GPC3 chimeric antigen receptor secretion

Abstract

A modified cell is provided that expresses (a) a chimeric antigen receptor targeting GPC3 and / or CD19, and (b) a multispecific antigen binding protein, variant or binding fragment thereof that binds to 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, which binds to an immune cell marker, wherein the first antigen binding protein, variant or binding fragment thereof, which binds 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 generating / generating cells as disclosed herein and methods of treating with cells as disclosed herein.

Description

Technical Field

[0001] This disclosure generally 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 that express chimeric antigen receptors targeting tumor antigens, and multispecific antigen-binding proteins, variants, or binding fragments that bind to one or more targets, 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 immune cell markers. Background Technology

[0002] It is projected that the global cancer incidence rate will rise to 28.4 million by 2040. Of all cancer cases in adults, 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. Because epithelial tissue is the most abundant tissue in the body, malignant tumors of epithelial tissue, also known as “carcinomas,” account for 80% to 90% of all solid tumor cases. For example, hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for approximately 90% of all liver cancer cases. Its mortality rate is very high, with a 5-year overall survival rate of only 18%. Since chemotherapy is no longer favored in HCC, the current standard of care for patients with metastatic or unresectable HCC with adequate liver function is the combination therapy of atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF). Subsequently, the definition of second-line treatment is less clear, and there is undoubtedly a significant unmet need in patients who have undergone atezolizumab-bevacizumab treatment. Breast cancer in women is another example, with ductal carcinoma and lobular carcinoma originating from epithelial tissue being the most common types of primary breast cancer, accounting for 90% of all breast cancer cases. Other examples include tumors found in the skin, ovaries (>90%), kidneys (>85%), lungs (>85%), stomach (approximately 89%), pancreas (>90%), head and neck (>90%), and prostate (>95%), most of which are cancerous. Although the chance of cure for "carcinoma in situ" is high, for most invasive or metastatic cancers, even with multiple treatment options available (such as surgery, chemotherapy, radiation therapy, and targeted therapy), the five-year survival rate is approximately 20-30%.

[0003] Compared to traditional treatment strategies, antibody and cell-based therapies are gaining increasing attention and R&D investment due to their potential as more promising standards of care for cancer treatment. Cell-based therapies, such as chimeric antigen receptor (CAR) T-cell therapy, have achieved great success in hematologic malignancies, but have shown limited efficacy in treating solid tumors. Among the complex challenges encountered by each approach, a fundamental problem lies in the heterogeneous distribution of tumor markers within the solid tumor cell population and antigen escape during treatment.

[0004] Immune escape from target antigen-negative cells is 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. Since tumor cells express a multi-surface antigen profile, one potential approach to overcome this challenge is to engineer CAR T cells with dual or multiple targets to combat tumor escape; for example, bispecific anti-CD19 / CD20 CAR T-cell therapy has shown early promise in treating relapsed or refractory B-cell lymphomas. However, finding multiple tumor-specific targets in solid tumors is extremely difficult. In fact, most tumor-associated antigens (TAAs) are also expressed at low levels in normal tissues, so the high efficacy of CARs may induce intolerable targeted non-tumor toxicity.

[0005] Therefore, there is a need for an alternative method to prevent cancer recurrence. There is a need for an alternative cell-based therapy. There is an urgent need for an alternative chimeric antigen receptor cell. Summary of the Invention

[0006] Phosphatidylinositol proteoglycan 3 (GPC3) is an oncogene protein that can be used as a highly tumor-specific target. It is widely expressed during embryonic development; however, its expression is strictly suppressed in most adult tissues. Elevated GPC3 expression has been reported in various tumor types, such as liver cancer, lung cancer, gastric cancer, ovarian cancer, and esophageal cancer. GPC3-targeting immunotherapies, such as CAR T-cell therapy, have demonstrated some therapeutic efficacy and overall safety in clinical practice. However, only a very limited number of patients achieve complete remission (CR). Metastasis and recurrence of the disease after treatment targeting these markers can largely be attributed to the fact that, although GPC3 is found at high levels in these cancers at diagnosis, not all tumor cells express GPC3. Re-emerging tumor masses often originate from the growth of antigen-negative cancer cells or escaped cancer cells whose antigen expression is subsequently downregulated or even lost after CAR T-cell therapy. Despite the very high reported complete remission (CR) rates, FDA-approved anti-CD19-guided CAR T-cell therapies such as "Kymriah" or "Yescarta" face the same antigen escape problem when treating various B-cell-origin leukemias and lymphomas. To overcome this challenge, the concept of dual-target CAR T-cells is frequently employed, and bispecific anti-CD19 / CD20 CAR T-cell therapies have shown early promise in treating relapsed or refractory B-cell lymphomas. However, in solid tumors, it is extremely difficult to find two or more highly specific tumor-specific antigens that can be targeted, as most tumor markers are also expressed in normal tissues, albeit at relatively low levels. Because CAR T-cells have the inherent ability to efficiently kill cancer cells, designing dual CARs targeting two or more antigens can potentially induce intolerable targeting-related non-tumor toxicities, and is therefore considered extremely dangerous for patients with solid tumors.

[0007] EpCAM (epithelial cell adhesion molecule) represents another class of biomarkers, widely expressed in almost all cancers. Cancer therapies targeting EpCAM have undergone over 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, both solitomab and catuxomab (anti-EpCAM BiTE) have shown dose-limiting toxicities and have failed to receive FDA approval.

[0008] Interestingly, EpCAM, a broad-spectrum biomarker for epithelial cells, has been found to co-express with GPC3 in many cancers. This disclosure develops a therapeutic approach that, instead of using a dual CAR setup, employs GPC3-targeting CAR T cells that secrete an anti-EpCAM bispecific T-cell connective (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 “GE CAR-BiTE T” (GPC3-targeting CAR T cells with anti-EpCAM BiTE secretion function).

[0009] CAR-BiTE T cells secreting anti-EpCAM BiTE first infiltrate and remain at the tumor site by recognizing specific tumor antigens expressed on tumor cells via CAR. Upon binding to the CAR target, 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 and activated, subsequently helping to clear tumor cells. Since GPC3 overexpression is tumor-specific, the expansion of anti-EpCAM BiTE is likely limited to the tumor site or its vicinity. Furthermore, because anti-GPC3 CAR T cells secrete recombinant anti-EpCAM BiTE antibodies inefficiently, further diffusion into normal tissues, along with the immediate presence of the required T cells, may reduce its targeted non-tissue toxicity. Additionally, EpCAM is defined as a cancer stem cell marker because it has been found to be expressed in both cancer progenitors and cancer stem cells. CAR T cell secretion of anti-EpCAM BiTE will help prevent cancer recurrence and relapse by simultaneously eliminating both cancer stem cells and progenitors.

[0010] As the experimental data disclosed herein show, GECAR-BiTE cells equipped with novel BiTE (Nb01-013A or Nb01-013B) exhibited superior performance in killing HepG2 and HT-29 cells, with potency comparable to MT110 BiTE. While MT110-secreting T cells also induced strong cytotoxicity against HeyA8 cells, GECAR-BiTE T cells using novel BiTE did not harm these target negative cells. Figure 5C , 5D(and 5E). Furthermore, anti-EpCAM BiTE can be injected directly into the tumor site for targeted therapy. The anti-EpCAM BiTE of this disclosure, secreted locally at the tumor site by CAR T cells (rather than systemic administration), limits its potential toxicity. This transforms a non-drugable (or difficult-to-drug) target into a dosable one.

[0011] In one respect, a modified cell is provided that expresses

[0012] (a) Chimeric antigen receptors targeting GPC3 and / or CD19, and

[0013] (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 binds to EpCAM (epithelial cell adhesion molecule), and a second antigen-binding protein, its variants, or binding fragments that bind to immune cell markers.

[0014] The first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region and / or a light chain variable region selected from the following:

[0015] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5, 1B8, 2B7 and 2D10)

[0016] CDR-H1, wherein CDR-H1 comprises:

[0017] GSIFSGND (SEQ ID NO: 25 - 2C4, hu2C4, 1A5, 2B7 and 2D10), or

[0018] GSSERFTS (SEQ ID NO: 29 - 1B8)

[0019] CDR-H2, wherein CDR-H2 comprises:

[0020] ITSGGST (SEQ ID NO: 26 - 2C4, hu2C4, 1A5, 2B7 and 2D10), or

[0021] ITNGGST (SEQ ID NO: 30 - 1B8), and

[0022] CDR-H3, wherein CDR-H3 comprises:

[0023] TNGRWSGDTYYAHH (SEQ ID NO: 27 - 2C4, hu2C4, 1A5, 2D10),

[0024] MAGTS (SEQ ID NO: 31 - 1B8), or

[0025] TNGRWSGDTYYAHL (SEQ ID NO: 33 - 2B7)

[0026] (ii) Heavy chain variable regions, comprising: (1B6, 1C1, 1C11, 1D4, and 1H6)

[0027] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0028] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0029] CDR-H3, wherein CDR-H3 contains ARSLGGRFRY (SEQ ID NO: 3),

[0030] (iii) Heavy chain variable region, which contains: (1E4)

[0031] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0032] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0033] CDR-H3, wherein CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO: 7),

[0034] (iv) Light chain variable regions comprising: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6)

[0035] CDR-L1, wherein CDR-L1 includes:

[0036] QSLLHSNGYNY (SEQ ID NO: 9 - 1B6, 1C1, 1C11 and 1H6),

[0037] QSLLHSNRYNY (SEQ ID NO: 17 - 1D4), or

[0038] QSISDF (SEQ ID NO: 19 - 1E4)

[0039] CDR-L2, wherein CDR-L2 includes:

[0040] LGS (SEQ ID NO: 10 - 1B6, 1C1, 1C11, 1D4 and 1H6), or

[0041] AAS (SEQ ID NO: 20 - 1E4), and

[0042] CDR-L3, wherein CDR-L3 includes:

[0043] MQALQTPYT (SEQ ID NO: 11 - 1B6, 1C1 and 1D4),

[0044] MQGLQSPWT (SEQ ID NO: 15 - 1C11),

[0045] QQSYIMPDT (SEQ ID NO: 21 - 1E4), or

[0046] MQGLQTPYT (SEQ ID NO: 23 - 1H6), and

[0047] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0048] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM include a heavy chain variable region comprising: (1A5, 1B8, 2B7, 2C4, 2D10, and hu2C4).

[0049] CDR-H1, wherein CDR-H1 comprises:

[0050] GSIFSGND (SEQ ID NO: 25 - 1A5, 2B7, 2C4, 2D10 and hu2C4), or

[0051] GSSERFTS (SEQ ID NO: 29 - 1B8)

[0052] CDR-H2, wherein CDR-H2 comprises:

[0053] ITSGGST (SEQ ID NO: 26 - 1A5, 2B7, 2C4, 2D10 and hu2C4), or

[0054] ITNGGST (SEQ ID NO: 30 - 1B8); and

[0055] CDR-H3, wherein CDR-H3 comprises:

[0056] TNGRWSGDTYYAHH (SEQ ID NO: 27 - 1A5, 2C4, 2D10 and hu2C4),

[0057] MAGTS (SEQ ID NO: 31 - 1B8), or

[0058] TNGRWSGDTYYAHL (SEQ ID NO: 33 - 2B7)

[0059] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0060] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a heavy chain variable region selected from the following:

[0061] (i) Heavy chain variable regions, which include: (2C4-VHH, hu2C4-VHH, 1A5-VHH and 2D10-VHH)

[0062] CDR-H1, wherein CDR-H1 contains GSIFSGND (SEQ ID NO: 25),

[0063] CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26), and

[0064] CDR-H3, wherein CDR-H3 comprises TNGRWSGDTYYAHH (SEQ ID NO: 27)

[0065] (ii) Heavy chain variable region, which contains: (1B8-VHH)

[0066] CDR-H1, wherein CDR-H1 contains GSSERFTS (SEQ ID NO: 29),

[0067] CDR-H2, wherein CDR-H2 comprises ITNGGST (SEQ ID NO: 30), and

[0068] CDR-H3, wherein CDR-H3 comprises MAGTS (SEQ ID NO: 31); and

[0069] (iii) Heavy chain variable region, which contains: (2B7-VHH)

[0070] CDR-H1, wherein CDR-H1 contains GSIFSGND (SEQ ID NO: 25),

[0071] CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26), and

[0072] CDR-H3, wherein CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO: 33),

[0073] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0074] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise heavy chain variable regions and / or light chain variable regions selected from the following:

[0075] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5 and 2D10)

[0076] CDR-H1, wherein CDR-H1 contains GSIFSGND (SEQ ID NO: 25),

[0077] CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26), and

[0078] CDR-H3, wherein CDR-H3 comprises TNGRWSGDTYYAHH (SEQ ID NO: 27)

[0079] (ii) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1H6)

[0080] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0081] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0082] CDR-H3, wherein CDR-H3 contains ARSLGGRFRY (SEQ ID NO: 3)

[0083] (iii) Heavy chain variable region, which contains: (1E4)

[0084] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0085] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0086] CDR-H3, wherein CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO: 7)

[0087] (iv) Light chain variable region, which comprises: (1B6 and 1C1)

[0088] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0089] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0090] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0091] (v) Light chain variable region, which contains: (1C11)

[0092] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0093] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0094] CDR-L3, wherein CDR-L3 comprises MQGLQSPWT (SEQ ID NO: 15)

[0095] (vi) Light chain variable region, which contains: (1D4)

[0096] CDR-L1, wherein CDR-L1 contains QSLLHSNRYNY (SEQ ID NO: 17),

[0097] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0098] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0099] (vii) Light chain variable region, which includes: (1E4)

[0100] CDR-L1, wherein CDR-L1 contains QSISDF (SEQ ID NO: 19),

[0101] CDR-L2, wherein CDR-L2 includes AAS (SEQ ID NO: 20), and

[0102] CDR-L3, wherein CDR-L3 includes QQSYIMPDT (SEQ ID NO: 21)

[0103] (viii) Light chain variable region, which contains: (1H6)

[0104] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0105] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0106] CDR-L3, wherein CDR-L3 comprises MQGLQTPYT (SEQ ID NO: 23)

[0107] (ix) Heavy chain variable region, which contains: (1B8-VHH)

[0108] CDR-H1, wherein CDR-H1 contains GSSERFTS (SEQ ID NO: 29),

[0109] CDR-H2, wherein CDR-H2 comprises ITNGGST (SEQ ID NO: 30), and

[0110] CDR-H3, wherein CDR-H3 comprises MAGTS (SEQ ID NO: 31); and

[0111] (x) Heavy chain variable region, which contains: (2B7-VHH)

[0112] CDR-H1, wherein CDR-H1 contains GSIFSGND (SEQ ID NO: 25),

[0113] CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26), and

[0114] CDR-H3, wherein CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO: 33)

[0115] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0116] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a heavy chain variable domain and / or a light chain variable domain selected from the following:

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

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

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

[0120] QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37 - hu2C4-VHH)

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

[0122] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4 - 1B6, 1C1, 1C11, 1D4 and 1H6)

[0123] (iv) Heavy-chain variable structural domains, which contain

[0124] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8 - 1E4),

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

[0126] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12 - 1B6 and 1C1)

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

[0128] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16 - 1C11)

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

[0130] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18 - 1D4)

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

[0132] DIQLTQSPSSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22 - 1E4)

[0133] (ix) Light chain variable structural domain, which contains

[0134] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24 - 1H6)

[0135] (x) Heavy-chain variable structural domains, which contain

[0136] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28- 1A5-VHH)

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

[0138] QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32 - 1B8-VHH)

[0139] (xii) Heavy-chain variable structural domain, which contains

[0140] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34 - 2B7-VHH)

[0141] (xiii) Heavy-chain variable structural domains, which contain

[0142] and

[0143] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has two or three amino acid substitutions.

[0144] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a single-domain heavy-chain variable domain having the following sequence:

[0145] (i) QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35 - clone 2C4-VHH), or

[0146] (ii) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37 - hu2C4-VHH), or

[0147] (iii) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28 - clone 1A5-VHH), or

[0148] (iv) QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32 - clone 1B8-VHH), or

[0149] Or

[0150] (vi)QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36 - clone 2D10-VHH),

[0151] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has two or three amino acid substitutions.

[0152] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a heavy chain variable region selected from the following:

[0153] (i) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4 and 1H6)

[0154] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0155] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0156] CDR-H3, wherein CDR-H3 contains ARSLGGRFRY (SEQ ID NO: 3)

[0157] (ii) Heavy chain variable region, which contains: (1E4)

[0158] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0159] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0160] CDR-H3, wherein CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO: 7); and

[0161] It contains a light chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6).

[0162] CDR-L1, wherein CDR-L1 includes:

[0163] QSLLHSNGYNY (SEQ ID NO: 9 - 1B6, 1C1, 1C11 and 1H6),

[0164] QSLLHSNRYNY (SEQ ID NO: 17 - 1D4), or

[0165] QSISDF (SEQ ID NO: 19 - 1E4)

[0166] CDR-L2, wherein CDR-L2 includes:

[0167] LGS (SEQ ID NO: 10-1B6, 1C1, 1C11, 1D4 and 1H6), or

[0168] AAS (SEQ ID NO: 20 - 1E4), and

[0169] CDR-L3, wherein CDR-L3 includes:

[0170] MQALQTPYT (SEQ ID NO: 11 - 1B6, 1C1 and 1D4),

[0171] MQGLQSPWT (SEQ ID NO: 15 - 1C11),

[0172] QQSYIMPDT (SEQ ID NO: 21 - 1E4), or

[0173] MQGLQTPYT (SEQ ID NO: 23 - 1H6)

[0174] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0175] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a heavy chain variable region and a light chain variable region selected from the following:

[0176] (i) Heavy chain variable region, which contains: (1B6 and 1C1)

[0177] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0178] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0179] CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3); and

[0180] The light chain variable region, which includes:

[0181] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0182] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0183] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0184] (ii) Heavy chain variable region, which contains: (1C11)

[0185] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0186] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0187] CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3); and

[0188] The light chain variable region, which includes:

[0189] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0190] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0191] CDR-L3, wherein CDR-L3 comprises MQGLQSPWT (SEQ ID NO: 15)

[0192] (iii) Heavy chain variable region, which contains: (1D4)

[0193] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0194] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0195] CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3); and

[0196] The light chain variable region, which includes:

[0197] CDR-L1, wherein CDR-L1 contains QSLLHSNRYNY (SEQ ID NO: 17),

[0198] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0199] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0200] (iv) Heavy chain variable region, which contains: (1H6)

[0201] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0202] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0203] CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3); and

[0204] The light chain variable region, which includes:

[0205] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0206] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0207] CDR-L3, wherein CDR-L3 comprises MQGLQTPYT (SEQ ID NO: 23); and

[0208] (v) Heavy chain variable region, which contains: (1E4)

[0209] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0210] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0211] CDR-H3, wherein CDR-H3 comprises AREVEGSSYDAFDI (SEQ ID NO: 7); and

[0212] The light chain variable region contains: (1E4)

[0213] CDR-L1, wherein CDR-L1 contains QSISDF (SEQ ID NO: 19),

[0214] CDR-L2, wherein CDR-L2 includes AAS (SEQ ID NO: 20), and

[0215] CDR-L3, wherein CDR-L3 includes QQSYIMPDT (SEQ ID NO: 21)

[0216] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0217] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a heavy chain variable domain and a light chain variable domain selected from the following:

[0218] (i) Heavy chain variable structural domain, which contains: (1B6 and 1C1)

[0219] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and

[0220] Light chain variable structural domain, which contains

[0221] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12)

[0222] (ii) Heavy-chain variable structural domains, which contain: (1C11)

[0223] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and

[0224] Light chain variable structural domain, which contains

[0225] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16)

[0226] (iii) Heavy chain variable structural domain, which contains: (1D4)

[0227] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and

[0228] Light chain variable structural domain, which contains

[0229] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18)

[0230] (iv) Heavy-chain variable structural domains, which contain: (1E4)

[0231] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), and

[0232] Light chain variable structural domain, which contains

[0233] DIQLTQSPSSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or

[0234] (v) Heavy-chain variable structural domain, which contains: (1H6)

[0235] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and

[0236] Light chain variable structural domain

[0237] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24), or

[0238] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has two or three amino acid substitutions.

[0239] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0240] (i)CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 72 - clone 2C4), or

[0241] (ii)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTAC ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74 - clone hu2C4-VHH);

[0242] (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41 - for clone 1B6, 1C1, 1C11, 1D4, 1H6), or

[0243] (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45 - clone 1E4); or

[0244] (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65 - Clone 1A5), or

[0245] (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), or

[0246] (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), or

[0247] (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73 - Clone 2D10), and / or

[0248] The light chain variable domain encoded by a nucleotide sequence comprising:

[0249] (i) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49 - Clone 1B6 and 1C1), or

[0250] (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53 - Clone 1C11), or

[0251] (iii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55 - Clone 1D4), or

[0252] (iv) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59 - Clone 1E4), or

[0253] (v)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCT ATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61 - clone 1H6),

[0254] Or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions.

[0255] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise heavy chain and / or light chain variable domains encoded by nucleotide sequences selected from:

[0256] (i) A heavy chain variable domain encoded by a nucleotide sequence comprising (1B6 and 1C1).

[0257] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0258] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0259] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49)

[0260] (ii) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1C1)

[0261] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0262] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0263] GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53)

[0264] (iii) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1D4)

[0265] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0266] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0267] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55)

[0268] (iv) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1H6)

[0269] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0270] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0271] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61); and

[0272] (v) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1E4)

[0273] CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGT ACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45), and / or

[0274] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0275] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCAT CGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59)

[0276] Or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions.

[0277] In some embodiments, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM comprise a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0278] (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCAGGGACCCAG (SEQ ID NO: 72) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0279] (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions; or

[0280] (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0281] (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGACCCAG (SEQ ID NO: 69) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0282] (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0283] (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTAC ACACTATGCAGACTCCGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto and / or has 10-20 nucleic acid substitutions,

[0284] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0285] (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO: 45) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has 10-20 nucleic acid substitutions.

[0286] In some implementations, the multispecific antigen-binding protein, its variants, or fragments are bispecific antibodies.

[0287] In some embodiments, the multispecific antigen-binding protein, its variants or fragments are immune cell connectors selected from T cell connectors, NK cell connectors, monocyte connectors and macrophage connectors.

[0288] In some embodiments, 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.

[0289] In some embodiments, the second antigen-binding protein, its variants, or binding fragments of the immune cell adaptor bind to an immune marker selected from CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0290] In some embodiments, the multispecific antigen-binding protein is an inducible bispecific T-cell connector comprising a heavy chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv).

[0291] In some implementations, the cells are selected, for example, from T cells, macrophages, monocytes, and NK cells.

[0292] In some embodiments, the cells are T cells, optionally CAR T cells.

[0293] In some implementations, the cells

[0294] i. Binds to GPC3 and secretes an inducible bispecific T cell connector targeting EpCAM and CD3 (GE CAR-BiTE T); or

[0295] ii. Binds to CD19 and secretes an inducible bispecific T cell connector (CD19 CAR-BiTE T) that targets EpCAM and CD3.

[0296] On the other hand, it provides polynucleotides that encode cells as described herein.

[0297] On the other hand, vectors for expressing polynucleotides as described herein are provided.

[0298] On the other hand, host cells containing vectors as described herein are provided.

[0299] In another aspect, methods for producing / generating cells as described herein are provided, including introducing polynucleotides as described herein into said cells.

[0300] In another aspect, compositions comprising cells as described herein are provided.

[0301] In another aspect, a method for treating a disease in a subject in need is provided, the method comprising administering to the subject cells or compositions as described herein, optionally the disease being a proliferative disease, optionally cancer.

[0302] definition

[0303] The term “antigen-binding protein” in this article is used in its broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies, as long as they exhibit the desired antigen-binding activity.

[0304] As used herein, the term "antibody" refers to both full-length (i.e., complete) antibodies (elements consisting of two heavy chains and two light chains) and their functionally active fragments (molecules containing an antigen-binding domain that specifically binds to an antigen, also known as antibody fragments or antigen-binding fragments). The characteristics of antibodies described herein also apply to antibody fragments unless the context otherwise requires. The term "antibody" encompasses both monovalent antibodies, which contain only one antigen-binding domain (e.g., a single-arm antibody consisting of interconnected full-length heavy and light chains, also known as a "half-antibody") and multivalent antibodies, which contain more than one antigen-binding domain, such as bivalent antibodies.

[0305] As used herein, the term “antigen-binding fragment” refers to a functionally active antibody-binding fragment, including but not limited to: Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibody, scFv, Fv, bivalent antibody, trivalent or tetravalent antibody, Bis-scFv, bisomic antibody, trisomic antibody, tetrasomic antibody, and epitope-binding fragments of any of the above.

[0306] As used herein, a “binding fragment” refers to a fragment that can bind to a target peptide or antigen with sufficient affinity, thereby characterizing the fragment as specific to the peptide or antigen.

[0307] The term "monoclonal antibody" (or "mAb") refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that each individual in a monoclonal antibody formulation is identical except for a small number of possible mutations (e.g., naturally occurring mutations). However, some differences in protein sequences related to post-translational modifications may exist between the various antibody molecules present in the composition (e.g., cleavage of C-terminal lysine residues in the heavy chain, deamidation of asparagine residues, and / or isomerization of aspartic acid residues). Unlike polyclonal antibody formulations, each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0308] As used in this article, the term "bibody antibody" 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.

[0309] As used herein, the term "trisomy antibody" (also known as Fab(scFv)2) refers to a Fab fragment in which the first scFv is attached to the C-terminus of the light chain and the second scFv is attached to the C-terminus of the heavy chain. As used herein, the term "tetrasomy antibody" refers to a form similar to a disomy antibody, which contains four Fvs and four Fv intermolecular heads.

[0310] The term "multivalent antibody" refers to an antibody that contains more than one antigen-binding domain, such as a bivalent antibody.

[0311] The term "Fv" refers to the two variable domains of a full-length antibody, such as a covariant variable domain, a homology pair, or an 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 short linker peptides of 10 to approximately 25 amino acids. The term "bis-scFv," as used herein, refers to a bispecific scFv.

[0312] As used herein, the term "dsscFv" or "disulfide-stable single-chain variable fragment" refers to a single-chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains and also contains an interdomain disulfide bond between the VH and VL domains.

[0313] 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.

[0314] As used herein, the term "Fab" refers to an antibody fragment comprising a light chain fragment containing a VL (variable light chain) domain and a constant domain (CL) of the light chain, and a VH (variable heavy chain) domain and a first constant domain (CH1) of the heavy chain. According to this disclosure, the dimer of Fab' forms F(ab')2, where dimerization can be achieved, for example, 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 subsequently 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 capacity like intact IgG, but smaller in size than 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 and can be used to achieve specific staining of primary antibody targets.

[0315] As used herein, the terms “(multiple) constant domains” or “constant region” are used interchangeably to refer to the (multiple) domains located outside the variable region of an antibody. Constant domains are identical across all antibodies of the same isotype, but differ between different isotypes. Typically, the constant region of the heavy chain, from the N-terminus to the C-terminus, is formed by a CH1-hinge-CH2-CH3-optional CH4 and contains three or four constant domains.

[0316] As used in this article, the term "DiFab" refers to two Fab molecules linked by their heavy chain C-terminus, or two Fab' molecules linked by one or more disulfide bonds in their hinge region.

[0317] The term "antigen-binding variant" refers to a polypeptide, such as an antibody having the desired properties described herein and comprising a VH and / or VL, wherein the VH and / or VL have at least about 80% amino acid sequence identity with the VH and / or VL of a reference antibody. Such antibody variants include, for example, antibodies with the addition or deletion of one or more amino acid residues in the VH and / or VL domains. Typically, the antibody variant will have at least about 80% amino acid sequence identity with the antibody described herein, alternatively at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. Optionally, the variant antibody has no more than one conserved amino acid substitution compared to the antibody sequence provided herein; alternatively, the variant antibody has 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.

[0318] The term "immune cells" refers to a specialized class of cells that play a crucial role in the body's defense against infection and foreign substances. They are part of the immune system and are responsible for recognizing 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.

[0319] As used herein, the term “specifically” in the context of antibodies is intended to refer to antibodies that recognize only their specific antigens, or antibodies that have a significantly higher binding affinity to their specific antigens compared to their binding to non-specific antigens, for example, binding affinity that is at least 5, 6, 7, 8, 9, or 10 times higher.

[0320] In the context of antibodies, the term "epitope" or "binding site" refers to the site (or portion) on an antigen that the complementary site of an antibody binds to or recognizes. Epitopes can be formed from consecutive amino acids (often called "linear epitopes") or from discontinuous amino acids formed by the ternary folding of a protein (often called "conformatory epitopes"). Epitopes formed from consecutive amino acids are generally preserved upon exposure to denaturing solvents, while epitopes formed by folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least three, and more commonly at least five to ten, amino acids with a unique spatial conformation. Epitopes are typically composed of chemically active surface groups of a molecule (e.g., amino acids, sugar side chains) and usually have specific three-dimensional structures and charge characteristics.

[0321] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main 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.

[0322] The term "chimeric antibody" (or its antigen-binding fragment) refers to 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 attached to a constant region of a different or altered class, effector function, and / or species, or to a completely different molecule that confers novel properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) a variable region or a portion thereof is altered, replaced, or exchanged by a variable region of a different or altered antigen specificity. For example, mouse antibodies can be modified by replacing their constant region with that of a human immunoglobulin. By replacing it with a human constant region, chimeric antibodies can retain their antigen-recognition specificity while exhibiting reduced antigenicity in humans compared to the original mouse antibody.

[0323] The term "chimeric antigen receptor" refers to receptor proteins that have been engineered to give T cells the new ability to target specific antigens. These receptors are chimeric because they combine antigen-binding and T cell activation functions into a single receptor. CAR T-cell therapy uses CAR-engineered T cells to treat cancer. In CAR T immunotherapy, T cells are modified to recognize cancer cells, thus targeting and destroying them more effectively. CAR T cells can be derived from T cells in a patient's own blood (autologous) or from T cells from another healthy donor (allogeneic). Once isolated from the body, these T cells are genetically engineered to express a specific CAR, enabling them to target antigens present on the surface of tumors. For safety reasons, CAR T cells are engineered to be specific for antigens expressed on tumors but not on healthy cells. CAR T cells destroy cells by stimulating cell proliferation, increasing their toxicity to other living cells (cytotoxicity), and increasing the 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, which each have different and interacting cytotoxic effects.

[0324] 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) whose frame region and CDR region in the variable region are both derived from human sequences. Antibodies or immunoglobulins are classified into several classes 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, human IgG constant region domains, particularly IgG1 and IgG3 isotypes, can be used. Alternatively, when antibody molecules are intended for therapeutic purposes and do not require antibody effector function, IgG2 and IgG4 isotypes can be used. Furthermore, if an 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 rest of the antibody with its human counterpart (i.e., the frame portion of the constant region and the variable region). Further frame region modifications can also be made within the human frame sequence and within the CDR sequence derived from another mammalian species. Humanized antibodies of this disclosure may contain amino acid residues encoded by non-human sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo, or conserved substitutions performed to promote stability or production). The definition of a humanized antibody explicitly 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 challenge but whose endogenous loci are disabled, such as Xeno mice immunized using human B-cell hybridoma technology.

[0325] As used herein, the term "recombinant humanized antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from host cells transformed to express humanized antibodies (e.g., transfected tumors), and antibodies prepared, expressed, created, or isolated by any other means involving splicing all or part of the human immunoglobulin gene, sequence, or other DNA sequences.

[0326] In this specification, the term "isolated" means that the antibody or polynucleotide (as the case may be) exists in a physical environment different from the physical environment in which it may 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 created synthetically. Isolated nucleic acids may comprise synthetic DNA (e.g., produced by 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.

[0327] The term "complementarity-determining region" ("CDR") refers to an amino acid sequence whose boundaries are defined using any of a variety of well-known schemes, including those described by Kabat (i.e., the "Kabat" numbering scheme), Al-Lazikani ("Chothia" numbering scheme), ImMunoGenTics ("IMGT" numbering scheme), etc. The term "complementarity-determining region" ("CDR") refers to a highly variable 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).

[0328] As used herein, the term "sequence identity" refers to the percentage of sequence identity determined by the antibody sequence with the largest alignment performed using the Kabat numbering convention. After alignment, if a target antibody region (e.g., the entire mature 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 target and reference antibody regions is calculated by dividing the number of positions in both regions occupied by the same amino acids by the total number of aligned positions in both regions (excluding vacancies), multiplied by 100 to convert to a percentage. In some instances, antigen-binding proteins contain sequences with at least 60% identity to any of the sequences disclosed herein. For example, an antigen-binding protein may comprise a sequence having an affinity of 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 ... 97%, 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, that differs from the sequences disclosed herein by about one, about two, about three, about four, about five, about six, about seven, about eight, about nine, about ten, or more amino acids or bases. 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.

[0329] In some instances, amino acid mutations are amino acid substitutions, and may include both conserved and / or non-conserved substitutions.

[0330] For example, “conservative substitution” can be performed based 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, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0331] As used herein, a “conservative substitution” is defined as the exchange of one amino acid for another listed in the same group of the six standard amino acid groups shown above. For example, exchanging Asp for Glu retains a negative charge in such a modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt the α-helix.

[0332] As used in this article, “non-conservative substitution” is defined as the exchange of one amino acid with another amino acid listed in different groups of the six standard amino acid groups (1) to (6) shown above.

[0333] 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-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, leucine, valine, hydroxyproline, sarcosine, citrulline, homocitrulline, sulfoalanine, 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 common amino acid analogs.

[0334] In some instances, amino acid mutations may be located in the CDR (e.g., CDR1, CDR2, or CDR3 region) of an antigen-binding protein. In another instance, amino acid changes may be located in the frame region (FR) (e.g., FR1, FR2, FR3, or FR4 region) of an antigen-binding protein.

[0335] 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.

[0336] The modification of the amino acid sequence can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis.

[0337] 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 chains of nucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0338] 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 intrinsic binding affinity, which reflects a 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.

[0339] As the term "K" is used in this article D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a K is obtained and expressed as molar concentration (M). d and K a These refer to the dissociation rate and binding rate of a specific antigen-antibody interaction, respectively. The Kc of the antibody... D The value can be determined using methods recognized in the art. As used herein, the term "low affinity" refers to K... D 100 nM or higher.

[0340] As used in this article, the term "moderate affinity" refers to K D The range is from 10 nM to 100 nM.

[0341] As used in this article, the term "high affinity" refers to K D The range is 1 to 10 nM.

[0342] As used in this article, the term "very high affinity" refers to K D It is 1 nM or lower.

[0343] 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.

[0344] As used herein, the term "multispecific" or "multispecific antibody" refers to an antibody having at least two binding domains (i.e., two or more binding domains, such as two or three binding domains) as described herein, wherein the at least two binding domains independently bind two different antigens or two different epitopes on the same antigen. Multispecific antibodies are typically monovalent for each specificity (antigen). Multispecific antibodies described herein include monovalent and multivalent (e.g., bivalent, trivalent, tetravalent) multispecific antibodies.

[0345] As used in this article, the term "bispecific" or "bispecific antibody" refers to an antibody that is specific to two antigens or that can bind to two target antigens / sites simultaneously.

[0346] As used herein, a “bispecific T-cell adaptor (BiTE)” refers to a class of artificial bispecific monoclonal antibodies that direct the host’s immune system (e.g., the cytotoxic activity of T cells) against target cells (e.g., cancer cells). BiTEs are fusion proteins consisting 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 an immune cell (e.g., binding to T cells via the CD3 receptor), and the other binds to a target (e.g., binding to tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs form a link between immune cells (e.g., T cells) and target cells (e.g., tumor cells). This 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 that enter the tumor cell and initiate apoptosis (e.g., perforin and granzyme).

[0347] In some instances, BiTE may refer to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In other instances, BiTE may also refer to a bispecific T-cell connector platform known in the art, which refers to a recombinant protein that simultaneously binds to two different antigens and has the ability to connect to immune cells (such as T cells).

[0348] 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 heavy-chain-only (VHH) nanobodies. As used herein, "heavy-chain-only nanobodies" refers to heavy-chain antibodies based on nanobodies. Heavy-chain antibodies are antibodies composed of two heavy chains and lack the two light chains typically present in antibodies.

[0349] As described herein, a “vector” is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell, for example, where a polypeptide encoded can be synthesized. Typically and preferably, a vector is a nucleic acid 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 leader sequence for secretion, a ribosome binding site, a polyadenylated sequence, a polylinker region for inserting a nucleic acid encoding the polypeptide to be expressed, and optional labeling elements.

[0350] Vectors are typically chosen 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). Vectors as described herein can be expression vectors and / or cloning vectors.

[0351] As used herein, the term "host cell" is intended to refer to the cell into which the expression vector has been introduced. It should be understood that such a term is intended to refer not only to the specific subject cell but also to the progeny of such cells. Because certain modifications may occur in the progeny due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0352] The terms “treating,” “treatment,” “therapy,” and their synonyms refer to therapeutic treatments and preventative or preventative measures aimed at preventing or alleviating medical conditions, including but not limited to diseases, symptoms, and ailments. Medical conditions also include the body’s response to a disease or ailment, such as inflammation. Those who require such treatment include those who already have a medical condition, those susceptible to a medical condition, or those who need preventative measures against a medical condition.

[0353] As used herein, the term "subject" 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 and is unlikely to have a medical condition. "Non-patient" includes healthy individuals, individuals who are not ill, and / or individuals who do not have 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 family Muridae and / or Leporidae, such as mice, rats, rabbits, etc.

[0354] As used herein, the terms “prevention” and / or “severity reduction” 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.

[0355] The term “and / or”, such as “X and / or Y”, is understood to mean “X and Y” or “X or Y”, and should be understood to explicitly support both meanings or either meaning.

[0356] Furthermore, in the descriptions herein, whenever the word "substantially" is used, it should be understood to include, but is not limited to, "completely" or "thoroughly." Additionally, whenever terms such as "comprising" or "comprise" are used, they are intended as non-restrictive descriptive language, meaning they broadly include the elements / components listed after such a term, except for other components not explicitly listed. For example, when using "comprising," referring to "one" feature also intends to refer to "at least one" of that feature. Terms such as "consisting" or "consist" can be considered, in appropriate context, as a subset of terms such as "comprising" or "comprise." Therefore, in embodiments disclosed herein that use terms such as "comprising" or "comprise," it should be understood that these embodiments provide instruction for corresponding embodiments using terms such as "consisting" or "consist." Furthermore, whenever terms such as “approximately” or “about” are used, they generally refer to reasonable changes, such as a change of + / - 5% in the published value, or a change of 4% in the published value, or a change of 3% in the published value, a change of 2% in the published value, or a change of 1% in the published value.

[0357] Furthermore, in the description herein, some values ​​may be disclosed in the form of ranges. Showing values ​​at the endpoints of a range is intended to illustrate preferred ranges. Whenever a range is described, it is intended to encompass and teach all possible subranges within that range, as well as individual numerical values. That is, the endpoints of a range should not be interpreted as fixed limitations. For example, describing a range of 1% to 5% is intended to specifically disclose 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 numerical values ​​within this range also include integers, fractions, and decimals. Furthermore, whenever a range is described, it is also intended to encompass and teach values ​​that have up to two additional decimal places or significant figures (where applicable) from the endpoints of the indicated numerical value. For example, describing a range of 1% to 5% is intended to specifically disclose ranges of 1.00% to 5.00% and 1.0% to 5.0% and all intermediate values ​​(e.g., 1.01%, 1.02%... 4.98%, 4.99%, 5.00% and 1.1%, 1.2%... 4.8%, 4.9%, 5.0%, etc.), encompassing these ranges. The specific disclosure above is intended to apply to any depth / breadth of the range.

[0358] As used herein, "at least 95% identical" means that the amino acid sequence has 95% or higher identity with the reference sequence over its full length, such as 96%, 97%, 98%, or 99%. The percentage of identity can be calculated using software programs.

[0359] Furthermore, when describing some embodiments, this disclosure may disclose methods and / or processes as steps in a specific order. However, unless otherwise required, it should be understood that the methods or processes described are not limited to the specific order of steps disclosed. Other orders of steps are 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 written order. The order of steps may vary and remains within the scope of this disclosure.

[0360] Furthermore, it should be understood that while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, in other alternative embodiments, one or more of these features / characteristics may be omitted, and this disclosure provides support for such omissions and these related alternative embodiments. Detailed Implementation

[0361] Exemplary, non-limiting embodiments of cells are disclosed, wherein the cells express a chimeric antigen receptor targeting GPC3 (or GE3) and / or CD19, and a multispecific antigen-binding protein, variant, or binding fragment that binds EpCAM and immune cell markers. Engineered cells expressing the polynucleotides described herein are also disclosed. Engineered cells expressing (a) a chimeric antigen receptor targeting a first antigen, and (b) a multispecific antigen-binding protein that binds EpCAM (epithelial cell adhesion molecule), optionally wherein the first target antigen in (a) is a disease-related antigen. Cells are engineered to minimize toxicity, side effects, and / or nonspecific cytotoxicity by maximizing the secretion of EpCAM BiTE at the target site, without being bound by any theoretical constraints.

[0362] For example, a target antigen is a molecule associated with a disease. The 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.

[0363] In some instances, chimeric antigen receptors target diseased cells. In some instances, diseased cells may include, but are not limited to, cells from proliferative diseases (e.g., tumors / cancers, inflammatory diseases). In some instances, diseased cells may be cells from cancer / tumors, optionally, cancer cells being solid tumors.

[0364] In some instances, solid tumors can include tumors of epithelial origin, such as carcinomas. In some instances, carcinomas can include, but are not limited to, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., squamous cell carcinoma of the lung), gastric cancer (e.g., gastric adenocarcinoma), breast cancer, skin cancer (e.g., melanoma), ovarian cancer (e.g., clear cell carcinoma of the ovary), kidney cancer, pancreatic cancer, head and neck cancer, prostate cancer, esophageal cancer, bladder cancer, colon cancer, and childhood cancers (e.g., hepatoblastoma, nephroblastoma, yolk sac tumor, etc.).

[0365] In some instances, tumor cells can include benign, precancerous, and malignant tumors. In other instances, tumor cells can include stem cells and progenitor cells.

[0366] In some instances, tumor cells may include human xenografts from animal models. In some instances, human xenografts may include Hep3B, HepG2, etc. In some instances, the human xenograft is a Hep3B xenograft and / or a HepG2 xenograft from a mouse model.

[0367] In some instances, the tumor cells may include tumor cell lines, such as, but not limited to, gastric adenocarcinoma cell lines (e.g., AGS), breast cancer cell lines (e.g., MDA-MB468), and hepatocellular carcinoma cell lines (e.g., Hep3B (GPC3)). 高 EpCAM 高 ), HepG2 (GPC3) 高 EpCAM 高 HT-29 (GPC3) 低 EpCAM 高 ), HeyA8 (GPC3) 阴性 EpCAM 阴性 ), Huh7 (GPC3) 高 EpCAM 高 )wait.

[0368] In some instances, the target antigen is an epithelial marker, which may include, but is not limited to, receptor tyrosine-protein kinase erbB-2 (HER2), phosphatidylinositol proteoglycan 3 (GPC3), Claudin 18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), delta-like canonical Notch ligand 3 (DLL3), carcinoembryonic antigen (CEA), mucin 1 (MUC1), mucin 16 (MUC16), CEA cell adhesion molecule 7 (CEACAM7), prominin-1 (CD133), differentiation cluster 147 (CD147), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), mesothelin (MSLN), mesenchymal-epithelial transition factor (c-Met), folate receptor α (FRα), etc.

[0369] In some instances, the chimeric antigen receptor binds to GPC3.

[0370] Unwilling to be bound by theory, GPC3 is a well-known carcinoembryonic protein that can serve as a highly tumor-specific target. It is widely expressed during embryonic development but is strictly suppressed in most adult tissues. Elevated GPC3 expression has been reported in various tumor types, such as liver cancer, lung cancer, gastric cancer, ovarian cancer, and esophageal cancer. Meanwhile, EpCAM is widely expressed in almost all cancers but also at low levels in normal epithelial cells. 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 shown dose-limiting toxicity and have failed to receive FDA approval.

[0371] Therefore, the inventors of this disclosure engineered CAR-T cells to secrete a second tumor-targeting molecule, namely an anti-EpCAM bispecific T-cell connector (BiTE). EpCAM is a well-known tumor-associated antigen that is typically overexpressed in almost all epithelial cell-derived solid tumors. However, neither CAR T cells nor systemically administered EpCAM-targeting BiTEs have been successful due to their high systemic toxicity, as EpCAM is also widely expressed in normal epithelial cells, albeit at lower levels. CAR-T cell secretion of anti-EpCAM BiTE localizes the anti-EpCAM BiTE at or near the tumor site, which not only reduces tumor escape but also significantly improves safety. Since anti-GPC3 CAR-T cells have previously demonstrated clinically validated efficacy and safety, this disclosure uses GPC3-targeting CAR-T cells as a carrier for anti-EpCAM BiTE. This disclosure demonstrates that GPC3-targeting CAR T cells secreting anti-EpCAM BiTE (named "GECAR-BiTE T") exhibit excellent efficacy in eradicating human xenograft tumors derived from hepatocellular carcinoma both in vitro and in vivo.

[0372] Therefore, in one respect, a cell is provided that expresses

[0373] (a) Chimeric antigen receptors targeting GPC3 and / or CD19, and

[0374] (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 binds to EpCAM (epithelial cell adhesion molecule), and a second antigen-binding protein, its variants, or binding fragments that bind to immune cell markers.

[0375] The first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region and / or a light chain variable region selected from the following:

[0376] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5, 1B8, 2B7 and 2D10)

[0377] CDR-H1, wherein CDR-H1 comprises:

[0378] GSIFSGND (SEQ ID NO: 25 - 2C4, hu2C4, 1A5, 2B7 and 2D10), or

[0379] GSSERFTS (SEQ ID NO: 29 - 1B8)

[0380] CDR-H2, wherein CDR-H2 comprises:

[0381] ITSGGST (SEQ ID NO: 26 - 2C4, hu2C4, 1A5, 2B7 and 2D10), or

[0382] ITNGGST (SEQ ID NO: 30 - 1B8), and

[0383] CDR-H3, wherein CDR-H3 comprises:

[0384] TNGRWSGDTYYAHH (SEQ ID NO: 27 - 2C4, hu2C4, 1A5, 2C4 and 2D10),

[0385] MAGTS (SEQ ID NO: 31 - 1B8), or

[0386] TNGRWSGDTYYAHL (SEQ ID NO: 33 - 2B7)

[0387] (ii) Heavy chain variable regions, comprising: (1B6, 1C1, 1C11, 1D4, and 1H6)

[0388] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0389] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0390] CDR-H3, wherein CDR-H3 contains ARSLGGRFRY (SEQ ID NO: 3),

[0391] (iii) Heavy chain variable region, which contains: (1E4)

[0392] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0393] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0394] CDR-H3, wherein CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO: 7),

[0395] (iv) Light chain variable regions comprising: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6)

[0396] CDR-L1, wherein CDR-L1 includes:

[0397] QSLLHSNGYNY (SEQ ID NO: 9 - 1B6, 1C1, 1C11 and 1H6),

[0398] QSLLHSNRYNY (SEQ ID NO: 17 - 1D4), or

[0399] QSISDF (SEQ ID NO: 19 - 1E4)

[0400] CDR-L2, wherein CDR-L2 includes:

[0401] LGS (SEQ ID NO: 10 - 1B6, 1C1, 1C11, 1D4 and 1H6), or

[0402] AAS (SEQ ID NO: 20 - 1E4), and

[0403] CDR-L3, wherein CDR-L3 includes:

[0404] MQALQTPYT (SEQ ID NO: 11 - 1B6, 1C1 and 1D4),

[0405] MQGLQSPWT (SEQ ID NO: 15 - 1C11),

[0406] QQSYIMPDT (SEQ ID NO: 21 - 1E4), or

[0407] MQGLQTPYT (SEQ ID NO: 23 - 1H6); and

[0408] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0409] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region comprising: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10).

[0410] CDR-H1, wherein CDR-H1 comprises:

[0411] GSIFSGND (SEQ ID NO: 25 - 2C4, hu2C4, 1A5, 2B7 and 2D10), or

[0412] GSSERFTS (SEQ ID NO: 29 - 1B8)

[0413] CDR-H2, wherein CDR-H2 comprises:

[0414] ITSGGST (SEQ ID NO: 26 - 2C4, hu2C4, 1A5, 2B7 and 2D10), or

[0415] ITNGGST (SEQ ID NO: 30 - 1B8); and

[0416] CDR-H3, wherein CDR-H3 comprises:

[0417] TNGRWSGDTYYAHH (SEQ ID NO: 27 - 2C4, hu2C4, 1A5, 2D10),

[0418] MAGTS (SEQ ID NO: 31 - 1B8), or

[0419] TNGRWSGDTYYAHL (SEQ ID NO: 33 - 2B7)

[0420] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0421] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region selected from the following:

[0422] (i) Heavy chain variable regions, which include: (2C4-VHH, hu2C4-VHH, 1A5-VHH and 2D10-VHH)

[0423] CDR-H1, wherein CDR-H1 contains GSIFSGND (SEQ ID NO: 25),

[0424] CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26), and

[0425] CDR-H3, wherein CDR-H3 comprises TNGRWSGDTYYAHH (SEQ ID NO: 27);

[0426] (ii) Heavy chain variable region, which contains: (1B8-VHH)

[0427] CDR-H1, wherein CDR-H1 contains GSSERFTS (SEQ ID NO: 29),

[0428] CDR-H2, wherein CDR-H2 comprises ITNGGST (SEQ ID NO: 30), and

[0429] CDR-H3, wherein CDR-H3 comprises MAGTS (SEQ ID NO: 31); and

[0430] (iii) Heavy chain variable region, which contains: (2B7-VHH)

[0431] CDR-H1, wherein CDR-H1 contains GSIFSGND (SEQ ID NO: 25),

[0432] CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26), and

[0433] CDR-H3, wherein CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO: 33),

[0434] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0435] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region and / or a light chain variable region selected from the following:

[0436] (i) Heavy chain variable regions, which include: (2C4, hu2C4, 1A5 and 2D10)

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

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

[0439] CDR-H3, wherein CDR-H3 comprises TNGRWSGDTYYAHH (SEQ ID NO: 27)

[0440] (ii) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1H6)

[0441] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0442] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0443] CDR-H3, wherein CDR-H3 contains ARSLGGRFRY (SEQ ID NO: 3)

[0444] (iii) Heavy chain variable region, which contains: (1E4)

[0445] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0446] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0447] CDR-H3, wherein CDR-H3 contains AREVEGSSYDAFDI (SEQ ID NO: 7)

[0448] (iv) Light chain variable region, which comprises: (1B6 and 1C1)

[0449] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0450] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0451] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0452] (v) Light chain variable region, which contains: (1C11)

[0453] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0454] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0455] CDR-L3, wherein CDR-L3 comprises MQGLQSPWT (SEQ ID NO: 15)

[0456] (vi) Light chain variable region, which contains: (1D4)

[0457] CDR-L1, wherein CDR-L1 contains QSLLHSNRYNY (SEQ ID NO: 17),

[0458] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0459] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0460] (vii) Light chain variable region, which includes: (1E4)

[0461] CDR-L1, wherein CDR-L1 contains QSISDF (SEQ ID NO: 19),

[0462] CDR-L2, wherein CDR-L2 includes AAS (SEQ ID NO: 20), and

[0463] CDR-L3, wherein CDR-L3 includes QQSYIMPDT (SEQ ID NO: 21)

[0464] (viii) Light chain variable region, which contains: (1H6)

[0465] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0466] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0467] CDR-L3, wherein CDR-L3 comprises MQGLQTPYT (SEQ ID NO: 23)

[0468] (ix) Heavy chain variable region, which contains: (1B8-VHH)

[0469] CDR-H1, wherein CDR-H1 contains GSSERFTS (SEQ ID NO: 29),

[0470] CDR-H2, wherein CDR-H2 comprises ITNGGST (SEQ ID NO: 30), and

[0471] CDR-H3, wherein CDR-H3 comprises MAGTS (SEQ ID NO: 31); and

[0472] (x) Heavy chain variable region, which contains: (2B7-VHH)

[0473] CDR-H1, wherein CDR-H1 contains GSIFSGND (SEQ ID NO: 25),

[0474] CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26), and

[0475] CDR-H3, wherein CDR-H3 contains TNGRWSGDTYYAHL (SEQ ID NO: 33)

[0476] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0477] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable domain and / or a light chain variable domain selected from the following:

[0478] (i) Heavy-chain variable structural domains, which contain

[0479] QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35 - 2C4-VHH)

[0480] (ii) Heavy-chain variable structural domains, which contain

[0481] QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37 - hu2C4-VHH)

[0482] (iii) Heavy-chain variable structural domains, which contain

[0483] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4 - 1B6, 1C1, 1C11, 1D4 and 1H6)

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

[0485] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8 - 1E4),

[0486] (v) Light chain variable structural domain, which contains

[0487] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12 - 1B6 and 1C1)

[0488] (vi) Light chain variable structural domain, which contains

[0489] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16 - 1C11)

[0490] (vii) Light chain variable structural domain, which contains

[0491] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18 - 1D4)

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

[0493] DIQLTQSPSSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22 - 1E4)

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

[0495] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24 - 1H6)

[0496] (x) Heavy-chain variable structural domains, which contain

[0497] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28- 1A5-VHH)

[0498] (xi) Heavy-chain variable structural domain, which contains

[0499] QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32 - 1B8-VHH)

[0500] (xii) Heavy-chain variable structural domain, which contains

[0501] QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34 - 2B7-VHH)

[0502] (xiii) Heavy-chain variable structural domains, which contain

[0503] and

[0504] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has two or three amino acid substitutions.

[0505] In some instances, the first antigen-binding protein that binds EpCAM, its variants, or binding fragments comprise a single-domain heavy-chain variable domain having the following sequence:

[0506] (i) QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35 - clone 2C4-VHH), or

[0507] (ii) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37 - hu2C4-VHH), or

[0508] (iii) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28 - clone 1A5-VHH), or

[0509] (iv) QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32 - clone 1B8-VHH), or

[0510] Or

[0511] (vi)QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36 - clone 2D10-VHH),

[0512] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has two or three amino acid substitutions.

[0513] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region selected from the following:

[0514] (i) Heavy chain variable region, which includes: (1B6, 1C1, 1C11, 1D4 and 1H6)

[0515] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0516] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0517] CDR-H3, wherein CDR-H3 contains ARSLGGRFRY (SEQ ID NO: 3)

[0518] (ii) Heavy chain variable region, which contains: (1E4)

[0519] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0520] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0521] CDR-H3, wherein CDR-H3 comprises AREVEGSSYDAFDI (SEQ ID NO: 7); and / or

[0522] It contains a light chain variable region, which includes: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6).

[0523] CDR-L1, wherein CDR-L1 includes:

[0524] QSLLHSNGYNY (SEQ ID NO: 9 - 1B6, 1C1, 1C11 and 1H6),

[0525] QSLLHSNRYNY (SEQ ID NO: 17 - 1D4), or

[0526] QSISDF (SEQ ID NO: 19 - 1E4)

[0527] CDR-L2, wherein CDR-L2 includes:

[0528] LGS (SEQ ID NO: 10-1B6, 1C1, 1C11, 1D4 and 1H6), or

[0529] AAS (SEQ ID NO: 20 - 1E4); and

[0530] CDR-L3, wherein CDR-L3 includes:

[0531] MQALQTPYT (SEQ ID NO: 11 - 1B6, 1C1 and 1D4),

[0532] MQGLQSPWT (SEQ ID NO: 15 - 1C11),

[0533] QQSYIMPDT (SEQ ID NO: 21 - 1E4), or

[0534] MQGLQTPYT (SEQ ID NO: 23 - 1H6)

[0535] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0536] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region and / or a light chain variable region selected from the following:

[0537] (i) Heavy chain variable region, which contains: (1B6 and 1C1)

[0538] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0539] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0540] CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3); and / or

[0541] The light chain variable region, which includes:

[0542] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0543] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0544] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0545] (ii) Heavy chain variable region, which contains: (1C11)

[0546] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0547] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0548] CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3); and / or

[0549] The light chain variable region, which includes:

[0550] CDR-L1, wherein CDR-L1 contains QSLLHSNGYNY (SEQ ID NO: 9),

[0551] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0552] CDR-L3, wherein CDR-L3 comprises MQGLQSPWT (SEQ ID NO: 15)

[0553] (iii) Heavy chain variable region, which contains: (1D4)

[0554] CDR-H1, wherein CDR-H1 contains GGTFSSYA (SEQ ID NO: 1),

[0555] CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2), and

[0556] CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3); and / or

[0557] The light chain variable region, which includes:

[0558] CDR-L1, wherein CDR-L1 contains QSLLHSNRYNY (SEQ ID NO: 17),

[0559] CDR-L2, wherein CDR-L2 includes LGS (SEQ ID NO: 10), and

[0560] CDR-L3, wherein CDR-L3 contains MQALQTPYT (SEQ ID NO: 11)

[0561] (iv) Heavy chain variable region, which contains: (1H6)

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

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

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

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

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

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

[0568] CDR-L3, wherein CDR-L3 comprises MQGLQTPYT (SEQ ID NO: 23); and

[0569] (v) Heavy chain variable region, which contains: (1E4)

[0570] CDR-H1, wherein CDR-H1 contains GDSISSNSVA (SEQ ID NO: 5),

[0571] CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6), and

[0572] CDR-H3, wherein CDR-H3 comprises AREVEGSSYDAFDI (SEQ ID NO: 7); and / or

[0573] The light chain variable region contains: (1E4)

[0574] CDR-L1, wherein CDR-L1 contains QSISDF (SEQ ID NO: 19),

[0575] CDR-L2, wherein CDR-L2 includes AAS (SEQ ID NO: 20), and

[0576] CDR-L3, wherein CDR-L3 includes QQSYIMPDT (SEQ ID NO: 21)

[0577] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0578] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable domain and / or a light chain variable domain selected from the following:

[0579] (i) Heavy chain variable structural domain, which contains: (1B6 and 1C1)

[0580] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and / or

[0581] Light chain variable structural domain, which contains

[0582] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12)

[0583] (ii) Heavy-chain variable structural domains, which contain: (1C11)

[0584] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and / or

[0585] Light chain variable structural domain, which contains

[0586] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16)

[0587] (iii) Heavy chain variable structural domain, which contains: (1D4)

[0588] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and / or

[0589] Light chain variable structural domain, which contains

[0590] DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18)

[0591] (iv) Heavy-chain variable structural domains, which contain: (1E4)

[0592] QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), and / or

[0593] Light chain variable structural domain, which contains

[0594] DIQLTQSPSSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or

[0595] (v) Heavy-chain variable structural domain, which contains: (1H6)

[0596] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), and / or

[0597] Light chain variable structural domain

[0598] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24), or

[0599] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has two or three amino acid substitutions.

[0600] In some instances, the first antigen-binding protein that binds EpCAM, its variants, or binding fragments contain a light chain constant domain having a sequence

[0601] (i)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13 - clone 1B6 - light chain constant domain), or

[0602] (ii)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (SEQ ID NO: 14 Cloning 1C1 - Light Chain Constant Domain)

[0603] Or a fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has two or three amino acid substitutions.

[0604] 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:

[0605] (i) Heavy chain variable region, which contains: (1B6 and 1C1)

[0606] CDR-H1, wherein CDR-H1 contains GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 38),

[0607] CDR-H2, wherein CDR-H2 comprises ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and

[0608] CDR-H3, wherein CDR-H3 comprises GCGAGATCGTTGGGTGGGAGATTTCGCTAC (SEQ ID NO: 40); and / or

[0609] The light chain variable region, which includes:

[0610] CDR-L1, wherein CDR-L1 contains CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO:46),

[0611] CDR-L2, wherein CDR-L2 includes TTGGGTTCT (SEQ ID NO: 47), and

[0612] CDR-L3, wherein CDR-L3 contains ATGCAAGCTCTACAAACTCCGTACACT (SEQ ID NO: 48)

[0613] (ii) Heavy chain variable region, which contains: (1C11)

[0614] CDR-H1, wherein CDR-H1 contains GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 38),

[0615] CDR-H2, wherein CDR-H2 comprises ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and

[0616] CDR-H3, wherein CDR-H3 comprises GCGAGATCGTTGGGTGGGAGATTTCGCTAC (SEQ ID NO: 40); and / or

[0617] The light chain variable region, which includes:

[0618] CDR-L1, wherein CDR-L1 contains CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO:46),

[0619] CDR-L2, wherein CDR-L2 includes TTGGGTTCT (SEQ ID NO: 47), and

[0620] CDR-L3, wherein CDR-L3 contains ATGCAAGGTCTACAAAGTCCCTGGACG (SEQ ID NO: 52)

[0621] (iii) Heavy chain variable region, which contains: (1D4)

[0622] CDR-H1, wherein CDR-H1 contains GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 38),

[0623] CDR-H2, wherein CDR-H2 comprises ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and

[0624] CDR-H3, wherein CDR-H3 comprises GCGAGATCGTTGGGTGGGAGATTTCGCTAC (SEQ ID NO: 40); and / or

[0625] The light chain variable region, which includes:

[0626] CDR-L1, wherein CDR-L1 contains CAGAGCCTCCTGCATAGTAATAGATACAACTAT (SEQ ID NO:54),

[0627] CDR-L2, wherein CDR-L2 includes TTGGGTTCT (SEQ ID NO: 47), and

[0628] CDR-L3, wherein CDR-L3 contains ATGCAAGCTCTACAAACTCCGTACACT (SEQ ID NO: 48)

[0629] (iv) Heavy chain variable region, which contains: (1H6)

[0630] CDR-H1, wherein CDR-H1 contains GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 38),

[0631] CDR-H2, wherein CDR-H2 comprises ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and

[0632] CDR-H3, wherein CDR-H3 comprises GCGAGATCGTTGGGTGGGAGATTTCGCTAC (SEQ ID NO: 40); and / or

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

[0634] CDR-L1, wherein CDR-L1 contains CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO:46),

[0635] CDR-L2, wherein CDR-L2 includes TTGGGTTCT (SEQ ID NO: 47), and

[0636] CDR-L3, wherein CDR-L3 comprises ATGCAAGGTCTACAGACTCCGTACACT (SEQ ID NO: 60); and

[0637] (v) Heavy chain variable region, which contains: (1E4)

[0638] CDR-H1, wherein CDR-H1 contains GGGGACAGTATCTCTAGTAACAGTGTTGCT (SEQ ID NO: 42),

[0639] CDR-H2, wherein CDR-H2 comprises ACATACTACAGGTCCAAGTGGTACAGT (SEQ ID NO: 43), and

[0640] CDR-H3, wherein CDR-H3 contains GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC (SEQ ID NO: 44); and / or

[0641] The light chain variable region contains: (1E4)

[0642] CDR-L1, wherein CDR-L1 contains CAGAGTATTAGCGACTTT (SEQ ID NO: 56),

[0643] CDR-L2, wherein CDR-L2 includes GCTGCATCG (SEQ ID NO: 57), and

[0644] CDR-L3, wherein CDR-L3 comprises TTACATTATGCCCGACACT (SEQ ID NO: 58)

[0645] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0646] In one embodiment, the antigen-binding protein, its variants, or fragments comprise a heavy-chain variable region encoded by a nucleic acid sequence selected from:

[0647] (i) Heavy chain variable regions, which include: (2C4-VHH, 1A5-VHH, 2D10-VHH and hu2C4-VHH)

[0648] CDR-H1, wherein CDR-H1 contains GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO: 62),

[0649] CDR-H2, wherein CDR-H2 comprises ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and

[0650] CDR-H3 comprising ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC (SEQID NO: 64)

[0651] (ii) Heavy chain variable region, which contains: (1B8-VHH)

[0652] CDR-H1, wherein CDR-H1 contains GGAAGCTCCGAAAGATTCACATCA (SEQ ID NO: 66),

[0653] CDR-H2, wherein CDR-H2 contains ATTACTAATGGTGGTAGCACA (SEQ ID NO: 67), and

[0654] CDR-H3, wherein CDR-H3 comprises ATGGCGGGTACGTCC (SEQ ID NO: 68); and

[0655] (iii) Heavy chain variable region, which contains: (2B7-VHH)

[0656] CDR-H1, wherein CDR-H1 contains GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO: 62),

[0657] CDR-H2, wherein CDR-H2 comprises ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and

[0658] CDR-H3, wherein CDR-H3 contains ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC (SEQ ID NO: 70)

[0659] Or a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

[0660] In some instances, the first antigen-binding protein that binds EpCAM, its variants, or binding fragments comprise a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0661] (i)CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 72 - clone 2C4), or

[0662] (ii)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTAC ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74 - clone hu2C4-VHH); or

[0663] (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41 - for clone 1B6, 1C1, 1C11, 1D4, 1H6), or

[0664] (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45 - clone 1E4); or

[0665] (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65 - Clone 1A5), or

[0666] (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), or

[0667] (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), or

[0668] (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73 - Clone 2D10), and / or

[0669] a light chain variable domain encoded by a nucleotide sequence comprising:

[0670] (vi) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49 - Clone 1B6 and 1C1), or

[0671] (vii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53 - Clone 1C11), or

[0672] (viii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55 - Clone 1D4), or

[0673] (ix) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59 - Clone 1E4), or

[0674] (x)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCT ATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61 - clone 1H6),

[0675] Or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions.

[0676] In some instances, the first antigen-binding protein that binds EpCAM, its variants, or binding fragments comprise a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0677] (i)GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTG GTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41 - for 1B6, 1C1, 1C11, 1D4, 1H6), or

[0678] (ii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45 - Clone 1E4); or

[0679] (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65 - Clone 1A5), or

[0680] (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), or

[0681] (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), or

[0682] (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 72 - Clone 2C4), or

[0683] (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73 - Clone 2D10), or

[0684] (viii)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74 - Clone hu2C4-VHH); and / or

[0685] The variable light domain encoded by a nucleotide sequence, said nucleotide sequence comprising:

[0686] (xi)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49 - Clone 1B6 and 1C1), or

[0687] (xii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53 - Clone 1C11), or

[0688] (xiii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55 - Clone 1D4), or

[0689] (xiv)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGC ATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59 - clone 1E4), or

[0690] (xv)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCT ATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61 - clone 1H6),

[0691] Or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions.

[0692] In some instances, the first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain heavy chain and / or light chain variable domains encoded by nucleotide sequences selected from:

[0693] (i) A heavy chain variable domain encoded by a nucleotide sequence comprising (1B6 and 1C1).

[0694] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0695] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0696] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49)

[0697] (ii) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1C1)

[0698] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0699] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0700] GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53)

[0701] (iii) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1D4)

[0702] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0703] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0704] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55)

[0705] (iv) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1H6)

[0706] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGT ACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41), and / or

[0707] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0708] GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61); and

[0709] (v) A heavy chain variable domain encoded by a nucleotide sequence comprising: (1E4)

[0710] CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGT ACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45), and / or

[0711] A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising:

[0712] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCAT CGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59)

[0713] Or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions.

[0714] In one embodiment, the antigen-binding protein, its variants, or fragments comprise a light chain constant domain encoded by a nucleotide sequence comprising:

[0715] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 50 - Clone 1B6 - Light chain constant domain), or

[0716] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT (SEQ ID NO: 51 - Clone 1C1 - Light chain constant domain), or

[0717] A sequence that is at least 60% identical to its own and / or has 10-20 nucleic acid substitutions.

[0718] In some instances, the first antigen-binding protein that binds EpCAM, its variants, or binding fragments comprise a heavy-chain variable domain encoded by a nucleotide sequence comprising:

[0719] (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCAGGGACCCAG (SEQ ID NO: 72) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0720] (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions; or

[0721] (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0722] (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGACCCAG (SEQ ID NO: 69) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0723] (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto and / or has 10 - 20 nucleic acid substitutions, or

[0724] (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto and / or has 10 - 20 nucleic acid substitutions,

[0725] GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or

[0726] (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO: 45) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has 10-20 nucleic acid substitutions.

[0727] In some instances, a 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 substitutions, insertions, deletions, and truncations. In some instances, the amino acid mutation is an amino acid substitution and may include conserved substitutions and / or non-conserved substitutions. In some instances, the mutation does not materially reduce the ability of the antigen-binding protein to specifically bind to a target. In some instances, the mutation does not materially reduce the ability of the antigen-binding protein to specifically bind to a target and does not functionally modulate the target (e.g., partially or completely neutralize it).

[0728] In some instances, the multispecific antigen-binding protein can 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 can be provided as a nanobody. In some instances, the multispecific antigen-binding protein can be provided as an Fc region. 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). In some embodiments, the bispecific T-cell adaptor (BiTE) is a nanobody containing only the heavy chain (VHH).

[0729] In some instances, multispecific antigen-binding proteins, their variants, or fragments are inducible bispecific immune cell adjuvants. In some instances, multispecific antigen-binding proteins are secreted by cells (optionally immune cells). In some instances, multispecific antigen-binding proteins, their variants, or fragments are bispecific T-cell adjuvants (BiTEs).

[0730] As used herein, a bispecific T-cell adaptor (BiTE) is a class of artificial bispecific monoclonal antibodies that direct the host's immune system (e.g., the cytotoxic activity of T cells) against target cells (e.g., cancer cells). BiTEs are fusion proteins consisting 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, and the other binds to the target (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs form a link between T cells and target cells (e.g., tumor cells). This allows T cells to exert cytotoxic activity against target cells (e.g., tumor cells) by producing proteins that enter tumor cells and initiate apoptosis (e.g., perforin and granzymes).

[0731] In some instances, BiTE may refer to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In other instances, BiTE may also refer to a bispecific T-cell connector platform known in the art, which refers to a recombinant protein that simultaneously binds to two different antigens and has the ability to connect to immune cells (such as T cells).

[0732] In some instances, the bispecific T-cell connector (BiTE) binds to two antigens; the first antigen is EpCAM; and the second antigen is an immune cell marker. In some instances, the second antigen binds to a protein, a variant thereof, or a binding fragment thereof, which binds to an immune marker selected from CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0733] In some instances, the multispecific antigen-binding protein is an inducible bispecific T-cell connector containing a heavy chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv).

[0734] In some instances, multispecific antigen-binding proteins, their variants, or fragments are inducible bispecific T-cell adaptors. In other instances, multispecific antigen-binding proteins are secreted by the cell.

[0735] In some instances, the bispecific T-cell connector (BiTE) binds to two antigens; the first antigen is EpCAM; and the second antigen is an immune cell marker. In some instances, the second antigen binds to a protein, a variant thereof, or a binding fragment thereof, which binds to an immune marker selected from CD3, NKG2D, CD4, CD8, CD16, CD64, etc.

[0736] In some instances, the multispecific antigen-binding protein is an inducible bispecific T-cell connector containing a heavy chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv).

[0737] In some instances, the 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 instances, the CD3 may include CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0738] 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.

[0739] In some instances, a cell can bind more than one host cell antigen. Therefore, in some instances, a cell can also bind one host cell antigen, two host cell antigens, three host cell antigens, four host cell antigens, and so on.

[0740] In some instances, engineered cells bind to target antigens and secrete BiTE targeting EpCAM and CD3 (CAR-BiTE T). In some instances, cells: i. bind to GPC3 and secrete an inducible bispecific T cell connector targeting EpCAM and CD3 (GE CAR-BiTE T); or ii. bind to CD19 and secrete an inducible bispecific T cell connector targeting EpCAM and CD3 (CD19 CAR-BiTE T). In some instances, cells bind to GPC3 and secrete BiTE targeting EpCAM and CD3 (GE CAR-BiTE T). In some instances, CAR T cells target GPC3 only (GE CAR-T).

[0741] Not wanting to be bound by theory, while CAR T 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 guided to the vicinity of the tumor, where they are activated and help clear tumor cells. By locally secreting anti-EpCAM BiTE by CAR T cells, it transforms an undrugable target into a druggable one, as it reduces the targeted non-tumor toxicity that might result from systemic delivery.

[0742] Since EpCAM is also defined as a cancer stem cell marker expressed on cancer progenitors and cancer stem cells, CAR T cells secreting anti-EpCAM BiTE will help prevent cancer recurrence and relapse by eliminating both cancer stem cells and progenitors.

[0743] As shown by the experimental data in this disclosure, GPC3-targeting CAR T cells secreting anti-EpCAM BiTE (GECAR BiTE T) have demonstrated excellent efficacy in eradicating human xenograft tumors derived from hepatocellular carcinoma both in vitro and in vivo.

[0744] In vitro data show that GE CAR-BiTE T cells kill Hep3B (GPC3) cells slightly faster than anti-GPC3 CAR alone (GE CAR T). 高 EpCAM 高 ) target cells ( Figure 2C This indicates that BiTE plays a crucial role in promoting target cell killing. Culture supernatant collected from GE CAR-BiTE T cells showed the ability to guide T cells to effectively mediate target cell killing. Figure 2D ).

[0745] In terms of in vivo data, in Hep3B xenografts, although both CAR T and GE CAR-BiTE T treatments reduced tumor growth, only mice treated with GE CAR-BiTE achieved complete tumor regression. Figure 3C , Figure 3D ), and also showed good safety, without causing weight loss in mice ( Figure 3B ). Figure 3F and Figure 3G This further demonstrated the superior efficacy of GE CAR-BiTE T cells expanded in a medium containing IL-7 and IL-15 in tumor control and long-term mouse survival. In HepG2 tumor xenografts, a more aggressive mouse model, tumors were successfully cured in all three mice treated with GE CAR-BiTE T cells, unlike the ineffectiveness of the CAR T cell-only treatment group. Figure 4D , Figure 4E ), and all the mice survived ( Figure 4G ), without any significant weight loss ( Figure 4F ).

[0746] A polypeptide comprising a multispecific antigen-binding protein is also disclosed. 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.

[0747] In some instances, multispecific antigen-binding proteins are bispecific immune cell connectors capable of simultaneously binding antigens and immune cells.

[0748] 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 an anti-EpCAM H-chain antibody variable region (i.e., VHH).

[0749] 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 CD3. In some instances, CD3 may include CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0750] 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).

[0751] In some instances, the immune cells are T cells, NK cells, macrophages, or monocytes. In other instances, the immune cells are T cells.

[0752] 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 an inducible BiTE, a non-inducible BiTE, or a constitutively expressed BiTE. In some instances, the bispecific T-cell adaptor (BiTE) binds to two antigens, where 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 contains a BiTE that bispecifically binds EpCAM and T cells. In some instances, the peptide contains a BiTE that bispecifically binds EpCAM and CD3. In some instances, the EpCAM targeted by the BiTE is modified by pairing anti-EpCAM VHH with anti-CD3 scFv.

[0753] In some instances, anti-EpCAM VHH is paired with anti-CD3 scFv, which may include clone Okt3 (Nb01-013A) or another anti-CD3 clone (MT110) (Nb01-013B) used in the art for anti-EpCAM.

[0754] On the other hand, polynucleotides encoding cell and / or polypeptide and / or multispecific antigen-binding proteins as described herein are provided.

[0755] Also disclosed is a polynucleotide comprising a sequence encoding an immune cell adaptor and a chimeric antigen receptor (CAR), wherein the immune cell adaptor is capable of bispecifically binding EpCAM (epithelial cell adhesion molecule) and immune cells, and wherein the CAR is capable of binding a first antigen.

[0756] In some instances, a polynucleotide is provided that contains a sequence encoding a chimeric antigen receptor capable of recognizing, binding to, and articulating GPC3-positive cells. In some instances, the primary antigen is GPC3. Therefore, in some instances, the CAR is capable of binding to GPC3.

[0757] In some instances, polynucleotides also contain sequences encoding one or more co-stimulatory domains, signal peptides, hinges, and / or signal transduction domains.

[0758] In some instances, co-stimulatory domains may include, but are not limited to, 4-1BB, CD28, CD27, OX-40, etc.

[0759] In some instances, the signal peptide can be, but is not limited to, IgH signal peptide, IgK signal peptide, CD8 signal peptide, etc.

[0760] 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, 4-1BB, etc.

[0761] In some instances, the sequence encoding the CAR encodes a GPC3 antigen-binding protein (or anti-GPC3 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-GPC3 scFv.

[0762] 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 an intracellular CD3 domain (signaling domain).

[0763] In some instances, the sequence encoding CAR encodes anti-GPC3 scFv, CD28 co-stimulatory domain, 4-1BB co-stimulatory domain, IgH signal peptide, IgH hinge, and CD3 signal transduction domain.

[0764] In some instances, the sequence encoding the immune cell adaptor 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 adaptor encodes an antigen-binding protein capable of binding EpCAM (anti-EpCAM antigen-binding protein) or fragments or variants thereof, as well as an anti-immune cell antigen-binding protein.

[0765] In some instances, sequences encoding immune cell adaptors encode single-stranded variable fragments (scFv) or single variable domains (VHH) located on the heavy strand.

[0766] In some instances, the sequence encoding the immune cell adaptor encodes anti-EpCAM scFv or anti-EpCAM VHH.

[0767] In some instances, the sequence encoding the immune cell adaptor encodes an anti-immune cell antigen-binding protein that binds to markers of immune cell activation. In some instances, these markers of immune cell activation may include, but are not limited to, CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0768] In some instances, the immunomodulatory marker may be in VHH or scFv form. In some instances, the sequence encoding the immune cell adaptor encodes an antigen-binding protein capable of binding to CD3 (anti-CD3 antigen-binding protein) or a fragment or variant thereof. In some instances, the sequence encoding the immune cell adaptor encodes a single-stranded variable fragment (scFv) or VHH form. In some instances, the sequence encoding the immune cell adaptor encodes an anti-CD3 scFv or an anti-CD3 single-domain VHH.

[0769] In some instances, immune cell adaptors may include His tags.

[0770] In some instances, the immune cell adaptor comprises an anti-EpCAM antigen-binding protein, a linker, an anti-CD3 scFv or an anti-CD3 single-domain VHH, and a His tag. In some instances, the linker is a cleavable linker, which may include, but is not limited to, P2A, T2A, F2A, etc.

[0771] In some instances, the immune cells are T cells, NK cells, macrophages, or monocytes. In other instances, the immune cells are T cells.

[0772] In some instances, the polynucleotide contains a sequence encoding an immune cell adaptor, specifically a sequence encoding a bispecific T cell adaptor (BiTE), such as an inducible BiTE, a non-inducible BiTE, or a constitutively expressed BiTE. In some instances, the polynucleotide contains a sequence encoding a BiTE that bispecifically binds to EpCAM and T cells. In some instances, the polynucleotide contains a sequence encoding an anti-GPC3 scFv CAR with a CD3 intracellular domain, and a sequence encoding a BiTE that binds to EpCAM and T cells.

[0773] Also disclosed is a vector for expressing polynucleotides or polypeptides as described herein. In some instances, the vector is selected from plasmids, viral particles, bacteriophages, baculoviruses, yeast plasmids, lipid-based vectors, polymer microspheres, liposomes, cell-based vectors, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, viral vectors, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, herpesviruses, vaccinia virus, foamy virus, cytomegalovirus, Semlikie Forest virus, poxvirus, pseudorabies virus, RNA viral vectors, DNA viral vectors, and vectors derived from a combination of plasmid and bacteriophage DNA. Further optionally, the polynucleotide is operatively linked to expression control sequences(s) to direct peptide synthesis. More optionally, the vector contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0774] In some instances, the vector is a lentiviral vector.

[0775] Host cells comprising vectors as disclosed herein or polynucleotides or polypeptides as disclosed herein are also disclosed. In some instances, the host cell comprises a cloning or expression vector as described above and / or a nucleic acid sequence encoding an antigen-binding protein, antibody, or binding fragment thereof as described herein.

[0776] The host cell can be any type of cell capable of being transformed or transfected with a nucleic acid or vector to produce an antigen-binding protein or a binding fragment / protein thereof encoded by it. Host cells containing a nucleic acid or 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 expression of the encoded sequence. Antibodies, antigen-binding proteins, or fragments or portions of antibodies can then be isolated from the cell.

[0777] The host cell can be a prokaryotic host cell (e.g., *Escherichia coli*) or a eukaryotic host cell (e.g., yeast, insect, or vertebrate cells). When cultured under appropriate conditions, the host cell expresses an antibody or a binding fragment thereof, which can then be collected from the culture medium (if the host cell secretes it) or directly from the host cell that produced it (if not secreted). The selection of a suitable host cell will depend on a variety of factors, such as the desired expression level, the peptide modifications required or necessary for activity (e.g., 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 is to undergo post-transcriptional modification (e.g., glycosylation and / or phosphorylation). Host cells can include bacterial cells, yeast cells, animal cells (e.g., mammalian cells), and / or plant cells.

[0778] Suitable mammalian host cells include CHO, myeloma, or hybridoma cells. Many are available from the American Type Culture Collection (ATCC, Manassas, Virginia). 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.

[0779] In one implementation, the host cell expresses / secretes antigen-binding proteins, variants thereof, or fragments thereof as disclosed herein.

[0780] In one aspect, a cell expressing / secreting an immune cell connective that is specific for EpCAM is provided.

[0781] In one embodiment, the cell is a stem cell, such as those selected from mesenchymal stem cells, neural stem cells, and pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). Therefore, 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.

[0782] In one implementation, the cells are immune cells.

[0783] In one embodiment, immune cells express / secrete an immune cell connective that is specific to EpCAM. In one embodiment, the immune cell connective is selected from T cell connectives, NK cell connectives, monocyte connectives, and macrophage connectives.

[0784] In one implementation, immune cells express / secrete a bispecific T-cell adaptor (BiTE) that is specific to EpCAM.

[0785] 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 as disclosed herein.

[0786] Surprisingly, the inventors have demonstrated that immune cells (e.g., CAR T cells) can secrete EpCAM BiTE. This was not previously known. 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., the site of a solid tumor), thereby minimizing toxicity and / or side effects.

[0787] 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.

[0788] 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 cell, a CAR NK cell, a CAR macrophage, or a CAR monocyte.

[0789] In some instances, immune cells can bind to more than one host cell antigen. Therefore, in some cases, immune cells can further bind to one host cell antigen, two host cell antigens, three host cell antigens, four host cell antigens, etc.

[0790] In another aspect, a method for producing / generating cells as described herein is provided, comprising introducing polynucleotides as described herein into said cells.

[0791] Methods for generating / creating cells as described herein are also disclosed, wherein one or more nucleic acids encoding host cell target antigens are fused in a vector via an adapter (e.g., 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 collected.

[0792] In some instances, the method includes introducing a vector, as described herein, into a cell. In some instances, the vector is introduced via viral transduction.

[0793] A composition comprising cells as described herein or a supernatant of said cells is also disclosed.

[0794] A pharmaceutical composition comprising (engineered) cells as described herein or a supernatant of said cells and a suitable pharmaceutical composition thereof.

[0795] In some instances, the composition is a preventative and / or therapeutic composition.

[0796] Pharmaceutically acceptable agents used in this pharmaceutical composition include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavoring and diluents, emulsifiers, suspending agents, solvents, fillers, extenders, buffers, delivery media, tension agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobial agents, and surfactants.

[0797] Compositions or pharmaceutical compositions as described herein are also disclosed for use in treatment / medicine / vaccination, and optionally, the compositions may also contain excipients and / or stabilizers.

[0798] In another aspect, a method for treating a disease in a subject in need is provided, the method comprising administering cells as described herein to the subject, optionally the disease being a proliferative disease.

[0799] A method for treating a disease in a subject in need is also disclosed, the method comprising administering to the subject engineered cells or compositions as described herein.

[0800] A method for preventing and / or reducing the severity of disease-related symptoms in subjects in need is also disclosed, the method comprising administering to the subject engineered cells or compositions as described herein.

[0801] In some instances, the cells or composition or pharmaceutical composition 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, and intramuscular routes.

[0802] The use of cells, as described herein, in the preparation of medicaments for the prevention and / or treatment of diseases is also disclosed.

[0803] Also disclosed are polynucleotides, antibodies, bispecific T-cell connectors, engineered cells, engineered immune cells, methods, compositions, or pharmaceutical compositions as described herein.

[0804] 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 (e.g., hepatocellular carcinoma), lung cancer (e.g., squamous cell carcinoma of the lung), stomach cancer (e.g., gastric adenocarcinoma), breast cancer, skin cancer (e.g., melanoma), ovarian cancer (e.g., clear cell carcinoma of the ovary), kidney cancer, pancreatic cancer, head and neck cancer, prostate cancer, esophageal cancer, bladder cancer, colon cancer, and childhood cancers (e.g., hepatoblastoma, nephroblastoma, yolk sac tumor, etc.).

[0805] Furthermore, in describing some embodiments, this disclosure may disclose methods and / or processes as steps in a specific order. However, unless otherwise required, it should be understood that the methods or processes described are not limited to the specific order of steps disclosed. Other orders of steps are 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 written order. The order of steps may vary and remains within the scope of this disclosure.

[0806] Furthermore, it should be understood that while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, in other alternative embodiments, one or more of these features / characteristics may be omitted, and this disclosure provides support for such omissions and these related alternative embodiments.

[0807] sequence

[0808] CDR uses bold (CDR1); bold and italic ( CDR2 ); or bold, italic, and underline ( CDR3 The highlighted residues / bases show the differences between the constant domains of the 1B6 and 1C1 light chains.

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816] Attached Figure Description

[0817] From the following discussion and, where applicable, in conjunction with the accompanying drawings, exemplary embodiments of this disclosure will be more readily understood and apparent to those skilled in the art. 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. These exemplary embodiments should not be construed as limiting the scope of this disclosure.

[0818] Figures 1A to 1H It was shown that anti-GPC3 CAR T (5C4) cells could suppress Hep3B xenografts in NSG mice, but could not completely eradicate them because the escaped tumor cells lost GPC3 expression. Figure 1A FACS analysis of tumor markers (GPC3 and EpCAM) expressed on Hep3B cells is shown. Figures 1B to 1DThe results of subcutaneous injection of two million Hep3B cells into the right abdomen of NSG mice (day -26) are shown. On day 0, mice were grouped according to tumor size and injected intravenously with ten million anti-GPC3 CAR T (5C4) cells or T-cell mimics via the tail vein (day 0). Figure 1B The percentage of CAR expression on anti-GPC3 CAR T (5C4) cells, as detected by flow cytometry analysis, is shown. Figure 1C The tumor size was shown and recorded every 3 to 7 days. Figure 1D The FACS analysis of tumor markers (GPC3 and EpCAM) expressed on tumors re-emerging in mice treated with anti-GPC3 CAR T (5C4) at 70 days post-CAR T cell infusion is shown. Figures 1E to 1H The results of subcutaneous injection of two million Hep3B cells into the right abdomen of NSG mice (day -21) are shown. On day 0, mice were grouped according to tumor size and four million sorted anti-GPC3 CAR T (5C4) cells or T-cell mimics enriched with CAR-positive populations were intravenously injected into these mice via the tail vein (day 0). Figure 1E The study showed that after lentiviral transduction, T cells expressing anti-GPC3 CAR T (5C4) were enriched by flow cytometry sorting, and the percentage of CAR expression on the sorted anti-GPC3 CAR T (5C4) cells was detected by flow cytometry analysis. Tumor size was measured and recorded for each mouse every 3 to 7 days. Figure 1F ) and weight ( Figure 1G ). Figure 1H The FACS analysis of tumor markers expressed on tumors re-emerging in three mice treated with anti-GPC3 CAR T (5C4) at 71 days after CAR T infusion is shown.

[0819] Figures 2A to 2D The construction and in vitro characterization of GE CAR-BiTE T cells are shown. Figure 2A The structural diagram of GE CAR-BiTE is shown. Figure 2B The percentage of CAR expression on anti-GPC3 CAR T (5C4) and GE CAR-BiTE T (using the “MT110” sequence as BiTE) cells, as detected by flow cytometry analysis, is shown. Figure 2C The percentage of cell lysis in human GE CAR-BiTE T cells mediated by time-process measurements using xCelligence impedance measurement (E:T ratio = 1:1) is shown. Figure 2DThe percentage of Hep3B cells mediated by naive T cells activated by anti-EpCAM BiTE is shown. Anti-EpCAM BiTE was secreted by human GE CAR-BiTE T cells, which were isolated from PBMCs of healthy donors. Time-process measurements were performed by xCelligence impedance measurement (E:T ratio = 6:1).

[0820] Figures 3A to 3G This demonstrates the complete eradication of large Hep3B xenografts in vivo by human GE CAR-BiTE T cells. Figures 3A to 3E The results of lentiviral transduction are shown, in which anti-GPC3 CAR T (5C4) cells, GE CAR-BiTE T cells, or T-mimicking cells were cultured and expanded in T-cell growth medium containing IL-2 (50 units per mL). Two million Hep3B cells were subcutaneously injected into the right abdomen of NSG mice (day -27). On day 0, the mice were regrouped according to measurable tumor size (3-5 mice per group), and eight million GE CAR-BiTE cells, CAR T (5C4) cells, or T-mimicking cells were intravenously injected into these mice via the tail vein. Figure 3A The percentage of CAR expression on anti-GPC3CAR T (5C4) and GE CAR-BiTE T (using the “MT110” sequence as BiTE) cells, as detected by flow cytometry, is shown. Weight of each mouse was measured and recorded every 3 to 7 days. Figure 3B ) and tumor size ( Figure 3C ). Figure 3D The tumor size of each individual mouse in each treatment group is shown. Figure 3E The study showed the removal of tumors in selected mice and the analysis of the expression of tumor markers GPC3 and EpCAM. Figure 3F and 3G The results of lentiviral transduction are shown, in which anti-GPC3 CAR T (5C4) cells, GE CAR-BiTE T cells, or T-mimicking cells were cultured and expanded in T-cell growth medium containing IL-7 (20 ng / ml) and IL-15 (5 ng / ml). Two million Hep3B (GPC3-positive, EpCAM-positive) cells were subcutaneously injected into the right abdomen of NSG mice (day -21). On day 0, the mice were regrouped according to measurable tumor size (3–6 mice per group), and eight million GE CAR-BiTE cells, CAR T (5C4) cells, or T-mimicking cells were intravenously injected into these mice via the tail vein. Figure 3F The tumor size of each mouse was measured and recorded every 3-4 days. Figure 3GSurvival curves for mice in the "Tumor Only" group (grey dashed line), "Mock T" group (grey solid line), "CAR T (5C4)" group (black dashed line), and "GE CAR-BiTE T" group (black solid line) are shown. Death was defined as either observed spontaneous death of the mouse or the mouse carrying a tumor larger than 2700 mm. 3 .

[0821] Figures 4A to 4G This demonstrates the complete eradication of HepG2 xenografts in vivo using human GE CAR-BiTE T cells. Figure 4A and 4B The results are shown after subcutaneous injection of one million HepG2 cells into the right abdomen of NSG mice (day -9). On day 0, the mice were regrouped according to measurable tumor size and injected intravenously with ten million CAR T (5C4) cells or T-cell analogs via the tail vein. Figure 4A The percentage of CAR expression on anti-GPC3 CAR T(5C4) cells, as detected by flow cytometry analysis, is shown. Figure 4B The tumor size of each mouse was measured and recorded every 3 to 7 days. Figure 4C to 4G The results are shown when one million HepG2 cells were subcutaneously injected into the right abdomen of NSG mice (day -4). On day 0, the mice were regrouped according to measurable tumor size and three million GE CAR-BiTE T cells, CAR T (5C4) cells, or T-cell mimics were intravenously injected into these mice via the tail vein. Figure 4C The percentage of CAR expression on anti-GPC3 CAR T (5C4) and GE CAR-BiTE T (using the “MT110” sequence as BiTE) cells, as detected by flow cytometry analysis, is shown. Figure 4D The tumor size of each mouse was measured and recorded every 3 to 7 days. Figure 4E The tumor size of each individual mouse in each treatment group is shown. Figure 4F The figure shows that the weight of each mouse was measured and recorded every 3 to 7 days. Figure 4G Survival curves for mice are shown. Death was defined as either observed spontaneous death of a mouse or a tumor exceeding 1700 mm in size. 3 .

[0822] Figures 5A to 5H This study demonstrated that GE CAR-BiTE T cells constructed using BiTE with anti-EpCAM VHH exhibited superior properties in terms of cytotoxic efficacy and specificity. Figure 5AThe structural forms of MT110 BiTE, Nb01-013ABiTE, or Nb01-013B BiTE antibodies secreted by GE CAR-BiTE cells are shown. Figure 5B FACS analysis of tumor markers (GPC3 and EpCAM) expressed on HepG2, HT-29, and HeyA8 cells is shown. CAR T (5C4) or GE CAR-BiTE T cells (MT110, Nb01-013A, or Nb01-013B) expression of HepG2 (GPC3 and EpCAM) was performed using xCelligence cellular impedance assay. Figure 5C ), HT-29 ( Figure 5D ) and HeyA8 ( Figure 5E The killing effect of interferon-r was measured and presented within 72 hours after co-culturing target cells and effector cells (E:T ratio = 2:1). The ELISA measurement of interferon-r was performed using the culture supernatant, which was prepared by co-culturing effector CAR T cells with HepG2 (… Figure 5F ), HT-29 ( Figure 5G ) and HeyA8 ( Figure 5H Cells were collected 48 hours after co-culturing.

[0823] Figure 6A and 6B Human GE CAR-BiTE T cells constructed using anti-EpCAM VHH-based BiTEs demonstrated superior tumor-killing efficacy in HepG2 xenografts in vivo. One million HepG2 cells were subcutaneously injected into the right ventricular region of 30 male NSG mice (day -4). On day 0, mice were regrouped based on measurable tumor size and intravenously injected with three million CAR T (5C4) cells, GE CAR-BiTE T cells (using “MT110,” “Nb01-013A,” or “Nb01-013B” as BiTEs), or simulated T cells via the tail vein. Figure 6A The tumor size of each individual mouse in each treatment group was measured and recorded every 3 to 7 days. Figure 6B The figures show that the weights of mice in different groups were measured and recorded every 3 to 7 days.

[0824] Figures 7A to 7D This study demonstrates that GE CAR-BiTE T cells constructed using anti-EpCAM VHH exhibit superior cytotoxicity, and that anti-EpCAM BiTE secreted by GE CAR-BiTE T cells can induce target cell killing independently of high GPC3 expression. Figure 7AThe structural forms of anti-GPC3 CAR T (5C4) or GE CAR-BiTE T cells (secreting anti-EpCAM BiTE anti-GPC3 CAR T cells, using “MT110”, “Nb01-013A”, or “Nb01-013B” as BiTE) or 19E CAR-BiTE T cells (secreting anti-EpCAM BiTE anti-CD19 CAR T cells, using “Nb01-013A” or “Nb01-013B” as BiTE) are shown. Pairs of anti-GPC3 CAR T or GE CAR-BiTE T cells or 19E CAR-BiTE T cells were analyzed using xCelligence cellular impedance analysis. Figure 7B Hep3B (GPC3) 高 EpCAM 高 ), ( Figure 7C HT-29 (GPC3) 低 EpCAM 高 )and( Figure 7D HeyA8 (GPC3) 阴性 EpCAM 阴性 The in vitro killing effect of cells was measured and presented within 72 hours after co-culturing target cells and effector cells (E:T ratio = 1:1).

[0825] Figure 8A The results show that human GE CAR-BiTE T cells constructed using anti-EpCAM VHH exhibit excellent tumor-killing effects in Hep3B xenografts in vivo. Figure 8A A schematic timeline of the in vivo Hep3B xenograft model is shown. Two million Hep3B cells were subcutaneously injected into the right abdomen of 50 male NSG mice (day -21). On day 0, the mice were regrouped according to measurable tumor size and injected intravenously via the tail vein with eight million CAR T (5C4) cells, GE CAR-BiTE T cells (anti-GPC3 CAR T cells secreting anti-EpCAM BiTE, using “MT110”, “Nb01-013A”, or “Nb01-013B” as BiTE), 19E CAR-BiTE T cells (anti-CD19 CAR T cells secreting anti-EpCAM BiTE, using “Nb01-013A” or “Nb01-013B” as BiTE), or simulated T cells. Ninety-two days after CAR T or CAR-BiTE T cell therapy, three surviving mice from the GE CAR-BiTE T (Nb01-013A) group were attacked with one million Hep3B cells (subcutaneously injected into the left abdomen), while five initial male NSG mice served as controls ("tumor only"). Figure 8B The tumor size of each mouse was measured and recorded every 3 to 7 days. The curves show the tumor size up to day 92. Figure 8C The tumor size of each individual mouse in each treatment group is shown up to day 92. Figure 8D The tumor size of the re-attacked tumor in the left abdomen is shown (starting from day 92).

[0826] Figures 9A to 9F This study demonstrates that human GE CAR-BiTE T cells constructed using anti-EpCAM VHH exhibit excellent in vivo T cell expansion in a Hep3B mouse xenograft model. In the same experiments as in Figure 8, buccal blood samples were collected weekly for flow cytometry analysis to monitor peripheral blood T cell counts, and cytokine levels were detected using Luminex® multiplex assays. Figure 9A The number of CD3-positive human T cells in the peripheral blood of selected mice was shown after 5 weeks of T cell infusion. Figure 9B The time-process analysis of CAR-positive human T cells in peripheral blood of mice in the CAR T (5C4) treatment group is shown. Figure 9C This study presents a time-progression analysis of CAR-positive human T cells in the peripheral blood of mice treated with GE CAR-BiTE T (Nb01-013A). Interferon-γ levels in the peripheral blood of selected mice 5 weeks after T cell infusion were measured using Luminex® multiplex assays. Figure 9D ), GM-CSF ( Figure 9E ) and perforin ( Figure 9F ) level.

[0827] Figure 10 Figures A through 10C show that human GE CAR-BiTE cells constructed using anti-EpCAM VHH-based BiTE exhibited excellent intratumoral T cell expansion in a Hep3B mouse xenograft model. In the same experiments as in Figure 8, selected mice were sacrificed 5 weeks after T cell infusion, the xenograft tumors were excised and isolated, and flow cytometry analysis was performed to determine the number of human T cells within the tumor. Figure 10 A shows the number of CD3-positive human T cells in mouse tumors of selected mice, expressed as cells per gram of tumor. Figure 10 B shows the number of CAR-positive human T cells in mouse tumors of selected mice, expressed as cells per gram of tumor. Figure 10 C shows the percentage of CAR-positive T cells in the human T cell population of selected mice in mouse tumors. Mice that showed effective tumor control were labeled.

[0828] Figure 11This diagram shows a comparison between CAR T cells enriched in tumors and CAR T cells dispersed in peripheral blood. In the same experiment as Figure 8, CAR T cells in the peripheral blood of selected mice (M18, M49, M38, and M40) with tumor control observed 5 weeks after T cell infusion were analyzed by flow cytometry, along with CAR T cells from excised tumors. The total number of CAR-positive T cells in the tumor was calculated by multiplying by the total weight of the tumor, while the total number of CAR-positive T cells in the peripheral blood was calculated by multiplying by the average blood volume of the experimental mice (approximately 2 ml).

[0829] Figure 12 The anti-EpCAM BiTE molecule Nb01-013A secreted by GE CAR-BiTE T cells was shown by ELISA detection. Figure 12 A shows the detection of anti-EpCAM BiTE molecules when anti-GPC3 CAR T (5C4) cells or GE CAR-BiTE T (Nb01-013A) cells were cultured in T cell growth medium containing IL-7 (20 ng / ml) and IL-15 (5 ng / ml). The initial cell density was 500,000 cells / mL, and the percentage of CAR expression in both cultures was approximately 60%. Cell culture supernatants were collected daily (24 h, 48 h, 72 h, 96 h) for ELISA to detect the amount of secreted anti-EpCAM BiTE molecule Nb01-013A. Figure 12 B shows that when GE CAR-BiTE T (Nb01-013A) cells are combined with Hep3B (GPC3) 高 EpCAM 高 HT-29 (GPC3) 低 EpCAM 高 ) or HeyA8 (GPC3) 阴性 EpCAM 阴性The detection of anti-EpCAM BiTE molecules when cells were co-cultured at a 2:1 E:T ratio. The percentage of CAR expression in GE CAR-BiTE T (Nb01-013A) cells used in all co-cultures was 8.5%. Supernatants were collected daily (24 hours, 48 ​​hours, and 72 hours) for ELISA to detect the amount of anti-EpCAM BiTE molecule Nb01-013A secreted by GE CAR-BiTE T (Nb01-013A) cells after co-culturing with different target cells. Briefly, ELISA plates were coated overnight with human EpCAM-Fc tag protein, blocked with casein for 2 hours, and then culture supernatant containing anti-EpCAM BiTE molecule Nb01-013A was added to the plates. After 1 hour of incubation, the plates were washed, and the bound BiTE molecules were detected by HRP-conjugated secondary antibody targeting the His tag.

[0830] Experimental Section

[0831] CAR T cells targeting GPC3 alone lead to antigen loss in an in vivo xenograft mouse model.

[0832] The in vivo therapeutic efficacy of 5C4 CAR T cells has been demonstrated in two independent experiments using an NSG mouse model transplanted with human hepatocellular carcinoma cells Hep3B (Figure 1). Hep3B cells in in vitro cultures have shown high expression levels of GPC3 and EpCAM (GPC3 and EpCAM). Figure 1A In the first experiment, 5C4 CAR T cells expressing 25.9% CAR were successfully prepared. Figure 1B CAR T cells demonstrated excellent tumor control, effectively inhibiting the growth of transplanted Hep3B tumors. Figure 1C However, after the tumor-free period, the tumor reappeared. Figure 1C ), and appears to have lost the antigen target GPC3, while EpCAM expression remains high ( Figure 1D In the second experiment, sorted, CAR-positive anti-GPC3 CART (5C4) cells (94.1%) were used. Figure 1E ) to treat the same Hep3B xenograft. Although treatment with these enriched CAR-expressing cells showed enhanced tumor control compared to the first experiment ( Figure 1F However, this still failed to stop the tumor from regrowth. These escaped tumor cells once again fertilized GPC3. 阴性 and EpCAM 阳性 This further reinforces the concept of multi-targeted approaches in the treatment of solid tumors. Furthermore, CAR T-cell therapy targeting GPC3 did not lead to weight loss in these mice, indicating low or no toxicity. Figure 1G ).

[0833] Construction and in vitro functional characterization of GE CAR-BiTE T construct

[0834] The GE CAR-BiTE T construct is designed to fuse the anti-EpCAM BiTE gene (using the "MT110" sequence) into an anti-GPC3 CAR (clone 5C4) lentiviral construct via a P2A cleavable linker. Figure 2A Lentiviral transduction and amplification ( Figure 2B The in vitro killing efficacy of CAR T cells was tested. Interestingly, GE CAR-BiTE T cells killed Hep3B (GPC3) cells. 高 EpCAM 高 The target cells were detected slightly faster than the anti-GPC3 CAR alone. Figure 2C This indicates that BiTE plays a crucial role in promoting target cell killing. The cytotoxic function of GE CAR-BiTE was further tested in another assay, which showed that only the culture supernatant collected from GE CAR-BiTE T cells mediated effective T cell-mediated target cell killing (initial T cells isolated from donor PBMCs). Figure 2D ).

[0835] Human GE CAR-BiTE T cells completely eradicate large Hep3B xenografts in vivo.

[0836] The tumor control efficacy of GE CAR-BiTE T was evaluated in NSG mice using Hep3B xenografts, with transduction rate assessed by flow cytometry. Figure 3A Although both CAR T (5C4) and GE-CAR-BiTE T treatments reduced tumor growth, only mice treated with GE-CAR-BiTE achieved complete tumor regression. Figure 3C , 3D ), and also showed good safety without causing weight loss in mice ( Figure 3B Further analysis showed that tumor cells surviving after anti-GPC3 CAR T(5C4) treatment lost GPC3 expression but still expressed EpCAM ( Figure 3E However, when the inventors analyzed cells isolated from residual tissue at the tumor transplantation site of representative mice treated with GE CAR-BiTE T cells, they were unable to detect any cells expressing GPC3 or EpCAM. Figure 3EThese double-negative cells were likely mouse stromal cells residing at the transplant site, and all Hep3B tumor cells were eradicated after GE CAR-BiTE T cell therapy. Next, the inventors slightly modified the T cell culture protocol, changing the IL-2 in the culture medium to a combination of IL-7 and IL-15, which is currently the widely accepted standard protocol for preparing CAR T cells in a clinical setting. Reproduced experiments using GE CAR-BiTE T cells expanded in a medium containing IL-7 and IL-15 reproduced the tumor control (… Figure 3F ) and long-term mouse survival ( Figure 3G The results show excellent efficacy in this area.

[0837] Human GE CAR-BiTE T cells completely eradicate HepG2 xenografts in vivo.

[0838] Similar to Hep3B, another HCC cell line, HepG2, also expresses high levels of GPC3 and EpCAM on its cell surface. However, compared to the Hep3B model, even when CAR T cells were infused at an earlier date after tumor inoculation (9 days in the HepG2 model compared to 21-27 days in the Hep3B model), anti-GPC3-targeted CAR T cells alone were relatively less effective in treating HepG2-derived xenografts. Figure 4A and 4B The inventors believe this is because HepG2 xenografts grow much faster in mice than Hep3B xenografts. The inventors are investigating whether GE CAR-BiTE T-cell therapy can be successful in this more aggressive mouse model. Lentiviral transduction ( Figure 4C Four days after HepG2 tumor cell inoculation, mice were infused with either CAR T (5C4) or GE CAR-BiTE T cells intravenously. Unlike the poorer response in the CAR T (5C4) treatment group, tumors in all three mice treated with GE CAR-BiTE T cells were successfully cured. Figure 4D , Figure 4E ), and all the mice survived ( Figure 4G No significant weight loss was observed until the experiment was voluntarily terminated on day 80. Figure 4F ).

[0839] Human GE CAR-BiTE cells using the novel BiTE technology have shown excellent cytotoxicity and specificity. sex.

[0840] The inventors noted that GE CAR-BiTE T cells using “MT110” typically exhibited lower T cell viability and poorer T cell expansion rates compared to CAR T (5C4) cells. Therefore, “MT110” was replaced with different versions of BiTE, wherein the anti-EpCAM arm was replaced with an internally identified anti-EpCAM VHH (clone 2C4) and paired with two different anti-CD3 scFv (clone Okt3 or the anti-CD3 clone used in “MT110”). These were named “Nb01-013A” and “Nb01-013B”, respectively. Figure 5A To compare their cytotoxic effects with GE CAR-BiTE T cells using "MT110", three representative cell lines were selected, including HepG2 (expressing high levels of GPC3 and EpCAM), HT-29 (expressing very low levels of GPC3 but high levels of EpCAM), and HeyA8 cells (not expressing GPC3 or EpCAM). Figure 5B Using these three cell lines expressing different levels of surface antigens, the results showed that GE CAR-BiTE cells equipped with novel BiTE (Nb01-013A or Nb01-013B) exhibited superior characteristics in killing HepG2 and HT-29 cells, with potency comparable to MT110 BiTE. Interestingly, although T cells secreting MT110 also induced strong cytotoxicity against HeyA8 cells, GE CAR-BiTE T cells using novel BiTE (Nb01-013A or Nb01-013B) did not attack these target-negative cells. Figure 5C , 5D (and 5E). This may be due to MT110's anti-EpCAM arm nonspecifically binding to different unknown cell surface proteins, or specifically binding to the EpCAM protein, whose expression level is extremely low, below the detection limit of flow cytometry. This may be attributed to MT110's high affinity for EpCAM. T cell activation-mediated target cell killing can also be reflected by parallel interferon-γ ELISA measurements ( Figure 5F , 5G and 5H).

[0841] In summary, the inventors have developed a novel CAR T-cell therapy for GPC3-positive solid tumors by locally secreting anti-EpCAM BiTE to provide additional targeting of EpCAM-positive cancer cells to CAR T cells targeting a single antigen, thus overcoming the challenge of tumor heterogeneity. By using CAR T cells that secrete anti-EpCAM BiTE, tumor clearance can be promoted by recruiting nearby bystander T cells (a population of T cells that do not express CAR), while avoiding the systemic toxicity that may be associated with other anti-EpCAM BiTE delivery methods, thereby maximizing the efficacy of CAR T-cell therapy. This disclosure illustrates the development of a prototype (GE CAR-BiTE), which also serves as a proof of concept. While the efficacy of GE CAR-BiTE has been demonstrated, this technology is not limited to CARs targeting GPC3 but can be extended to many other tumor antigens that can serve as CAR targets for various types of cancer occurring in multiple organs.

[0842] Human GE CAR-BiTE cells using VHH BiTE molecules demonstrated tumor killing activity in a HepG2 mouse xenograft model. It exhibits excellent properties in terms of wound healing efficacy.

[0843] After constructing GECAR-BiTE T cells using different anti-EpCAM BiTE molecules (“MT110”, “Nb01-013A”, and “Nb01-013B”), the inventors continued to test their tumor control efficacy using in vivo xenograft models. The HepG2 xenograft model was tested first (Figure 6). Although CAR T (5C4) failed to control tumor growth, all three groups of GECAR-BiTE T cell therapy showed some in vivo tumor control efficacy. Figure 6A ), without significant weight loss ( Figure 6B Tumor shrinkage was observed in 40% (2 / 5 mice), 60% (3 / 5 mice), and 80% (4 / 5 mice) of the “MT110,” “Nb01-013A,” and “Nb01-013B” groups, respectively. Figure 6A ).

[0844] Human GE CAR-BiTE cells using the VHH BiTE molecule showed superior performance in killing cells expressing EpCAM. Different CAR antigen-independent in vitro cytotoxicity.

[0845] Next, use Hep3B (GPC3) 高 EpCAM 高 HT-29 (GPC3) 低 EpCAM 高 ) and HeyA8 (GPC3) 阴性 EpCAM 阴性The study tested GECAR-BiTE T cells using different anti-EpCAM BiTE (“MT110”, “Nb01-013A”, and “Nb01-013B”) and anti-CD19 CAR T cells secreting anti-EpCAM BiTE (“Nb01-013A” or “Nb01-013B”) (named “19E CAR-BiTE T cells”). Figure 7A (It has the effect of killing bacteria in vitro.)

[0846] In the in vitro killing assay using the xCelligence RTCA system, a 1:1 E:T ratio was used (adjusted to 10% CAR expression level). All GE CAR-BiTE T cells, including GE CAR-BiTE T (MT110) cells, did not show HeyA8 (GPC3) expression at low E:T ratios. 阴性 EpCAM 阴性 Cell killing effect ( Figure 7D However, in killing EpCAM-positive cells Hep3B ( Figure 7B ) and HT-29 ( Figure 7C Strong cytotoxicity was observed in 19E CAR-BiTE T cells (using "Nb01-013A" as BiTE) against EpCAM-positive Hep3B (GPC3) cells. 高 EpCAM 高 )( Figure 7B ) and HT-29 (GPC3 低 EpCAM 高 )( Figure 7C Both showed partial killing, indicating that under the current in vitro conditions, the amount of anti-EpCAM BiTE secreted by 19E CAR-BiTE T cells (Nb01-013A) exceeded the BiTE-mediated cell-killing threshold and was independent of the expression level of CAR-specific antigens. Conversely, 19E CAR-BiTE T cells (using “Nb01-013B” as BiTE) showed partial killing only against HT-29 (GPC3) cells. 低 EpCAM 高 The cells showed minimal killing effect, indicating that "Nb01-013B" BiTE is far less effective than "Nb01-013A" BiTE in mediating T cell killing. Figure 7C ).

[0847] Human GE CAR-BiTE cells using VHH BiTE molecules showed superior efficacy in a Hep3B mouse xenograft model. Different CAR antigen-dependent tumor killing efficacy.

[0848] Based on this observation, it was speculated whether in vivo tumor control depends on or is independent of CAR-specific antigen expression (Figure 8). Interestingly, Hep3B xenografts from two mice treated with 19E CAR-BiTE T cells showed similar growth rates to the control groups (“tumor-only” and “T-mimicking”), suggesting that, unlike in vitro, the in vivo tumor control efficacy of CAR-BiTE T cells depends on CAR-specific antigen expression. In this experiment, the inventors did not observe the effect of GE CAR-BiTET (MT110) cells in controlling Hep3B xenografts, whereas this was always observed in previous experiments. This is because when the humanitarian endpoint (tumor size exceeding 3000 mm) is reached... 3 When the mice were euthanized, the in vivo expansion of GE CAR-BiTE T (MT110) cells had not yet reached the threshold point for significant tumor control. This may be due to the different T cell donors used to generate CAR T cells in different experiments. However, GE CAR-BiTE T cells generated using the same T cell donor (using "Nb01-013A" or "Nb01-013B" as BiTE) showed stronger tumor control efficacy, indicating that replacing the anti-EpCAM arm with our anti-EpCAM 2C4-VHH significantly improved the in vivo expansion and in vivo killing efficacy of GE CAR-BiTE T cells. All nine test mice in the GE CAR-BiTE T (Nb01-013A) mouse group achieved effective tumor control. In addition, three mice survived in a healthy state for more than 90 days after treatment. Figure 8B , Figure 8C On day 92 following GE CAR-BiTE T (Nb01-013A) cell therapy, a re-challenge test was conducted using 1 million Hep3B cells, which were subcutaneously injected into the left abdomen of the remaining three surviving mice. Five initial NSG mice were also included as a "tumor-only" control. Approximately 20 days later, all initial mice injected with 1 million Hep3B cells began to show tumor growth, reaching an average size of 445.3 mm on day 133 (41 days after subcutaneous injection of 1 million Hep3B cells). 3 However, no tumor growth was observed in the left abdomen of any of the three mice in the GE CAR-BiTE T (Nb01-013A) group that underwent re-attack. Figure 8D ).

[0849] Human GE CAR-BiTE cells using VHH BiTE molecules showed superior efficacy in a Hep3B mouse xenograft model. Different T-cell proliferation in the body.

[0850] To monitor the in vivo expansion and cytotoxic function of CAR T cells, the inventors collected buccal blood samples weekly for flow cytometry analysis of T cell counts and Luminex® multiplex assay analysis of cytokine levels (Figure 9). From week 4 (day 28 post-T cell therapy), significant expansion of human T cells was detectable in mouse blood, with the number continuously increasing before reaching peak levels and then decreasing. Figure 9B , Figure 9C As expected, when comparing CD3-positive T cell levels in different groups 5 weeks after T cell infusion, T cell expansion was significantly higher in mice in the GE CAR-BiTE T (Nb01-013A) and GE CAR-BiTE T (Nb01-013B) groups than in the GE CAR-BiTE T (MT110) group. Figure 9A In addition, the serum cytokines secreted by human T cells in the first two groups of mice, such as human interferon-γ (…),… Figure 9D ), GM-CSF ( Figure 9E ) and perforin ( Figure 9F The levels of CAR-T (5C4) cells were comparable to those in the CAR-T (5C4) group mice, and significantly higher than those in the GE CAR-BiTE T (MT110) group and three control groups, including the mimic T, 19E CAR-BiTE T (Nb01-013A), and 19E CAR-BiTE T (Nb01-013B). Due to efficient T cell expansion in the mice, all tumors in the CAR-T (5C4) and GE CAR-BiTE T (Nb01-013A) groups, and five tumors in the GE CAR-BiTE T (Nb01-013B) group, reached 3000 mm. 3 Previously all shrank ( Figure 8C ).

[0851] Human GE CAR-BiTE cells using VHH BiTE molecules showed superior efficacy in a Hep3B mouse xenograft model. Abnormal local T cell expansion within the tumor.

[0852] On days 33-35, all mice in the “tumor only,” “simulated T,” “GE CAR-BiTE T (MT110),” “19E CAR-BiTE T (Nb01-013A),” and “19E CAR-BiTE T (Nb01-013B)” groups were euthanized because their tumor sizes were mostly close to the humanitarian endpoint. The inventors selected one to two mice from each group to analyze tumor-infiltrating human T cells from the excised tumors. For example, in the CAR T (5C4) and GE CAR-BiTE T (Nb01-013A) groups, all treated mice showed good tumor growth control; therefore, the inventors randomly selected one mouse from each of these groups for FACS analysis, while the remaining eight mice were kept for continuous monitoring. However, for the GE CAR-BiTE T (Nb01-013B) group, not all mice showed tumor shrinkage at the analysis date. Therefore, the inventors included a total of five mice for FACS analysis, in which effective tumor control was observed in M38 and M40, but not in M34, M37, and M39. Interestingly, the inventors observed that human T cells (CD3-positive cells) were present in M18, M49, M38, and M40. Figure 10 A) and CAR-positive T cells ( Figure 10 B) Abundant accumulation indicates that effective intratumoral CAR T cell expansion is necessary for effective tumor control activity. Furthermore, a moderate to high percentage of anti-GPC3 CAR T cells was detectable in the M18 (CAR T group) and all GE CAR-BiTE T group mice (M29, M49, M34, M37, M38, M39, and M40), but not in the 19E CAR-BiTE T group (M27, M22, and M23) (CAR-positive T cell population less than 10%). Figure 10 (C) This indicates that CAR-positive T cells only accumulate in vivo when CAR T cells encounter their CAR-targeting antigen.

[0853] More interestingly, when the inventors compared the number of tumors removed and the total number of CAR-positive T cells in the peripheral blood of four mice that showed effective tumor control ( Figure 11 It was observed that the number of CAR-positive T cells located in the tumor was significantly higher than the number of CAR-positive T cells circulating in the peripheral blood.

[0854] Anti-EpCAM camel VHH BiTE secreted by GE CAR-BiTE T cells could be detected in cell cultures.

[0855] Anti-GPC3 CAR T (5C4) and GE CAR-BiTE T (Nb01-013A) cells were cultured in T cell growth medium containing IL-7 and IL-15 at an initial cell density of 500,000 cells / mL. Cell culture supernatant was collected daily for four consecutive days, and ELISA was performed to detect the amount of secreted anti-EpCAM BiTE molecule Nb01-013A. The results clearly showed that the amount of secreted Nb01-013A molecules increased over time, indicating continuous secretion and accumulation of anti-EpCAM VHH BiTE (5C4) in GE CAR-BiTE T (Nb01-013A) cell cultures. Figure 12 A).

[0856] Interestingly, in another experiment, GE CAR-BiTE T (Nb01-013A) cells were co-cultured with different target cells, including Hep3B (GPC3). 高 EpCAM 高 HT-29 (GPC3) 低 EpCAM 高 ) and HeyA8 (GPC3) 阴性 EpCAM 阴性 The percentage of CAR expression in GE CAR-BiTE T (Nb01-013A) cells used in all co-cultures was 8.5%. Culture supernatants were collected daily from the co-cultures over three days, and ELISA showed that Nb01-013A secretion only increased over time after co-culturing with Hep3B cells, indicating that GE CAR-BiTE T (Nb01-013A) cells only increased when co-cultured with GPCs. 高 CAR-positive T cell populations will only expand when target cells are present. Figure 12 B).

[0857] In summary, this disclosure demonstrates that the use of VHH BiTE molecules constructed from anti-EpCAM VHH clone 2C4 (“Nb01-013A” and “Nb01-013B”) in GE CAR-BiTE T cells achieves more efficient and stronger tumor-killing effects than GE CAR-BiTE T cells using “MT110”, with the best efficacy observed when “Nb01-013A” is used as the BiTE molecule.

[0858] application

[0859] The modified cell implementation schemes disclosed herein utilize 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.

[0860] The technology described in this TDF can have the following applications:

[0861] 1. Anti-GPC3 CAR T cells that secrete anti-EpCAM BiTE can be used to treat cancers that express GPC3, such as hepatocellular carcinoma, clear cell carcinoma of the ovary (OCCC), squamous cell carcinoma of the lung, melanoma, and some childhood cancers (hepatoblastoma, nephroblastoma, and yolk sac tumor), as well as certain gastric cancers.

[0862] 2. CAR T cells secreting anti-EpCAM BiTE can be configured to target other tumor-specific antigens, including but not limited to HER2, Claudin18.2, ROR1, DLL3, CEA, MUC1, MUC16, CEACAM7, CD133, CD147, PSCA, PSMA, MSLN, c-Met, and FRα. These anti-EpCAM BiTE-secreting 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.

[0863] This disclosure improves the efficacy of current single-target CAR T-cell therapy for solid tumors by using 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 targeted non-tumor 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.

[0864] Those skilled in the art will understand that other changes and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of this disclosure as broadly described. For example, features of different exemplary embodiments may be mixed, combined, interchanged, merged, adopted, modified, included, etc., across different exemplary embodiments in the description herein. Therefore, this embodiment should be considered illustrative rather than restrictive in all respects.

Claims

1. A modified cell that expresses (a) Chimeric antigen receptors targeting GPC3 or CD19, and (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. The first antigen-binding protein, its variants, or binding fragments that bind EpCAM contain a heavy chain variable region and / or a light chain variable region selected from the following: (i) Heavy chain variable region, which contains: CDR-H1, wherein CDR-H1 comprises: GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it, or GSSERFTS (SEQ ID NO: 29) CDR-H2, wherein CDR-H2 comprises: ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it, or ITNGGST (SEQ ID NO: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-H3, wherein CDR-H3 comprises: TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. MAGTS (SEQ ID NO: 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, or TNGRWSGDTYYAHL (SEQ ID NO: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. (ii) Heavy chain variable region, which contains: CDR-H1, wherein CDR-H1 comprises GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2, wherein CDR-H2 comprises IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-H3, wherein CDR-H3 comprises ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. (iii) Heavy chain variable region, which includes: CDR-H1, wherein CDR-H1 comprises GDSISSNSVA (SEQ ID NO: 5) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2, wherein CDR-H2 comprises TYYRSKWYS (SEQ ID NO: 6) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-H3, wherein CDR-H3 comprises AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. (iv) Light chain variable region, which includes: CDR-L1, wherein CDR-L1 includes: QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. QSLLHSNRYNY (SEQ ID NO: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, 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. CDR-L2, wherein CDR-L2 includes: LGS (SEQ ID NO: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it, or AAS (SEQ ID NO: 20-1E4) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-L3, wherein CDR-L3 includes: MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. MQGLQSPWT (SEQ ID NO: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. QQSYIMPDT (SEQ ID NO: 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, or MQGLQTPYT (SEQ ID NO: 23) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it.

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

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

4. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy chain variable region comprising: CDR-H1, wherein CDR-H1 comprises: GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it, or GSSERFTS (SEQ ID NO: 29) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2, wherein CDR-H2 comprises: ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it, or ITNGGST (SEQ ID NO: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it; and CDR-H3, wherein CDR-H3 comprises: TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. MAGTS (SEQ ID NO: 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, or TNGRWSGDTYYAHL (SEQ ID NO: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it.

5. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy chain variable region selected from: (i) Heavy chain variable region, which contains: CDR-H1, wherein CDR-H1 comprises GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-H3, wherein CDR-H3 comprises TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. (ii) Heavy chain variable region, which contains: CDR-H1, wherein CDR-H1 comprises GSSERFTS (SEQ ID NO: 29) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2, wherein CDR-H2 comprises ITNGGST (SEQ ID NO: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-H3, wherein CDR-H3 comprises MAGTS (SEQ ID NO: 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it; and (iii) Heavy chain variable region, which includes: CDR-H1, wherein CDR-H1 comprises GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. CDR-H2, wherein CDR-H2 comprises ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, and CDR-H3, wherein CDR-H3 comprises TNGRWSGDTYYAHL (SEQ ID NO: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with it.

6. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy chain variable domain and / or a light chain variable domain selected from the following: (i) Heavy-chain variable structural domains, which contain QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (ii) Heavy-chain variable structural domains, which contain QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (iii) Heavy-chain variable structural domains, which contain EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (iv) Heavy-chain variable structural domains, which contain QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (v) Light chain variable structural domain, which contains DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (vi) Light chain variable structural domain, which contains EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGTDFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (vii) Light chain variable structural domain, which contains DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (viii) Light chain variable structural domain, which contains DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYAASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (ix) Light chain variable structural domain, which contains DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (x) Heavy-chain variable structural domain, which contains QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (xi) Heavy-chain variable structural domain, which contains QVQLQESGGGLVQPGGSLRLSCAASGSSERFTSVAWYRQAPGKERELVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCMAGTSWGQGTQ (SEQ ID NO: 32), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (xii) Heavy-chain variable structural domain, which contains QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions. (xiii) Heavy-chain variable structural domains, which contain QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions.

7. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy chain variable domain and / or a light chain variable domain selected from the following: (i) Heavy-chain variable structural domains, which contain: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or fragments, variants, or sequences thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions, and / or Light chain variable structural domain, which contains DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (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 with it and / or having two or three amino acid substitutions. (ii) Heavy-chain variable structural domains, which include: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or fragments, variants, or sequences thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions, and / or Light chain variable structural domain, which contains 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 with it and / or having two or three amino acid substitutions. (iii) Heavy-chain variable structural domains, which include: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or fragments, variants, or sequences thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions, and / or Light chain variable structural domain, which contains DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (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 with it and / or having two or three amino acid substitutions. (iv) Heavy-chain variable structural domains, which contain: QVQLQQSGPGLVKPSQTLSLTCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), or fragments, variants, or sequences thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions, and / or Light chain variable structural domain, which includes: 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 with it and / or having two or three amino acid substitutions, or (v) Heavy-chain variable structural domains, which contain: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARSLGGRFRYWGQGTL (SEQ ID NO: 4), or fragments, variants, or sequences thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it and / or having two or three amino acid substitutions, and / or Light chain variable structural 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 with it and / or having two or three amino acid substitutions.

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

9. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy chain variable domain encoded by a nucleotide sequence or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, said nucleotide sequence comprising: (i) Heavy chain variable region, which contains: CDR-H1, wherein CDR-H1 contains GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO: 62), CDR-H2, wherein CDR-H2 comprises ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3, the CDR-H3 includes ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC (SEQ IDNO: 64); (ii) Heavy chain variable region, which contains: CDR-H1, wherein CDR-H1 contains GGAAGCTCCGAAAGATTCACATCA (SEQ ID NO: 66), CDR-H2, wherein CDR-H2 contains ATTACTAATGGTGGTAGCACA (SEQ ID NO: 67), and CDR-H3, wherein CDR-H3 comprises ATGGCGGGTACGTCC (SEQ ID NO: 68); and (iii) Heavy chain variable region, which includes: CDR-H1, wherein CDR-H1 contains GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO: 62), CDR-H2, wherein CDR-H2 comprises ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 comprising ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC (SEQ ID NO: 70).

10. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy-chain variable domain encoded by a nucleotide sequence comprising: (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCAGGGACCCAG (SEQ ID NO: 72) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions; or (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO: 45) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions; or (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGACCCAG (SEQ ID NO: 69) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (vii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGACCCAG (SEQ ID NO: 71) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, and / or The light chain variable domain is encoded by a nucleotide sequence comprising: (xvi)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (xvii)GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (xviii)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (xix)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (xx)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions.

11. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy chain and / or light chain variable domain encoded by a nucleotide sequence selected from: (vi) A heavy-chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, and / or A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 49) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions. (vii) A heavy-chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, and / or A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising: GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAAGTCCCTGGACGTTCGGCCAAGGGACCAAG (SEQ ID NO: 53) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions. (viii) A heavy-chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, and / or A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 55) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions. (ix) A heavy-chain variable domain encoded by a nucleotide sequence comprising: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, and / or A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCGTACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions; and (x) A heavy-chain variable domain encoded by a nucleotide sequence comprising: CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO: 45) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, and / or A light chain variable domain encoded by a nucleotide sequence, the nucleotide sequence comprising: GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTACATTATGCCCGACACTTTTGGCCAGGGGACGAAA (SEQ ID NO: 59) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has 10-20 nucleic acid substitutions.

12. The cell according to any one of the preceding claims, wherein the first antigen-binding protein, its variant, or binding fragment that binds EpCAM comprises a heavy-chain variable domain encoded by a nucleotide sequence comprising: (i) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCTG (SEQ ID NO: 74) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions; or (ii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCAGGGACCCAG (SEQ ID NO: 72) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 65) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCAGTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGTATGGCGGGTACGTCCTGGGGCCAGGGACCCAG (SEQ ID NO: 69) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto and / or has 10 - 20 nucleic acid substitutions, or (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCACTGGGGCCAGGGGACCCAG (SEQ ID NO: 73) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto and / or has 10 - 20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGTTGGGTGGGAGATTTCGCTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to and / or has 10-20 nucleic acid substitutions, or (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATG (SEQ ID NO:45) or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to it and / or has 10-20 nucleic acid substitutions.

13. The cell according to any one of the preceding claims, wherein the multispecific antigen-binding protein, its variants or fragments are bispecific antibodies.

14. The cell according to any one of the preceding claims, wherein the multispecific antigen-binding protein, its variants or fragments are immune cell connectors, said immune cell connectors being selected from T cell connectors, NK cell connectors, monocyte connectors and macrophage connectors.

15. The cell according to any one of the preceding claims, wherein the multispecific antigen-binding protein, its variants or fragments are bispecific T cell connectors (BiTEs), such as inducible BiTEs, non-inducible BiTEs or constitutively expressed BiTEs.

16. The cell according to any one of the preceding claims, wherein the second antigen-binding protein, its variants or binding fragments bind an immune marker selected from CD3, NKG2D, CD4, CD8, CD16 and CD64.

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

18. The cell according to any one of the preceding claims, wherein the cell is selected from T cells, macrophages, monocytes and NK cells.

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

20. The cell according to any one of the preceding claims, wherein the cell i. Binding to GPC3 and secreting an inducible bispecific T cell adjuvant that targets EpCAM and CD3 (GE CAR-BiTE T); or ii. Bind to CD19 and secrete an inducible bispecific T cell connector that targets EpCAM and CD3 (CD19 CAR-BiTE T).

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

22. A vector expressing the polynucleotide according to claim 21.

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

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

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

26. A method of treating a disease in a subject in need, the method comprising administering cells according to any one of claims 1 to 20 or a composition according to claim 25 to the subject, optionally, the disease being a proliferative disease, optionally cancer.

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