] CDR-H3, which contains gcgagatcgttgggtgggagatttcgctac (SEQ ID NO:40); and / or

[0641] The light chain variable region, which includes:

[0642] CDR-L1 contains cagagcctcctgcatagtaatggatacaactat (SEQ ID NO:46).

[0643] CDR-L2, which contains ttgggttct (SEQ ID NO:47), and

[0644] CDR-L3 contains atgcaaggtctacaaagtccctggacg (SEQ ID NO:52).

[0645] (iii) Heavy chain variable region, which contains: (1D4)

[0646] CDR-H1 contains ggaggcaccttcagcagctatgct (SEQ ID NO:38).

[0647] CDR-H2, which contains atcatccctatctttggtacagca (SEQ ID NO:39), and

[0648] CDR-H3, which contains gcgagatcgttgggtgggagatttcgctac (SEQ ID NO:40); and / or

[0649] The light chain variable region, which includes:

[0650] CDR-L1 contains cagagcctcctgcatagtaatagatacaactat (SEQ ID NO:54).

[0651] CDR-L2, which contains ttgggttct (SEQ ID NO:47), and

[0652] CDR-L3 contains atgcaagctctacaaactccgtacact (SEQ ID NO:48).

[0653] (iv) Heavy chain variable region, which contains: (1H6)

[0654] CDR-H1 contains ggaggcaccttcagcagctatgct (SEQ ID NO:38).

[0655] CDR-H2, which contains atcatccctatctttggtacagca (SEQ ID NO:39), and

[0656] CDR-H3, which contains gcgagatcgttgggtgggagatttcgctac (SEQ ID NO:40); and / or

[0657] The light chain variable region, which includes:

[0658] CDR-L1 contains cagagcctcctgcatagtaatggatacaactat (SEQ ID NO:46).

[0659] CDR-L2, which contains ttgggttct (SEQ ID NO:47), and

[0660] CDR-L3, which contains atgcaaggtctacagactccgtacact (SEQ ID NO:60); and

[0661] (v) Heavy chain variable region, which contains: (1E4)

[0662] CDR-H1 contains ggggacagtatctctagtaacagtgttgct (SEQ ID NO:42).

[0663] CDR-H2, which contains acatactacaggtccaagtggtacagt (SEQ ID NO:43), and

[0664] CDR-H3, which contains gcaagagaagttgagggcagcagctatgatgcttttgatatc (SEQ ID NO:44); and / or

[0665] The light chain variable region contains: (1E4)

[0666] CDR-L1 contains cagagtattagcgacttt (SEQ ID NO:56).

[0667] CDR-L2, which contains gctgcatcg (SEQ ID NO:57), and

[0668] CDR-L3 contains ttacattatgcccgacact (SEQ ID NO:58);

[0669] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0670] In one implementation, the antigen-binding protein, its variants, or fragments comprise a heavy-chain variable region encoded by a nucleic acid sequence selected from the group consisting of:

[0671] (i) Heavy chain variable regions, which include: (2C4-VHH, 1A5-VHH, 2D10-VHH and hu2C4-VHH)

[0672] CDR-H1 contains ggaagcatcttcagtggcaatgac (SEQ ID NO:62).

[0673] CDR-H2, which contains atactagcggtggtagtaca (SEQ ID NO:63), and

[0674] CDR-H3, which contains acaaacggaagatggtcaggcgatacttactatgcccatcac (SEQ ID NO:64)

[0675] (ii) Heavy chain variable region, which contains: (1B8-VHH)

[0676] CDR-H1 contains ggaagctccgaaagattcacatca (SEQ ID NO:66).

[0677] CDR-H2, which contains atactaatggtggtagcaca (SEQ ID NO:67), and

[0678] CDR-H3, which contains atggcgggtacgtcc (SEQ ID NO:68); and

[0679] (iii) Heavy chain variable region, which contains: (2B7-VHH)

[0680] CDR-H1 contains ggaagcatcttcagtggcaatgac (SEQ ID NO:62).

[0681] CDR-H2, which contains atactagcggtggtagtaca (SEQ ID NO:63), and

[0682] CDR-H3 contains acaaacggaagatggtcaggcgatacttactatgcccatctc (SEQ ID NO:70).

[0683] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0684] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0685] (i)CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:72 - clone 2C4),

[0686] (ii)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO:74 - Clone hu2C4-VHH);

[0687] (iii)GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41 - For clones 1B6, 1C1, 1C11, 1D4, 1H6), or

[0688] (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45 - Clone 1E4);

[0689] (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:65 - Clone 1A5),

[0690] (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO:69 - Clone 1B8),

[0691] (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO:71 - Clone 2B7),

[0692] (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:73 - Clone 2D10), or

[0693] and / or

[0694] The light chain variable domain is encoded by a nucleotide sequence comprising:

[0695] (vi) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:49 - Clone 1B6 and 1C1), or

[0696] (vii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO:53 - Clone 1C11), or

[0697] (viii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:55 - Clone 1D4), or

[0698] (ix)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGC ATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO:59 - clone 1E4), or

[0699] (x)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCT ATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:61 - clone 1H6),

[0700] Or a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or has 10-20 nucleic acid substitutions.

[0701] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0702] (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:72 - Clone 2C4), or

[0703] (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO:74 - Clone hu2C4-VHH); or

[0704] (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41 - for clone 1B6, 1C1, 1C11, 1D4, 1H6), or

[0705] (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45 - clone 1E4); or

[0706] (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:65 - Clone 1A5), or

[0707] (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO:69 - Clone 1B8), or

[0708] (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO:71 - Clone 2B7), or

[0709] (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:73 - Clone 2D10), and / or

[0710] The light chain variable domain is encoded by the following nucleotide sequence, which comprises:

[0711] (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:49 - Clone 1B6 and 1C1), or

[0712] (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO:53 - Clone 1C11), or

[0713] (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:55 - Clone 1D4), or

[0714] (iv) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO:59 - Clone 1E4), or

[0715] (v)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCT ATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:61 - clone 1H6),

[0716] Or a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or has 10-20 nucleic acid substitutions.

[0717] In some instances, the first antigen-binding protein that binds to EpCAM, its variants, or binding fragments contain heavy-chain and / or light-chain variable domains encoded by nucleotide sequences selected from the group consisting of:

[0718] (i) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1B6 and 1C1)

[0719] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and / or

[0720] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0721] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:49)

[0722] (ii) a heavy chain variable domain encoded by a nucleotide sequence comprising: (1C11)

[0723] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and / or

[0724] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0725] GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO:53)

[0726] (iii) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1D4)

[0727] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and / or

[0728] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0729] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:55)

[0730] (iv) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1H6)

[0731] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41), and / or

[0732] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0733] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:61); and

[0734] (v) A heavy chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises: (1E4)

[0735] CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGT ACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45), and / or

[0736] A light chain variable domain encoded by the following nucleotide sequence, wherein the nucleotide sequence comprises:

[0737] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCAT CGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO:59)

[0738] Or a sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or has 10-20 nucleic acid substitutions.

[0739] In one embodiment, the antigen-binding protein, its variants, or fragments comprise a light chain constant domain encoded by a nucleotide sequence comprising:

[0740] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO:50 - Clone 1B6 light chain constant domain), or

[0741] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT (SEQ ID NO:51 - Clone 1C1 - Light chain constant domain), or

[0742] It has at least 60% identity with its sequence and / or has 10-20 nucleic acid substitutions.

[0743] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind to EpCAM contain a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0744] (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCAGGGACCCAG (SEQ ID NO:72) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0745] (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO:74) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions; or

[0746] (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:65) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0747] (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGACCCAG (SEQ ID NO:69) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0748] (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGACCCAG (SEQ ID NO:71) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0749] (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTAC ACACTATGCAGACTCCGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO:73) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with and / or having 10-20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGAGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions, or

[0750] (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it and / or having 10-20 nucleic acid substitutions.

[0751] In some instances, the multispecific antigen-binding protein may comprise an amino acid sequence having one or more amino acid mutations relative to any of the sequences disclosed herein. In some instances, 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 relative to any of the sequences disclosed herein. In some instances, the one or more amino acid mutations may be independently selected from substitution, insertion, deletion, and truncation. In some instances, the amino acid mutation is an amino acid substitution and may include conserved and / or non-conserved substitutions. In some instances, the mutation does not significantly reduce the ability of the antigen-binding protein to specifically bind to a target. In some instances, the mutation does not significantly reduce the ability of the antigen-binding protein to specifically bind to a target and does not functionally modulate (e.g., partially or completely neutralize) the target.

[0752] In some instances, the multispecific antigen-binding protein may be a bispecific, trispecific, or tetraspecific antigen-binding protein, etc. In some instances, the multispecific antigen-binding protein is a bispecific antigen-binding protein. In some instances, the multispecific antigen-binding protein, its variants, or fragments are bispecific antibodies. In some instances, the multispecific antigen-binding protein may be provided as a nanobody. In some instances, the multispecific antigen-binding protein may be provided as an Fc region. In some instances, the multispecific antigen-binding protein is an inducible bispecific T-cell adjuvant comprising a heavy-chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv). In some instances, the bispecific T-cell adjuvant (BiTE) is a nanobody (VHH) having only a heavy chain.

[0753] In some instances, the multispecific antigen-binding protein, its variants, or fragments are inducible bispecific immune cell adjuvants. In some instances, the multispecific antigen-binding protein is secreted by cells (optionally immune cells). In some instances, the multispecific antigen-binding protein, its variants, or fragments are bispecific T-cell adjuvants (BiTE).

[0754] As used in this article, bispecific T-cell adaptors (BiTEs) refer to a class of artificial bispecific monoclonal antibodies that direct the host's immune system, such as T cells, to exert cytotoxic activity against target cells, such as cancer cells. BiTEs are fusion proteins composed of two single-chain variable fragments (scFvs) of different antibodies or amino acid sequences from four different genes on a single polypeptide chain of approximately 55 kDa. One scFv binds to T cells via the CD3 receptor, while the other binds to a target of interest (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs establish a link between T cells and target cells, such as tumor cells. This allows T cells to exert cytotoxic activity against target cells, such as tumor cells, by producing proteins like perforin and granzymes, which enter tumor cells and initiate apoptosis.

[0755] In some instances, BiTE can refer to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In other instances, BiTE can also refer to a bispecific T-cell adjuvant platform known in the art, which refers to a recombinant protein that simultaneously binds to two different antigens and has the ability to adjuvant immune cells (such as T cells).

[0756] In some instances, bispecific T-cell adjuvants (BiTEs) bind to two antigens; the first antigen is EpCAM; and the second antigen is an immune cell marker. In some instances, the second antigen-binding protein, its variants, or binding fragments bind to an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, CD64, etc.

[0757] In some instances, the multispecific antigen-binding protein is an inducible bispecific T-cell adaptor containing a heavy chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv).

[0758] In some instances, a bispecific T-cell adaptor (BiTE) binds to two antigens; the first antigen is an epithelial cell adhesion molecule (EpCAM); and the second antigen is CD3. In some examples, CD3 may include CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0759] In some instances, the modified / engineered immune cells can 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 instances, the modified / engineered immune cells are T cells, NK cells, or macrophages. In some instances, the modified / engineered immune cells are T cells.

[0760] In some instances, immune cells bind to HER2 and secrete an inducible bispecific T-cell adjuvant (HECAR-BiTE T) that targets EpCAM and CD3.

[0761] Not wanting to be bound by theory, while CAR cells directly kill target cells, anti-EpCAM BiTE exerts its cytotoxicity by recruiting nearby immune cells, such as T cells. In this way, bystander T cells are physically directed to the vicinity of the tumor, simultaneously activated and aiding in the elimination of tumor cells. By applying local secretion of anti-EpCAM BiTE by CAR-T cells, this invention transforms an undrugable target into a druggable one, as it reduces the non-tumor-targeting toxicity that may result from systemic delivery. Since EpCAM is also defined as a cancer stem cell marker expressed on cancer progenitors and cancer stem cells, the secretion of anti-EpCAM BiTE by CAR cells will consequently contribute to the prevention of cancer recurrence and relapse by eliminating cancer stem cells and progenitors.

[0762] Furthermore, as the experimental data disclosed herein demonstrate, HER2-targeting CAR-T cells secreting anti-EpCAM BiTE (HE CAR-BiTE T) exhibited superior efficacy in killing human tumors derived from gastric or breast cancer in vitro compared to HE CAR-T cells alone. Figure 1D and Figure 1E Compared to HE CAR-T alone, HE CAR-BiTE T clones showed higher AGS (HER2) in both 4D5 and F5. 高 EpCAM 高 ) and MDA-MB468 (HER2 极低 EpCAM 高 Percentage of cells lysed in MDA-MB468 (HER2) 极低 EpCAM 高 ()( Figure 1E In comparison, clones F5 and 4D5 showed minimal or reduced cell lysis in MDA-MB468 cells compared to their respective HE CAR-BiTE T cells. This indicates 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 this invention target only host cells when EpCAM expression levels are high, thus avoiding targeting cells with low EpCAM expression. Clone 4D5 of HE CAR-T cells could not tolerate low levels of interferon-γ secretion (… Figure 1F In the case of ) to initiate highly efficient killing of AGS cells ( Figure 1D This indicates that the cytotoxic function of HE CAR-BiTE T cells against clone 4D5 is primarily contributed by secreted anti-EpCAM BiTE. This disclosure also demonstrates that the modified cells disclosed herein achieve more efficient and durable tumor control compared to treatment with anti-HER2 CAR T cells alone (see Figure 2).

[0763] In some instances, the cells disclosed herein can bind to immune cells in the target environment, including but 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 instances, the immune cell is a T cell, a macrophage, a monocyte, or an NK cell. In some instances, the immune cell is a T cell.

[0764] Therefore, in some instances, the cells bind to HER2 and secrete multispecific antigen-binding proteins (e.g., HE CAR-BiTE T) that bind to both EpCAM and CD3.

[0765] Also disclosed are polypeptides comprising multispecific antigen-binding proteins. In some instances, the multispecific antigen-binding protein is a bispecific antibody. In some instances, the polypeptide comprises a multispecific antigen-binding protein that binds to EpCAM (epithelial cell adhesion molecule) and immune cells.

[0766] In some instances, the multispecific antigen-binding protein is a bispecific cell conjugate capable of binding both an antigen and an immune cell.

[0767] In some instances, the peptide contains an anti-EpCAM antigen-binding protein. In some instances, the peptide contains a single-domain anti-EpCAM antibody, optionally containing an anti-EpCAM H-chain antibody variable region (i.e., VHH).

[0768] In some instances, the peptide contains an anti-immune cell antigen-binding protein. In some instances, the peptide contains an anti-immune cell antigen-binding protein that binds to markers of immune cell activation. In some instances, the markers of immune cell activation are CD3, NKG2D, CD4, CD8, CD16, CD64, etc. In some instances, the peptide binds to CD3. In some instances, CD3 may include CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0769] In some instances, the peptide contains an anti-CD3 antigen-binding protein. In some instances, the peptide contains a single-chain variable fragment of an anti-CD3 antibody (anti-CD3 scFv).

[0770] In some instances, the immune cells are T cells, NK cells, macrophages, or monocytes. In other instances, the immune cells are T cells.

[0771] In some instances, the peptide is a bispecific antibody / antigen-binding protein. In some instances, the bispecific antibody / antigen-binding protein is a bispecific T-cell adaptor (BiTE), such as inducible BiTE, non-inducible BiTE, or constitutively expressed BiTE. In some embodiments, the bispecific T-cell adaptor (BiTE) binds to two antigens, wherein the first antigen is EpCAM and the second antigen is an immune cell marker. In some instances, the second antigen is an immune cell marker involved in immune cell activation. In some instances, the second antigen targeted by the bispecific T-cell adaptor (BiTE) may include, but is not limited to, CD3, NKG2D, CD28, CD16, CD64, etc. In some instances, the peptide comprises a BiTE that bispecifically binds to EpCAM and T cells. In some instances, the peptide comprises a BiTE that bispecifically binds to EpCAM and CD3. In some instances, EpCAM targeted by the BiTE is modified with an anti-EpCAM VHH paired with anti-CD3 scFv.

[0772] In some embodiments, anti-EpCAM VHH is paired with anti-CD3 scFv, which may include clone Okt3 (Nb01-013A).

[0773] On the other hand, polynucleotides encoding the cells and / or peptides and / or multispecific antigen-binding proteins described herein are provided.

[0774] It also discloses a polynucleotide containing a sequence encoding an immune cell adjuvant and a chimeric antigen receptor (CAR), wherein the immune cell adjuvant 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.

[0775] In some instances, a polynucleotide comprising a sequence encoding a chimeric antigen receptor is provided, which is capable of recognizing, binding to, and conjugating HER2-positive cells. In some instances, the primary antigen is HER2. Therefore, in some instances, the CAR is capable of binding to HER2.

[0776] In some instances, polynucleotides also contain sequences encoding one or more co-stimulatory domains, signal peptides, hinges, and / or signal transduction domains.

[0777] In some instances, co-stimulatory domains may include, but are not limited to, 4-1BB, CD28, CD27, OX-40, etc.

[0778] In some instances, the signal peptide can be, but is not limited to, IgH signal peptide, IgK signal peptide, CD8 signal peptide, etc.

[0779] In some instances, the hinge may be, but is not limited to, the IgH hinge, the hinge and / or transmembrane domain of immunoglobulin-like proteins (such as IgA, IgD, IgE, IgG, IgM, etc.), CD28, CD8, 4-1 BB, etc.

[0780] In some instances, the sequence encoding the CAR encodes a HER2 antigen-binding protein (or anti-HER2 antigen-binding protein), or a fragment or variant thereof. In some instances, the sequence encoding the CAR encodes a single-stranded variable fragment. In some instances, the sequence encoding the CAR encodes an anti-HER2 scFv.

[0781] In some instances, the sequence encoding the CAR also encodes an immune cell signaling domain. In some instances, the signaling domain may include, but is not limited to, intracellular domains of CD3, TCFζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3ε, CD3η, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some instances, the CD3 intracellular domain may be one or more of CD3ζ, CD3ε, CD3γ, and CD3δ. In some instances, the sequence encoding the CAR also encodes the CD3 intracellular domain (signaling domain).

[0782] In some instances, the sequence encoding CAR encodes anti-HER2 scFv, CD28 co-stimulatory domain, 4-1BB co-stimulatory domain, IgH signal peptide, IgH hinge, and CD3 signal transduction domain.

[0783] In some instances, the sequence encoding the immune cell adjuvant encodes a multispecific antigen-binding protein. In some instances, the multispecific antigen-binding protein is a bispecific antibody. In some instances, the sequence encoding the immune cell adjuvant encodes an antigen-binding protein or fragments or variants thereof capable of binding EpCAM (anti-EpCAM antigen-binding protein) as well as an anti-immune cell antigen-binding protein.

[0784] In some instances, the sequence encoding an immune cell adjuvant encodes a single-stranded variable fragment (scFv) or a single variable domain (VHH) located on the heavy strand.

[0785] In some instances, the sequence encoding the immune cell adaptor encodes anti-EpCAM scFv or anti-EpCAM VHH.

[0786] In some instances, the sequence encoding the immune cell adaptor encodes an anti-immune cell antigen-binding protein that binds to an immune cell activation marker. In some instances, the immune cell activation marker may include, but is not limited to, CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0787] In some instances, the immune activation marker may be in VHH or scFv form. In some instances, the sequence encoding the immune cell adjuvant encodes an antigen-binding protein (anti-CD3 antigen-binding protein) or a fragment or variant thereof capable of binding to CD3. In some instances, the sequence encoding the immune cell adjuvant encodes a single-stranded variable fragment (scFv) or VHH form. In some instances, the sequence encoding the immune cell adjuvant encodes an anti-CD3 scFv or an anti-CD3 single-domain VHH.

[0788] In some instances, immune cell adjuvants may contain a His tag.

[0789] In some instances, immune cell adaptors contain anti-EpCAM antigen-binding proteins, adaptors, anti-CD3 scFv or anti-CD3 single-domain VHH, and His tags. In some instances, the adaptor is a cleavable adaptor, which may include, but is not limited to, P2A, T2A, F2A, etc.

[0790] In some instances, the immune cells are T cells, NK cells, macrophages, or monocytes. In other instances, the immune cells are T cells.

[0791] In some instances, the polynucleotide containing the sequence encoding an immune cell adjuvant is a sequence encoding a bispecific T cell adjuvant (BiTE) (such as inducible BiTE, non-inducible BiTE, or constitutively expressed BiTE). In some instances, the polynucleotide contains a sequence encoding a BiTE that binds bispecifically to EpCAM and T cells. In some instances, the polynucleotide contains a sequence encoding an anti-HER2 scFv CAR with a CD3 intracellular domain, and a sequence encoding a BiTE that binds to EpCAM and T cells.

[0792] In yet another aspect, vectors for expressing the polynucleotides or polypeptides described herein are provided. In some instances, the vectors are selected from the group consisting of: plasmids, viral particles, bacteriophages, baculoviruses, yeast plasmids, lipid-based vectors, polymeric microspheres, liposomes and cell-based mediators, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, viral mediators, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, herpesviruses, vaccinia virus, foamy virus, cytomegalovirus, Semliki Forest virus, poxvirus, pseudorabies virus, RNA viral vectors, DNA viral vectors, and vectors derived from combinations of plasmids and bacteriophage DNA. Further optionally, the polynucleotide is operatively linked to one or more expression control sequences to direct peptide synthesis. Even further optionally, the vector contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0793] In some instances, the vector is a lentivirus vector.

[0794] In some implementations, the host cell contains a clone or expression vector as described above and / or a nucleic acid sequence encoding an antigen-binding protein, antibody, or binding fragment as described above.

[0795] The host cell can be any type of cell capable of being transformed or transfected with a nucleic acid or a vector to produce an antigen-binding protein or a binding fragment / protein thereof encoded by it. Host cells containing a nucleic acid or a vector can be used to produce an antigen-binding protein or a binding fragment / protein thereof, or a portion thereof (e.g., a heavy chain sequence or light chain sequence encoded by the nucleic acid or vector). After the nucleic acid or vector is introduced into the cell, the cell is cultured under conditions suitable for the expression of the encoding sequence. Antibodies, antigen-binding proteins, or fragments or portions of antibodies can then be isolated from the cell.

[0796] The host cell can be a prokaryotic host cell (such as *Escherichia coli*) or a eukaryotic host cell (such as yeast cells, insect cells, or vertebrate cells). When cultured under suitable conditions, the host cell expresses the antibody or a binding fragment thereof, which can then be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produced it (if it does not secrete it). The selection of a suitable host cell will depend on various factors, such as the desired expression level, the peptide modifications required or necessary for activity, such as glycosylation or phosphorylation, and the ease with which it folds into a biologically active molecule. The selection of the host cell will depend in part on whether the antibody or its binding fragment will be post-transcribed (e.g., glycosylation and / or phosphorylation). Host cells may include bacterial cells, yeast cells, animal cells such as mammalian cells, and / or plant cells.

[0797] Suitable mammalian host cells include CHO, myeloma, 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 number CCL61), human embryonic kidney (HEK) 293 or 293T cells (ATCC number CRL1573), 3T3 cells (ATCC number CCL92), or PER.C6 cells. Other cell types used for antibody expression include lymphocyte lines such as NSO myeloma cells and SP2 cells, and COS cells.

[0798] In one implementation, the host cell expresses / secretes the antigen-binding proteins, variants thereof, or fragments disclosed herein.

[0799] On the one hand, it provides cells that express / secrete EpCAM-specific immune cell adaptors.

[0800] In one embodiment, the cell is a stem cell, for example selected from the group consisting of mesenchymal stem cells, neural stem cells, and pluripotent stem cells (such as induced pluripotent stem cells (iPSCs)). Thus, in one embodiment, the stem cell is a mesenchymal stem cell. In one embodiment, the stem cell is a neural stem cell. In one embodiment, the stem cell is a pluripotent stem cell, such as an iPSC.

[0801] In one implementation, the cell is an immune cell.

[0802] In one embodiment, immune cells express / secrete an EpCAM-specific immune cell adjuvant. In one embodiment, the immune cell adjuvant is selected from the group consisting of T cell adjuvants, NK cell adjuvants, monocyte adjuvants, and macrophage adjuvants.

[0803] In one implementation, immune cells express / secrete a bispecific T-cell adaptor (BiTE) specific to EpCAM.

[0804] In one embodiment, immune cells express / secrete bispecific T-cell adaptors (BiTEs), such as inducible, non-inducible, or constitutive BiTEs comprising antigen-binding proteins, variants, or fragments thereof disclosed herein.

[0805] Surprisingly, the inventors have determined that immune cells, such as CAR T cells, are capable of secreting EpCAMBiTE. This was previously unknown. Advantageously, by engineering immune cells to express the EpCAM immune connective, the EpCAM immune connective is allowed to be secreted at the target site (e.g., at the site of a solid tumor), thereby minimizing toxicity and / or side effects.

[0806] In some instances, 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, monocytes, etc. In one embodiment, the immune cell is an NK cell. In one embodiment, the immune cell is a macrophage. In one embodiment, the immune cell is a dendritic cell. In one embodiment, the immune cell is a monocyte.

[0807] In one embodiment, the immune cell is a T cell. In one embodiment, the immune cell is a CAR T cell, such as an anti-GPC3, anti-HER2, or anti-CD19 CAR T cell. Therefore, in one embodiment, the CAR T cell is an anti-GPC3 CAR T cell. In one embodiment, the CAR T cell is an anti-HER2 CAR T cell. In one embodiment, the CAR T cell is an anti-CD19 CAR T cell. In one embodiment, the immune cell is a CAR T-, CAR NK-, CAR macrophage-, or CAR monocyte-.

[0808] In some instances, immune cells can bind to more than one host cell antigen. Therefore, in some embodiments, immune cells can also bind to one host cell antigen, two host cell antigens, three host cell antigens, four host cell antigens, etc.

[0809] In yet another aspect, methods for producing / generating the cells described herein are provided, which include introducing the polynucleotides described herein into cells. In some instances, the cells are immune cells.

[0810] Methods for producing / generating the cells described herein are also disclosed, wherein one or more nucleic acids encoding host cell target antigens are fused in a vector via an adapter (such as a P2A cleavable adapter); and wherein the vector is transformed or transfected into host cells. In some instances, the culture supernatant of the host cells transformed or transfected with the vector is harvested.

[0811] In some instances, the method involves introducing the vector described herein into cells. In some instances, the vector is introduced via viral transduction.

[0812] Compositions comprising cells or cell supernatants as described herein are also disclosed.

[0813] Pharmaceutical compositions comprising (engineered) cells or cell supernatants as described herein are also disclosed.

[0814] In some instances, the composition is a preventative and / or therapeutic composition.

[0815] Pharmaceutically acceptable agents used in the pharmaceutical compositions of the present invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavoring agents and diluents, emulsifiers, suspending agents, solvents, fillers, buffers, delivery media, tension agents, solubilizers, wetting agents, complexing agents, buffers, antimicrobial agents and surfactants.

[0816] Also disclosed are compositions or pharmaceutical compositions as described herein for use in treatments / medications / vaccines, optionally including excipients and / or stabilizers.

[0817] In yet another aspect, a method for treating a disease in a subject in need is provided, the method comprising administering to the subject cells as described herein, optionally, the disease being a proliferative disease.

[0818] Methods for treating diseases in subjects in need are also disclosed, which include administering engineered cells or compositions as described herein to the subjects.

[0819] Methods for preventing and / or reducing the severity of symptoms caused by a disease in subjects in need are also disclosed, which include administering engineered cells or compositions as described herein to the subjects.

[0820] In some instances, cells or compositions or pharmaceutical compositions will be administered to the subject via one or more routes of administration (including but not limited to local, intravascular, intravenous, oral, subcutaneous, intra-arterial, intrathecal, intraperitoneal, intranasal, intradermal, intramuscular, etc.).

[0821] The use of the cells described herein in the preparation of medicaments for the prevention and / or treatment of diseases is also disclosed.

[0822] It also discloses polynucleotides, antibodies, bispecific T-cell adaptors, engineered cells, engineered immune cells, methods, compositions, or pharmaceutical compositions as described herein.

[0823] In some instances, the disease is a proliferative disease. In some instances, the disease is a tumor or cancer. In some instances, the disease is cancer, and may include, but is not limited to, liver cancer (such as hepatocellular carcinoma), lung cancer (such as squamous cell carcinoma of the lung), stomach cancer (such as gastric adenocarcinoma), breast cancer, skin cancer (such as melanoma), ovarian cancer (such as clear cell carcinoma of the ovary), kidney cancer, pancreatic cancer, head and neck cancer, prostate cancer, esophageal cancer, bladder cancer, colon cancer, childhood cancers (such as hepatoblastoma, nephroblastoma, yolk sac tumor, etc.), etc.

[0824] Furthermore, when describing some embodiments, this disclosure may have disclosed methods and / or processes in a specific order of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific order of steps disclosed. Other orders of steps are also possible. The specific order of steps disclosed herein should not be construed as an undue limitation. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the order they are written. The order of steps may be changed, and still remain within the scope of this disclosure.

[0825] Furthermore, it should be understood that while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may be omitted in other alternative embodiments, and this disclosure provides support for such omissions and these related alternative embodiments.

[0826] sequence

[0827] CDRs are displayed in bold (CDR1), in bold and italics (CDR2), or in bold, italics, and underline (CDR3). The highlighted residues / bases show the differences between the constant domains of the 1B6 and 1C1 light chains.

[0828] 1. Amino acid sequence

[0829] 1.1. Variable domain of heavy chain in human IgG1

[0830] 1B6, 1C1, 1C11, 1D4, 1H6 (SEQ ID NO:4)

[0831] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL

[0832] 1E4 (SEQ ID NO:8)

[0833] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM

[0834] 1.2. Variable domain of human IgG1 light chain

[0835] 1B6 (SEQ ID NO:12)

[0836] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK

[0837] 1B6 (Light Chain Constant Structural Domain) (SEQ ID NO:13)

[0838] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC

[0839] 1C1 (SEQ ID NO:12)

[0840] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK

[0841] 1C1 (Light chain constant domain) (SEQ ID NO:14)

[0842] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC

[0843] 1C11 (SEQ ID NO:16)

[0844] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK

[0845] 1D4 (SEQ ID NO:18)

[0846] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK

[0847] 1E4 (SEQ ID NO:22)

[0848] DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK

[0849] 1H6 (SEQ ID NO:24)

[0850] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK

[0851] 1.3. Single-domain heavy-chain variable domain of llama VHH

[0852] 2C4-VHH (SEQ ID NO:35)

[0853] QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ

[0854] 1A5-VHH (SEQ ID NO:28)

[0855] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ

[0856] 1B8-VHH (SEQ ID NO:32)

[0857] QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ

[0858] 2B7-VHH (SEQ ID NO:34)

[0859] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ

[0860] 2D10-VHH (SEQ ID NO:36)

[0861] QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ

[0862] 1.4. Single-domain heavy-chain variable domain of humanized llama VHH

[0863] hu2C4-VHH (SEQ ID NO:37)

[0864] QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL

[0865] 1.5. Anti-EpCAM Okt3 BiTE

[0866] Nb01-013A (SEQ ID NO:75)

[0867] Notes

[0868] 2C4-VHH, 2C4-VHH (CDR1), 2C4-VHH (CDR2), 2C4-VHH (CDR3)

[0869] Okt3-VH

[0870] Okt3-VL

[0871] G4S or (G4S)3 connector

[0872] 6x His tags (for the purpose of detecting BiTE by ELISA or Western blotting)

[0873] QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQVTVSSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINHHHHHH

[0874] 2. Nucleotide sequence

[0875] 2.1 Heavy chain variable domain of human IgG1

[0876] 1B6, 1C1, 1C11, 1D4, 1H6 (SEQ ID NO:41)

[0877] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG

[0878] 1E4 (SEQ ID NO:45)

[0879] CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG

[0880] 2.2. Light chain variable domain of human IgG1

[0881] 1B6 (SEQ ID NO:49)

[0882] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG

[0883] 1B6 (light chain constant domain) (SEQ ID NO:50)

[0884] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0885] 1C1 (SEQ ID NO:49)

[0886] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG

[0887] 1C1 (Light chain constant domain) (SEQ ID NO:51)

[0888] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT

[0889] 1C11 (SEQ ID NO:53)

[0890] GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG

[0891] 1D4 (SEQ ID NO:55)

[0892] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG

[0893] 1E4 (SEQ ID NO:59)

[0894] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA

[0895] 1H6 (SEQ ID NO:61)

[0896] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG

[0897] 2.3. Single-domain variable heavy domain of llama VHH

[0898] 1A5 (SEQ ID NO:65)

[0899] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG

[0900] 1B8 (SEQ ID NO:69)

[0901] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG

[0902] 2B7 (SEQ ID NO:71)

[0903] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG

[0904] 2C4 (SEQ ID NO:72)

[0905] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG

[0906] 2D10 (SEQ ID NO:73)

[0907] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG

[0908] 2.4. Single - domain heavy - chain variable domain of humanized llama VHH

[0909] hu2C4 - VHH (SEQ ID NO:74)

[0910] CAGGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACAT ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG Attached Figure Description

[0911] Exemplary embodiments of this disclosure will be better understood and apparent to those skilled in the art from the following discussion and, if applicable, from the accompanying drawings. It should be understood that other modifications may be made without departing from the scope of the invention. The exemplary embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more embodiments to form new exemplary embodiments. The exemplary embodiments should not be construed as limiting the scope of this disclosure.

[0912] Figure 1A The construction and structural diagram of HE CAR-BiTE T (clone 4D5 or F5) using anti-EpCAM VHH BiTE (Nb01-013A) are shown. Figure 1B FACS analysis of tumor markers (HER2 and EpCAM) expressed on AGS and MDA-MB468 cells is shown. Figure 1C The percentage of CAR expression on anti-HER2 CAR T (4D5 or F5) and HECAR-BiTE T (4D5 or F5) cells, as detected by flow cytometry analysis, is shown. Figure 1D The percentage of AGS cells mediated by anti-HER2 CAR T (4D5 or 5F) and HE CAR-BiTE T (4D5 or F5) cells is shown when time-course measurements are performed using xCelligence impedance spectroscopy (E:T ratio = 2:1). Figure 1EThe percentage of MDA-MB468 cells mediated by anti-HER2 CAR T (4D5 or 5F) and HE CAR-BiTE T (4D5 or F5) cells is shown when time-course measurements are performed using xCelligence impedance spectroscopy (E:T ratio = 1:1). Figure 1F The bar chart shows the interferon-r and IL-2 measurements by ELISA, as measured using the culture supernatant collected after co-culturing effector CAR T cells with AGS cells for 36 hours. Figure 1G The bar chart shows the interferon-r and IL-2 measurements by ELISA, as measured using the culture supernatant collected after co-culturing effector CAR T cells with MDA-MB468 cells for 36 hours.

[0913] Figure 2 shows that human HE CAR-BiTE T cells constructed using anti-EpCAM VHH exhibited excellent tumor killing in in vivo AGS xenografts. 1.3 million AGS (Her2) xenografts were used. 高 EpCAM 高 Cells were subcutaneously injected into the right flank of female NSG mice (day -9). On day 0, mice were regrouped based on measurable tumor size and injected intravenously via the tail vein with 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 simulated T cells. Figure 2A The tumor size of each mouse was measured and recorded every 3 to 7 days. Figure 2B The tumor size of each individual mouse in each treatment group was measured and recorded every 3 to 7 days. Figure 2C The data shows that the weight of each mouse was measured and recorded every 3 to 7 days.

[0914] Figure 3This study demonstrates the detection of the anti-EpCAM BiTE molecule Nb01-013A secreted by HE CAR-BiTE T cells via ELISA. Anti-HER2 CAR T cells (4D5 or F5) or anti-HER2 CAR T cells secreting anti-EpCAM BiTE (Nb01-013A) were cultured in T cell growth medium containing IL-7 (20 ng / ml) and IL-15 (5 ng / ml) (designated "HECAR-BiTE T (4D5 or F5)"). The initial cell density was 1.5 million / ml, with CAR expression ranging from 36.8% to 57.2%. Cell culture supernatants were collected at 24 h and 48 h for ELISA to detect the amount of secreted anti-EpCAM BiTE molecule Nb01-013A. In short, human EpCAM-Fc-tagged protein was used to coat ELISA plates overnight. After blocking with casein for 2 hours, culture supernatant containing the anti-EpCAM BiTE molecule Nb01-013A was added to the plates. After incubation for 1 hour, the plates were washed, and the bound BiTE molecules were detected using a secondary antibody conjugated with HRP targeting the His tag.

[0915] Experimental Section

[0916] Construction and in vitro functional characterization of HE CAR-BiTE T construct

[0917] In this invention, the inventors fused the anti-EpCAM BiTE gene (using the sequence “Nb01-013A”) with an anti-HER2 CAR (from trastuzumab clone 4D5 or our internal anti-HER2 clone F5) lentiviral construct using a P2A cleavable adapter to construct the “HE CAR-BiTE T” ( Figure 1A After lentiviral transduction and amplification ( Figure 1C ), AGS (HER2) 高 EpCAM 高 ) and DA-MB468 (HER2 极低 EpCAM 高 )cell( Figure 1B Both were used to test the in vitro killing efficacy of CAR T cells. HE CAR-BiTE T cells showed that they could kill AGS cells at a faster rate than anti-HER2 CAR alone. Figure 1D This indicates that BiTE is involved in target cell killing. Surprisingly, anti-HER2 CAR T cells constructed from clone 4D5 could not secrete interferon-γ at low levels ( Figure 1F In the case of ) to initiate highly efficient killing of AGS cells ( Figure 1DThis indicates that the cytotoxic function of HECAR-BiTE T cells (clone 4D5) is primarily attributed to the secretion of anti-EpCAM BiTE. More interestingly, anti-HER2 CAR T cells (clone 4D5) showed resistance to MDA-MB468 (HER2). 极低 EpCAM 高 While HER2-mediated killing of cells at comparable levels, anti-HER2 CAR-T cells constructed using our patented clone F5 exhibited high specificity by not killing cells expressing very low levels of HER2. Figure 1E and Figure 1G ).

[0918] In summary, we have developed a novel CAR T-cell therapy targeting cancer (solid tumors originating from epithelial cells) in which CAR-T cells targeting a single antigen possess the additional ability to target EpCAM-positive cancer cells, overcoming the challenge of tumor heterogeneity through local secretion of anti-EpCAM BiTE. By using CAR T cells that secrete anti-EpCAM BiTE, we can also 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 promote tumor clearance while avoiding the systemic toxicity that may result from other methods of delivering anti-EpCAM BiTE. We have demonstrated the development of a prototype (HE CAR-BiTE), which also serves as a proof of concept. HE CAR-BiTE has been shown to work successfully, but our technology is not limited to HER2-targeting CARs and can be extended to many other tumor antigens that will be used as CAR targets for various types of cancer occurring in multiple organs.

[0919] Human HE CAR-BiTE cells showed superior killing efficacy in a mouse xenograft model.

[0920] Anti-EpCAM BiTE “Nb01-013A” was constructed by pairing anti-EpCAM VHH clone 2C4 with anti-human CD3 agonist clone Okt3. After testing the in vitro killing efficacy of HE CAR-BiTE T cells (“Nb01-013A” used as BiTE) using different CAR clones (4D5 or F5), we continued with in vivo testing using AGS (Her2) cells. 高 EpCAM 高 Xenograft models were used to test their tumor control efficacy (Figure 2). Consistent with in vitro data, AGS appeared to be resistant to 4D5 CAR-T. Although CAR T (4D5) failed to control tumor growth, other groups treated with CAR T (F5) or CAR-BiTE T cells showed some in vivo tumor control efficacy. Figure 2A) without significant weight loss Figure 2C CAR T (F5) treatment showed effective control of tumor growth in all 5 mice. However, after 5-6 weeks of tumor shrinkage, the remaining tumors began to regrow and reached a size exceeding 1000 mm. 3 This indicates that the tumor originated from Her2 阴性 Escape from the cell population. Effective tumor control was observed in 3 mice from the HECAR-BiTE T (4D5) group and 2 mice from the HECAR-BiTE T (F5) group, and these mice remained tumor-free for up to 181 days after T cell infusion. Figure 2B This shows that clearing Her2... 阴性 EpCAM 阳性 Tumor control is prolonged due to tumor cells.

[0921] Anti-EpCAM llama VHHBiTE secreted by HE CAR-BiTE T cells could be detected in cell cultures.

[0922] Nb01-013A BiTE molecules can also be detected in in vitro cultured HE CAR-BiTE T (4D5) and HE CAR-BiTE T (F5) cells. Anti-HER2 CAR T (4D5 or F5) cells or HE CAR-BiTE T (4D5 or F5) cells were cultured in T cell growth medium containing IL-7 and IL-15 at an initial cell density of 1.5 million / ml. Cell culture supernatants were collected at 24 h and 48 h, and the amount of secreted anti-EpCAM BiTE molecule Nb01-013A was detected by ELISA. For both HE CAR-BiTE T (4D5) and HE CAR-BiTE T (F5), the amount of secreted Nb01-013A molecules increased over time, indicating continuous secretion and accumulation of anti-EpCAM VHH BiTE from both cultures. Figure 3 ).

[0923] In summary, we have demonstrated that using the BiTE molecule constructed from anti-EpCAM VHH (Nb01-013A) in HE CAR-BiTE T cells achieves more efficient and durable tumor control compared to anti-Her2 CAR T cell therapy alone.

[0924] application

[0925] The modified cell implementation scheme disclosed herein utilizes the following factors: 1) tumor-directed T cell expansion, and 2) tumor-restricted secretion of anti-EpCAM BiTE, which is not achievable through conventional systemic delivery methods. The anti-EpCAM BiTE-secreting CAR T cells disclosed herein can be extended to other cancer types to establish a broader range of next-generation CAR T cell therapies.

[0926] The technology described in the current TDF may have the following applications:

[0927] 1. Anti-HER2 CAR T cells secreting anti-EpCAM BiTE can be used to treat HER2 expression. These anti-EpCAM BiTE CAR T cells can be used to target various 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.

[0928] 2. CAR T cells secreting anti-EpCAM BiTE can be configured to target other tumor-specific antigens, including but not limited to GPC3, Claudin18.2, ROR1, DLL3, CEA, MUC1, MUC16, CEACAM7, CD133, CD147, PSCA, PSMA, MSLN, c-Met, FRα, etc.

[0929] This disclosure improves the efficacy of current single-target CAR-T cell therapy for solid tumors by leveraging anti-EpCAM BiTE to overcome tumor heterogeneity and treatment-related tumor escape. Furthermore, by applying locally secreted anti-EpCAM BiTE from CAR T cells, a druggable target is transformed into a druggable one, as this reduces the non-tumor-targeting toxicity that may result from systemic delivery. Moreover, by targeting cancer progenitors and cancer stem cells expressing EpCAM, it further contributes to the complete eradication of tumors and the prevention of tumor recurrence.

[0930] Those skilled in the art will understand that other changes and / or modifications can be made to the embodiments disclosed herein without departing from the spirit or scope of this disclosure, which is broadly described herein. For example, features of different exemplary embodiments described herein may be mixed, combined, interchanged, incorporated, adopted, modified, included, or similarly used among different exemplary embodiments. Therefore, the present embodiments are to be considered illustrative rather than restrictive in all respects.

Claims

1. A modified cell expressing (a) a chimeric antigen receptor targeting HER2, and (b) a multi-specific antigen binding protein, variant or binding fragment thereof that binds one or more targets, comprising a first antigen binding protein, variant or binding fragment thereof that binds to EpCAM (epithelial cell adhesion molecule) and a second antigen binding protein, variant or binding fragment thereof that binds to an immune cell marker, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising: a CDR-H1 comprising: GSIFSGND (SEQ ID NO:25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or GSSERFTS (SEQ ID NO:29) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, a CDR-H2 comprising: ITSGGST (SEQ ID NO:26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or ITNGGST (SEQ ID NO:30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising: TNGRWSGDTYYAHH (SEQ ID NO:27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, MAGTS (SEQ ID NO:31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or TNGRWSGDTYYAHL (SEQ ID NO:33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; (ii) a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; (iii) a heavy chain variable region comprising: a CDR-H1 comprising GDSISSNSVA (SEQ ID NO: 5) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, a CDR-H2 comprising TYYRSKWYS (SEQ ID NO: 6) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and a CDR-H3 comprising AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; (iv) a light chain variable region comprising: a CDR-L1 comprising: QSLLHSNGYNY (SEQ ID NO: 9) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, QSLLHSNRYNY (SEQ ID NO: 17) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, or QSISDF (SEQ ID NO:19) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. a CDR-L2 comprising: LGS (SEQ ID NO: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or AAS (SEQ ID NO:20) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-L3 comprising: MQALQTPYT (SEQ ID NO: 11) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, MQGLQSPWT (SEQ ID NO: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, QQSYIMPDT (SEQ ID NO:21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or MQGLQTPYT (SEQ ID NO:23) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

2. The cell of claim 1, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising: a CDR-H1 comprising GSIFSGND (SEQ ID NO: 25) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; (ii) a heavy chain variable region comprising: CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; (iii) a heavy chain variable region comprising: CDR-H1 comprising GDSISSNSVA (SEQ ID NO: 5) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising TYYRSKWYS (SEQ ID NO: 6) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (iv) a light chain variable region comprising: CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 comprising MQALQTPYT (SEQ ID NO: 11) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (v) a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, a CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and a CDR-L3 comprising MQGLQSPWT (SEQ ID NO: 15) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (vi) a light chain variable region comprising: a CDR-L1 comprising QSLLHSNRYNY (SEQ ID NO: 17) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, a CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and a CDR-L3 comprising MQALQTPYT (SEQ ID NO: 11) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (vii) a light chain variable region comprising: a CDR-L1 comprising QSISDF (SEQ ID NO: 19) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, a CDR-L2 comprising AAS (SEQ ID NO: 20) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and a CDR-L3 comprising QQSYIMPDT (SEQ ID NO: 21) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto (viii) a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, a CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and a CDR-L3 comprising MQGLQSPWT (SEQ ID NO: 15) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-L3 comprising MQGLQTPYT (SEQ ID NO:23) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto (ix) a heavy chain variable region comprising: CDR-H1 comprising GSSERFTS (SEQ ID NO:29) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising ITNGGST (SEQ ID NO:30) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising MAGTS (SEQ ID NO:31) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and (x) a heavy chain variable region comprising: CDR-H1 comprising GSIFSGND (SEQ ID NO:25) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising ITSGGST (SEQ ID NO:26) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising TNGRWSGDTYYAHL (SEQ ID NO:33) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

3. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable region and / or a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising: CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising IIPIFGTA (SEQ ID NO:2) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising ARSLGGRFRY (SEQ ID NO:3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or a light chain variable region comprising: CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 comprising MQALQTPYT (SEQ ID NO: 11) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (ii) a heavy chain variable region comprising: CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or a light chain variable region comprising: CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-L3 comprising MQGLQSPWT (SEQ ID NO: 15) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (iii) a heavy chain variable region comprising: CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-H3 comprising the sequence of ARSLGGRFRY (SEQ ID NO:3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or a heavy chain variable region comprising: CDR-H1 comprising the sequence of GGTFSSYA (SEQ ID NO: 1) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising the sequence of IIPIFGTA (SEQ ID NO:2) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising the sequence of ARSLGGRFRY (SEQ ID NO:3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or a heavy chain variable region comprising: CDR-H1 comprising the sequence of GDSISSNSVA (SEQ ID NO:5) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising the sequence of RYWSNSLY (SEQ ID NO:6) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising the sequence of ARSLGGRFRY (SEQ ID NO:3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and / or a heavy chain variable region comprising: CDR-H1 comprising the sequence of GDSISSNSVA (SEQ ID NO:5) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising the sequence of RYWSNSLY (SEQ ID NO:6) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising the sequence of ARSLGGRFRY (SEQ ID NO:3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and (v) a heavy chain variable region comprising: CDR-H1 comprising the sequence of GDSISSNSVA (SEQ ID NO:5) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising the sequence of RYWSNSLY (SEQ ID NO:6) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and CDR-H3 comprising the sequence of ARSLGGRFRY (SEQ ID NO:3) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. CDR-H2 comprising TYYRSKWYS (SEQ ID NO: 6) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and CDR-H3 comprising AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and / or a heavy chain variable region comprising: CDR-H1 comprising GSSERFTS (SEQ ID NO: 29) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto (ii) a heavy chain variable region comprising: CDR-H1 comprising GSSERFTS (SEQ ID NO: 29) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, GSIFSGND (SEQ ID NO:25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or GSSERFTS (SEQ ID NO:29) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and ITSGGST (SEQ ID NO:26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or ITNGGST (SEQ ID NO:30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto TNGRWSGDTYYAHH (SEQ ID NO:27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, MAGTS (SEQ ID NO: 31 - 1B8) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, or TNGRWSGDTYYAHL (SEQ ID NO:33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

4. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable region comprising: CDR-H1 comprising GSSERFTS (SEQ ID NO: 29) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto 5. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising: CDR-H1 comprising GSSERFTS (SEQ ID NO: 29) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto (ii) a heavy chain variable region comprising: CDR-H1 comprising GSSERFTS (SEQ ID NO: 29) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto CDR-H2 comprising ITNGGST (SEQ ID NO: 30) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, and CDR-H3 comprising MAGTS (SEQ ID NO: 31) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto; and (iii) a heavy chain variable region comprising: CDR-H1 comprising GSIFSGND (SEQ ID NO: 25) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto, and CDR-H3 comprising TNGRWSGDTYYAHL (SEQ ID NO: 33) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

6. The cell of any one of the preceding claims, wherein 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) a heavy chain variable domain comprising QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (ii) a heavy chain variable domain comprising QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (iii) a heavy chain variable domain comprising EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (iv) a heavy chain variable domain comprising QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (v) a light chain variable domain comprising DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (vi) a light chain variable domain comprising EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (vii) a light chain variable domain comprising DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (viii) a light chain variable domain comprising DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (ix) a light chain variable domain comprising DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (x) a heavy chain variable domain comprising QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (xi) a heavy chain variable domain comprising QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or (xii) a heavy chain variable domain comprising QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or (xiii) a heavy chain variable domain comprising QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions.

7. The cell of any one of the preceding claims, wherein 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) a heavy chain variable domain comprising: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or a light chain variable domain comprising: DIQMTQSPSSLSASVGDRVTITCQASQSISSYLAWYQQKPGKAPKLLIYGASNLESGFIPDFKVHNAKNTVYLQMNSLKPEDTAVYYNYLRFSGVPSRFSGSGTKLE (SEQ ID NO: 5) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions. DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (ii) a heavy chain variable domain comprising: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or a light chain variable domain comprising: EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, (iii) a heavy chain variable domain comprising: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or a light chain variable domain comprising: EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or (iv) a heavy chain variable domain comprising: QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or a light chain variable domain comprising: DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, or (v) a heavy chain variable domain comprising: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, and / or a light chain variable domain DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO:24) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions.

8. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a single domain heavy chain variable domain having the sequence of: (i) QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, or (ii) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, or (iii) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, or (iv) QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, or (v) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions, or (vi) QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36) or a fragment, variant or sequence thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 2 or 3 amino acid substitutions.

9. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable domain encoded by a nucleotide sequence comprising: (i) a heavy chain variable region comprising: a CDR-H1 comprising GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and a CDR-H3 comprising ACAAACGGAAGATGGTCAGGCGATACTT ACTATGCCCATCAC (SEQ ID NO: 64) (ii) a heavy chain variable region comprising: a CDR-H1 comprising GGAAGCTCCGAAAGATTCACATCA (SEQ ID NO: 66), a CDR-H2 comprising ATTACTAATGGTGGTAGCACA (SEQ ID NO: 67), and a CDR-H3 comprising ACAAACGGAAGATGGTCAGGCGATACTT ACTATGCCCATCAC (SEQ ID NO: 64) CDR-H3 comprising ATGGCGGGTACGTCC (SEQ ID NO: 68); and (iii) a heavy chain variable region comprising: CDR-H1 comprising GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO: 62), CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 comprising ACAAACGGAAGATGGTCAGGCGATACTT ACTATGCCCATCTC (SEQ ID NO: 70); or a fragment or variant or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

10. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable domain encoded by a nucleotide sequence comprising: (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 72) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, or (ii) a heavy chain variable region comprising: CDR-H1 comprising GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO: 62), CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 comprising ACAAACGGAAGATGGTCAGGCGATACTT ACTATGCCCATCTC (SEQ ID NO: 70); or a fragment or variant or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleic acid substitutions thereof, or (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleic acid substitutions thereof, or (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleic acid substitutions thereof, or (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO: 45) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, or (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO: 69) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or having 10-20 nucleic acid substitutions, or (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or having 10-20 nucleic acid substitutions, or (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or having 10-20 nucleic acid substitutions thereto, and / or a light chain variable domain encoded by a nucleotide sequence comprising: (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or having 10-20 nucleic acid substitutions thereto, or (ii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or having 10-20 nucleic acid substitutions thereto, or (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or having 10-20 nucleic acid substitutions, or (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or having 10-20 nucleic acid substitutions, or (iv) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, or (v) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions.

11. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof 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) a heavy chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or a light chain variable domain encoded by a nucleotide sequence comprising: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:49) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, (ii) a heavy chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or a light chain variable domain encoded by a nucleotide sequence comprising: GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO:53) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, (iii) a heavy chain variable domain encoded by a nucleotide sequence comprising: GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO:53) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or a light chain variable domain encoded by a nucleotide sequence comprising: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:55) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, (iv) a heavy chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or a light chain variable domain encoded by a nucleotide sequence comprising: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO:61) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or (v) a heavy chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions, and / or a heavy chain variable domain encoded by a nucleotide sequence comprising: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO:59) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions.

12. The cell of any one of the preceding claims, wherein the first antigen binding protein, variant or binding fragment thereof that binds to EpCAM comprises a heavy chain variable domain encoded by a nucleotide sequence comprising: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO:59) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto and / or having 10-20 nucleic acid substitutions. (i) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleotide substitutions thereof, or (ii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 72) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleotide substitutions thereof, or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleotide substitutions thereof, or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or having 10-20 nucleic acid substitutions, or (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO: 69) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or having 10-20 nucleic acid substitutions, or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleotide substitutions thereof, or (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleotide substitutions thereof, or (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 75) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or with 10-20 nucleotide substitutions thereof, or (vii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO:41) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or having 10-20 nucleic acid substitutions thereof, or (viii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45) or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity and / or having 10-20 nucleic acid substitutions thereof.

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

14. The cell of any one of the preceding claims, wherein the multispecific antigen binding protein, variant or fragment thereof is an immune cell engager selected from the group comprising a T cell engager, an NK cell engager, a monocyte engager and a macrophage engager.

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

16. The cell of any one of the preceding claims, wherein the second antigen binding protein, variant or binding fragment thereof binds an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16 and CD6.

17. The cell of 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 of any one of the preceding claims, wherein the cell is an immune cell, for example selected from the group consisting of a T cell, a macrophage, a monocyte and an NK cell.

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

20. The cell of any one of the preceding claims, wherein the cell binds to HER2 and secretes an inducible immune cell engager targeting EpCAM and CD3 (HE CAR-BiTE T).

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

22. A vector expressing the polynucleotide of claim 21.

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

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

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

26. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the cell of any one of claims 1 to 20 or the composition of claim 25, optionally the disease is a proliferative disease, optionally a cancer.

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