Secretion of GPC3 chimeric antigen receptor

Abstract

Modified cells are provided that express a multispecific antigen-binding protein, a variant or a variant thereof, that binds to one or more targets, comprising (a) a chimeric antigen receptor targeting GPC3 and / or CD19, and (b) a first antigen-binding protein, a variant or a binding fragment thereof that binds to EpCAM (epithelial cell adhesion molecule), and a second antigen-binding protein, a variant or a binding fragment thereof that binds to an immune cell marker, wherein the first antigen-binding protein, a variant or a binding fragment thereof that binds to EpCAM includes a heavy chain variable region and / or a light chain variable region containing the sequence disclosed herein. Methods for producing / generating the cells disclosed herein and therapeutic methods using the cells disclosed herein are also disclosed.

Description

[Technical Field]

[0001] This disclosure broadly relates to modified cells specific to antigen-binding proteins, chimeric antigen receptors, and epithelial cell adhesion molecules (EpCAM). In particular, this disclosure relates to cells expressing multi-specific antigen-binding proteins, variants, or binding fragments that bind to one or more targets, including a chimeric antigen receptor that targets a tumor antigen, and 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 an immune cell marker. [Background technology]

[0002] The global cancer rate is projected to increase to 28.4 million cases by 2040. Approximately 90% of all adult cancer cases are solid tumors. Solid tumors, such as liver cancer, lung cancer, stomach cancer, and breast cancer in women, are among the leading causes of cancer-related death. Malignant tumors of epithelial tissue, also known as "carcinomas" because epithelial tissue is the most abundant tissue in the body, account for 80-90% of solid tumor cases. For example, hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for about 90% of all liver cancer cases. It has a high mortality rate, with a five-year overall survival rate of only 18%. Because chemotherapy is no longer preferred for HCC, the current standard treatment for metastatic or unresectable patients with adequate liver function is a combination of atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF). Subsequent second-line treatments are not clearly defined, and there is certainly a very large unmet need in patients after Atezolizumab-Bev. Breast cancer in women is one example, with ductal and lobular carcinomas of epithelial origin 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 (~89%), pancreas (>90%), head and neck (>90%), and prostate (>95%), the majority of which are carcinomas. While "carcinoma in situ" has a high chance of cure, the 5-year survival rate for most invasive or metastatic cancers is only around 20-30%, even with the availability of multiple treatment modalities such as surgery, chemotherapy, radiation therapy, and targeted therapy.

[0003] Compared to conventional treatment strategies, antibody therapies and cell-based therapies are attracting increasing attention and investment in their development as potentially more promising standard treatments for cancer. Cell-based therapies such as chimeric antigen receptor (CAR) T have achieved great success in hematological malignancies, but have shown limited efficacy in the treatment of solid tumors. Among the complex challenges encountered by each approach, one of the essential problems lies in the heterogeneous distribution of tumor markers in solid tumor cell populations and antigen escape during treatment.

[0004] Immune escape of target antigen-negative cells emerges as a major mechanism of cancer relapse. Consequently, the insufficient efficacy of single-antigen-targeting CAR-T cells contributes to resistance to CAR T-cell therapy. Because tumor cells express a diverse repertoire of surface antigens, a potential approach to overcome this challenge is to design dual or multi-targeted CAR T cells to combat tumor escape, as exemplified by bispecific anti-CD19 / CD20 CAR T-cell therapy, which has shown early promise for treating relapsed or refractory B-cell lymphoma. Nevertheless, finding multiple tumor-specific targets in solid tumors is extremely difficult. In fact, most solid tumor-associated antigens (TAAs) are also expressed at low levels in normal tissues, and therefore, the highly potent properties of CARs can induce unacceptable toxicity outside the targeted tumor.

[0005] Therefore, there is a need to provide alternative methods to prevent cancer relapse. Cell-based alternative therapies are needed. The provision of alternative chimeric antigen receptor cells is urgently required. [Overview of the project]

[0006] Glypican 3 (GPC3) is an oncofoetal protein that can be used as a highly tumor-specific target. Glypican 3 is widely expressed during embryonic development; however, its expression is strictly suppressed in most adult tissues. Elevated GPC3 expression has been reported in various types of tumors, including those of the liver, lung, stomach, ovaries, esophagus, and many others. Immunotherapy targeting GPC3, such as CAR T-cell therapy, has been clinically proven to have some therapeutic efficacy and to be generally safe. However, only a very limited number of patients have achieved complete remission (CR). Disease metastasis and recurrence after treatment targeting these markers may be largely due to the fact that not all tumor cells express GPC3, even though it is present at high levels in these cancers at diagnosis. Reappearing tumor masses often originate from the proliferation of antigen-negative cancer cells or from cancer cells that subsequently reduced, or even lost, antigen expression after CAR T-cell therapy. While very high complete remission (CR) rates have been reported, CAR T-cell therapies using anti-CD19 targeted "Kymriah" or "Yescarta," approved by the FDA for the treatment of various B-cell-derived leukemias and lymphomas, also faced the same problem of antigen escape. To overcome this challenge, the idea of ​​dual-targeted CAR T cells has often been applied, and bispecific anti-CD19 / CD20 CAR T-cell therapy has long been considered promising for treating relapsed or refractory B-cell lymphoma. However, since most tumor markers are expressed in normal tissue, albeit at relatively low levels, finding two or more targetable antigens with high tumor specificity in solid tumors is extremely difficult. Because CAR T cells have the inherent characteristic of being highly capable of killing cancer cells, dual-CAR designs that multitarget two or more antigens are considered extremely dangerous for patients with solid tumors because they can induce unacceptable toxicity outside the tumor.

[0007] EpCAM (epidermal cell adhesion molecule) is another category of biomarkers that are widely expressed in almost all cancers. Cancer therapies targeting EpCAM have been in clinical development for over a decade. However, because EpCAM is also expressed at low levels in normal epithelium, anti-EpCAM CAR T cells have been shown to be highly toxic to normal tissue. For the same reason, both solitomab and catumaxomab (anti-EpCAM BiTE) showed dose-limiting toxicity and did not receive FDA approval.

[0008] Interestingly, as a broad-spectrum marker for epithelial cells, EpCAM has been found to co-express with GPC3 in many cancers. Instead of using a dual-CAR configuration, this disclosure describes the development of a therapeutic approach using GPC3-targeted CAR T cells that secrete an anti-EpCAM bispecific T cell engager (BiTE). This ensures that the presence of anti-EpCAM BiTE is confined to the tumor site, maximizing its contribution to tumor eradication and prevention of tumor escape while avoiding systemic toxicity. These CAR T cells were named "GE CAR-BiTE T" (GPC3-targeted CAR T cells with anti-EpCAM BiTE secretion).

[0009] CAR-BiTE T cells that secrete anti-EpCAM BiTE first infiltrate and retain the tumor site via CAR recognition of specific tumor antigens expressed on tumor cells. Upon association with the CAR target, the CAR T cells will be activated and will begin to proliferate while 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. This approach causes bystander T cells to be physically directed and activated in close proximity to the tumor, and subsequently assist in the clearance of tumor cells. Since GPC3 overexpression is tumor-specific, the proliferation of anti-EpCAM BiTE is likely limited to the tumor site or its vicinity. In addition, because the secretion of recombinant anti-EpCAM BiTE antibodies by anti-GPC3 CAR T cells is not very efficient, further diffusion into normal tissue, along with the need for just-in-time T cell presence, will likely reduce its extra-tissue toxicity to its target. Furthermore, since EpCAM is known to be expressed in cancer progenitor cells and cancer stem cells, it is also defined as a cancer stem cell marker. The secretion of anti-EpCAM BiTE by CAR T cells will, by eliminating cancer stem cells and progenitor cells, incidentally contribute to the prevention of cancer relapse and recurrence.

[0010] As shown by the experimental data of the present disclosure, GE CAR-BiTE cells equipped with the novel BiTE (Nb01-013A or Nb01-013B) showed excellent properties of killing HepG2 cells and HT-29 cells with the same ability as MT110 BiTE. T cells secreting MT110 also induced strong cytotoxicity against HeyA8 cells, but GE CAR-BiTE T cells using the new BiTE missed these target-negative cells (Figures 5C, 5D, and 5E). Furthermore, anti-EpCAM BiTE can be injected into the tumor site for targeted therapy. The anti-EpCAM BiTE of the present disclosure, which is secreted only locally (not systemically administered) to the tumor site by CAR T cells, limits its potential toxicity. This transforms undruggable (or difficult) targets into druggable targets.

[0011] In one aspect, (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 that binds to an immune cell marker Modified cells expressing are provided, The first antigen-binding protein, variant or binding fragment thereof that binds to EpCAM is: (i) ·GSIFSGND (SEQ ID NO: 25 - 2C4, hu2C4, 1A5, 2B7, and 2D10), or ·GSSERFTS (SEQ ID NO: 29 - 1B8) including CDR-H1, ·ITSGGST (SEQ ID NO: 26 - 2C4, hu2C4, 1A5, 2B7, and 2D10), or ·ITNGGST (SEQ ID NO: 30 - 1B8) including CDR-H2, and ·TNGRWSGDTYYAHH (SEQ ID NO: 27 - 2C4, hu2C4, 1A5, 2D10), ·MAGTS (Sequence ID 31-1B8), or ·TNGRWSGDTYYAHL (Sequence ID 33-2B7) Including CDR-H3, Heavy chain variable regions including: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10) (ii) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6, 1C1, 1C11, 1D4 and 1H6) (iii) CDR-H1 (SEQ ID NO: 5) containing GDSISSNSVA, CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4) (iv) QSLLHSNGYNY (Sequence IDs 9-1B6, 1C1, 1C11 and 1H6), · QSLLHSNRYNY (Sequence ID 17-1D4), or • QSISDF (Sequence ID 19-1E4) Including CDR-L1, · LGS (Sequence IDs 10-1B6, 1C1, 1C11, 1D4 and 1H6), or • AAS (Sequence ID 20-1E4) Including CDR-L2, and • MQALQTPYT (Sequence IDs 11-1B6, 1C1 and 1D4) • MQGLQSPWT (Sequence ID 15-1C11) · QQSYIMPDT (Sequence ID 21-1E4), or • MQGLQTPYT(Sequence ID 23-1H6) CDR-L3 Light chain variable regions including: (1B6, 1C1, 1C11, 1D4, 1E4 and 1H6); and Or a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. It includes a heavy chain variable region and / or a light chain variable region selected from the group consisting of the following.

[0012] In some embodiments, a first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM includes a heavy chain variable region, the heavy chain variable region being: (1A5, 1B8, 2B7, 2C4, 2D10, and hu2C4) · GSIFSGND (sequence numbers 25-1A5, 2B7, 2C4, 2D10 and hu2C4), or ·GSSERFTS (Sequence ID 29-1B8) CDR-H1, including · ITSGGST (Sequence IDs 26-1A5, 2B7, 2C4, 2D10 and hu2C4), or • ITNGGST (Sequence ID: 30-1B8) CDR-H2 including; and ·TNGRWSGDTYYAHH (Sequence IDs 27-1A5, 2C4, 2D10 and hu2C4), ·MAGTS (Sequence ID 31-1B8), or ·TNGRWSGDTYYAHL (Sequence ID 33-2B7) CDR-H3, including Or a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Includes.

[0013] In some embodiments, a first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM includes a heavy chain variable region, the heavy chain variable region being: (i) CDR-H1 containing GSIFSGND (sequence number 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID 27). Heavy chain variable regions including: (2C4-VHH, hu2C4-VHH, 1A5-VHH, and 2D10-VHH) (ii) CDR-H1 containing GSSERFTS (SEQ ID NO: 29), CDR-H2 containing ITNGGST (SEQ ID NO: 30), and CDR-H3 containing MAGTS (Sequence ID 31) Heavy chain variable region including: (1B8-VHH); and (iii) CDR-H1 containing GSIFSGND (sequence number 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID 33) Heavy chain variable region including: (2B7-VHH) Or a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Selected from the group consisting of .

[0014] In some embodiments, a first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM includes a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and / or light chain variable region being: (i) CDR-H1 containing GSIFSGND (sequence number 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (Sequence ID 27) Heavy chain variable regions including: (2C4, hu2C4, 1A5, and 2D10) (ii) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6, 1C1, 1C11, 1D4, 1H6) (iii) CDR-H1 containing GDSISSNSVA (Sequence ID 5), CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4) (iv) CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable regions including: (1B6 and 1C1) (v) CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) Light chain variable region including: (1C11) (vi) CDR-L1 containing QSLLHSNRYNY (Sequence ID 17) CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable region including: (1D4) (vii) CDR-L1 containing QSISDF (Sequence ID 19) CDR-L2 containing AAS (Sequence ID 20), and CDR-L3 containing QQSYIMPDT (SEQ ID NO: 21) Light chain variable region including: (1E4) (viii) CDR-L1 containing QSLLHSNGYNY (Sequence ID 9) CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) Light chain variable region including: (1H6) (ix) CDR-H1 containing GSSERFTS (SEQ ID NO: 29), CDR-H2 containing ITNGGST (SEQ ID NO: 30), and CDR-H3 containing MAGTS (Sequence ID 31) Heavy chain variable region including: (1B8-VHH); and (x) CDR-H1 containing GSIFSGND (sequence number 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHL (Sequence ID 33) Heavy chain variable region including: (2B7-VHH) Or a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Selected from the group consisting of .

[0015] In some embodiments, a first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a heavy chain variable domain and / or a light chain variable domain, wherein the heavy chain variable domain and / or light chain variable domain are: (i)QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ(Sequence ID 35-2C4-VHH) Heavy chain variable domains including (ii) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH WGQGTL(Sequence ID 37-hu2C4-VHH) Heavy chain variable domains including (iii)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence IDs 4-1B6, 1C1, 1C11, 1D4 and 1H6) Heavy chain variable domains including (iv)QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQGTM (Sequence ID 8-1E4) Heavy chain variable domains including (v)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK (Sequence IDs 12-1B6 and 1C1) Light chain variable domains including (vi)EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT FGQGTK(Sequence ID 16-1C11) Light chain variable domains including (vii)DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK(Sequence ID 18-1D4) Light chain variable domains including (viii)DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT FGQGTK(Sequence ID 22-1E4) Light chain variable domains including (ix)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT FGQGTK (Sequence ID 24-1H6) Light chain variable domains including (x)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ(Sequence ID 28-1A5-VHH) Heavy chain variable domains including (xi)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ(Sequence ID 32-1B8-VHH) Heavy chain variable domains including (xii)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL WGQGTQ(Sequence ID 34-2B7-VHH) Heavy chain variable domains including (xiii)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ(Sequence ID 36-2D10-VHH) Heavy chain variable domains including; and Alternatively, a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. Selected from the group consisting of .

[0016] In some embodiments, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID 35-Clone 2C4-VHH), or (ii) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH WGQGTL (Sequence ID 37-hu2C4-VHH), or (iii)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID 28 - Clone 1A5-VHH), or (iv)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ (Sequence ID 32-clone 1B8-VHH), or (v)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL WGQGTQ (Sequence ID 34-Clone 2B7-VHH), or (vi)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID 36-Clone 2D10-VHH), Alternatively, 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. It includes a single-domain heavy chain variable domain having the following sequence.

[0017] In some embodiments, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6, 1C1, 1C11, 1D4 and 1H6) (ii) CDR-H1 containing GDSISSNSVA (SEQ ID NO: 5) CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4) It includes a heavy chain variable region selected from the group consisting of; Furthermore • QSLLHSNGYNY (Sequence IDs 9-1B6, 1C1, 1C11, and 1H6) · QSLLHSNRYNY (Sequence ID 17-1D4), or • QSISDF (Sequence ID: 19-1E4) CDR-L1 · LGS (Sequence IDs 10-1B6, 1C1, 1C11, 1D4 and 1H6), or • AAS (Sequence ID 20-1E4) Including CDR-L2, and • MQALQTPYT (Sequence IDs 11-1B6, 1C1 and 1D4) • MQGLQSPWT (Sequence ID 15-1C11) • QQSYIMPDT (Sequence ID 21-1E4), or • MQGLQTPYT(Sequence ID 23-1H6) CDR-L3 Or, a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Light chain variable region including: (1B6, 1C1, 1C11, 1D4, 1E4 and 1H6).

[0018] In some embodiments, a first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM includes a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being: (i) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6 and 1C1); and CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable region including (ii) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable region including: (1C11); and CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) Light chain variable region including (iii) CDR-H1 containing GGTFSSYA (Sequence ID 1) CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable region including: (1D4); and CDR-L1 containing QSLLHSNRYNY (Sequence ID 17), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable region including (iv) CDR-H1 containing GGTFSSYA (SEQ ID NO: 1) CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable region including: (1H6); and CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) Light chain variable region including; Furthermore (v) CDR-H1 containing GDSISSNSVA (SEQ ID NO: 5), CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4); and CDR-L1 containing QSISDF (SEQ ID NO: 19), CDR-L2 containing AAS (Sequence ID 20), and CDR-L3 containing QQSYIMPDT (SEQ ID NO: 21) Light chain variable region including: (1E4) Or a fragment, variation, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. It includes heavy chain variable regions and light chain variable regions selected from the group consisting of the following.

[0019] In some embodiments, a first antigen-binding protein, a variant thereof, or a binding fragment that binds to EpCAM comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain and the light chain variable domain are: (i)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1B6 and 1C1), and DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK(SEQ ID NO: 12) Light chain variable domains including (ii)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1C11), and EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT FGQGTK(SEQ ID NO: 16) Light chain variable domains including (iii)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1D4), and DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK(Sequence ID 18) Light chain variable domains including (iv)QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGRT YYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQGTM (Sequence ID 8) Heavy chain variable domains including: (1E4), and DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT FGQGTK(SEQ ID NO: 22) Light chain variable domains including, or (v)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1H6), and DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT FGQGTK(SEQ ID NO: 24) Light chain variable domain, or Or, a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical and / or has two or three amino acid substitutions. Selected from the group consisting of .

[0020] In some embodiments, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID 72-Clone 2C4), or (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCA CTGGGGCCAGGGGACCCTG(Sequence ID 74-clone hu2C4-VHH); or (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGGAGATTCGCTAC TGGGGCCAGGGAACCCTG (in the case of SEQ ID NO: 41 - 1B6, 1C1, 1C11, 1D4, 1H6 clones), or (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (SEQ ID NO: 45 - clone 1E4); or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 65 - Clone 1A5), or (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), or (vii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), or (viii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID 73 - Clone 2D10) It includes a heavy chain variable domain encoded by a nucleotide sequence containing and / or The light chain variable domain is: (i)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 49 - clones 1B6 and 1C1), or (ii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID 53-Clone 1C11), or (iii)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 55-Clone 1D4), or (iv)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence ID 59-Clone 1E4), or (v)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 61 - Clone 1H6), Alternatively, a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it, and / or a sequence having 10 to 20 nucleic acid substitutions. It is encoded by a nucleotide sequence containing [the specified character].

[0021] In some embodiments, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)GAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID 41) Heavy chain variable domains encoded by nucleotide sequences including: (1B6 and 1C1), and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence No. 49) Light chain variable domain encoded by a nucleotide sequence containing (ii)GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID 41) Heavy chain variable domain encoded by a nucleotide sequence containing: (1C11), and / or GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID 53) Light chain variable domain encoded by a nucleotide sequence containing (iii)GAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence No. 41) Heavy chain variable domain encoded by a nucleotide sequence containing: (1D4), and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 55) Light chain variable domain encoded by a nucleotide sequence containing (iv) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID 41) A heavy chain variable domain encoded by a nucleotide sequence containing: (1H6), and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 61) Light chain variable domains encoded by nucleotide sequences including; and (v) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (Sequence No. 45) Heavy chain variable domain encoded by a nucleotide sequence containing: (1E4), and / or GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence No. 59) Light chain variable domain encoded by a nucleotide sequence containing Alternatively, a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it, and / or a sequence having 10 to 20 nucleic acid substitutions. It includes heavy chain and / or light chain variable domains encoded by a nucleotide sequence selected from the group consisting of the following:

[0022] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 72), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (SEQ ID NO: 74), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions; or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 65), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (iv)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (Sequence ID 69), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (Sequence ID 71), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (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 having 10 to 20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGGAGATTCGCTAC TGGGGCCAGGGAACCCTG (SEQ ID NO: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC Sequence TGGGGCCAAGGGACAATG (SEQ ID NO: 45), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. It contains a heavy chain variable domain encoded by a nucleotide sequence that includes [the specified nucleotide].

[0023] In some embodiments, the multispecific antigen-binding protein, its variants, or fragments are bispecific antibodies.

[0024] In some embodiments, the multispecific antigen-binding protein, its variants, or fragments are immune cell engagers selected from the group including T cell engagers, NK cell engagers, monocyte engagers, and macrophage engagers.

[0025] In some embodiments, the highly specific antigen-binding protein, its variants, or fragments are bispecific T cell engagers (BiTEs), such as inducible BiTEs, non-inducible BiTEs, or constitutively expressed BiTEs.

[0026] In some embodiments, a second antigen-binding protein of an immune cell engager, its variant or binding fragment, binds to an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0027] In some embodiments, the polyspecific antigen-binding protein is an inducible bispecific T cell engager containing a heavy-chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv).

[0028] In some embodiments, the cells are selected from a group consisting of, for example, T cells, macrophages, monocytes, and NK cells.

[0029] In some embodiments, the cells are T cells, optionally CAR T cells.

[0030] In some embodiments, the cells are i. It secretes inducible bispecific T cell engagers that bind to GPC3 and target EpCAM and CD3 (GE CAR-BiTE T); or ii. It binds to CD19 and secretes inducible bispecific T cell engagers that target EpCAM and CD3 (CD19 CAR-BiTE T).

[0031] In another embodiment, polynucleotides encoding cells are provided as described herein.

[0032] In yet another embodiment, a vector expressing polynucleotides as described herein is provided.

[0033] In yet another embodiment, a host cell containing a vector as described herein is provided.

[0034] In yet another embodiment, a method for producing / generating cells as described herein is provided, comprising introducing polynucleotides into cells as described herein.

[0035] In yet another embodiment, a composition comprising cells as described herein is provided.

[0036] In yet another embodiment, a method is provided for treating a disease in a subject requiring it, the method comprising administering a cell or composition described herein to a subject, optionally, the disease being a proliferative disorder, optionally, cancer.

[0037] definition In this specification, the term "antigen-binding protein" is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies, as long as they exhibit the desired antigen-binding activity.

[0038] As used herein, the term “antibody” refers to a whole (i.e., full-length) antibody (i.e., comprising two heavy chains and two light chain elements) and its functionally active fragment (i.e., a molecule containing an antigen-binding domain that specifically binds to an antigen, also called an antibody fragment or antigen-binding fragment). The characteristics described herein with respect to antibodies also apply to antibody fragments unless otherwise indicated in the context. The term “antibody” encompasses monovalent antibodies, i.e., antibodies containing only one antigen-binding domain (e.g., a one-armed antibody containing interconnected full-length heavy chains and full-length light chains, also called a “half-antibody”), and multivalent antibodies, i.e., antibodies containing more than one antigen-binding domain, e.g., bivalent antibodies.

[0039] As used herein, the term “antigen-binding fragment” refers to a functionally active antibody-binding fragment, including but not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies, scFv, Fv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, triabody, tetrabody, and any of the epitope-binding fragments described above.

[0040] As used herein, "binding fragment" refers to a fragment that can bind to a target peptide or antigen with sufficient affinity to characterize the fragment as being specific to the peptide or antigen.

[0041] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous population of antibodies; that is, each individual in a monoclonal antibody preparation is identical except for possible variations (e.g., spontaneous variations) that may be present in trace amounts. Certain differences in protein sequence related to post-translational modifications (e.g., cleavage of the heavy chain C-terminal lysine, deamide of asparagine residues, and / or isomerization of aspartic acid residues) may still exist among the various different antibody molecules present in the composition. In contrast to polyclonal antibody preparations, each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0042] As used herein, the term "diabody" refers to two Fv pairs, a first VH / VL pair, and a further VH / VL pair having two Fv linkers such that the VH of the first Fv is connected to the VL of the second Fv, and the VL of the first Fv is connected to the VH of the second Fv.

[0043] As used herein, the term “tribody” (also called Fab(scFv)2) refers to a Fab fragment having a first scFv attached to the C-terminus of the light chain and a second scFv attached to the C-terminus of the heavy chain. As used herein, the term “tetrabody” refers to a form similar to a diabody, containing four Fvs and four inter-Fv linkers.

[0044] The term "multivalent antibody" refers to an antibody that contains more than one antigen-binding domain, such as a bivalent antibody.

[0045] The term "Fv" refers to a pair of variable domains in a full-length antibody, such as a cognate pair or affinity maturation variable domain, i.e., a VH and VL pair. The term "scFv" refers to a single-chain variable fragment, which is a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin, linked by a short linker peptide of 10 to approximately 25 amino acids. As used herein, the term "bis-scFv" refers to a bispecific scFv.

[0046] As used herein, the terms “dsscFv” or “disulfide-stabilized single-chain variable fragment” refer to a single-chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains and further contains interdomain disulfide bonds between the VH and VL domains.

[0047] The term "DVD-Ig" (also known as dual V-domain IgG) refers to a full-length antibody that has four additional variable domains, one at the N-terminus of each heavy chain and one at the N-terminus of each light chain.

[0048] As used herein, the term “Fab” refers to an antibody fragment comprising a light chain fragment, including the VL (variable light chain) domain and constant domain (CL) of the light chain, and the VH (variable heavy chain) domain and first constant domain (CHI) of the heavy chain. Dimerization of Fab' according to this disclosure creates F(ab')2, for example, where dimerization may be via a hinge. The term “F(ab')” refers to a monovalent fragment of a single light chain homodimer obtained by pepsin digestion of IgG, followed by reduction of the light chain disulfide bond. As used herein, the term “F(ab')2” refers to a fragment of IgG prepared by pepsin digestion of IgG. Since the F(ab')2 fragment is a disulfide-linked homodimer of two light chain dimers, it retains divalent epitope bonds like the whole IgG, but is smaller in size compared to the whole IgG because it lacks a heavy chain. The F(ab')2 and F(ab') fragments do not bind to immunoglobulin receptors on cells and may be useful in achieving specific staining of the primary antibody target.

[0049] As used herein, the terms “constant domain” or “constant region” are used interchangeably to refer to the antibody domain located outside the variable region. The constant domain is identical in all antibodies of the same isotype, but differs from isotype to isotype. Typically, the constant region of a heavy chain is formed from the N-terminus to the C-terminus, containing three or four constant domains, consisting of CH1-hinge-CH2-CH3-optionally CH4.

[0050] As used herein, the term “DiFab” refers to two Fab molecules linked via the C-terminus of a heavy chain, or two Fab' molecules linked via one or more disulfide bonds in their hinge region.

[0051] The term “antigen-binding variant” refers to a polypeptide, for example, an antibody comprising VH and / or VL having the desired features described herein and having at least about 80% amino acid sequence identity with the VH and / or VL of a reference antibody. Such antibody variants include, for example, antibodies in which one or more amino acid residues are added to or deleted from the VH and / or VL domains. Typically, antibody variants will have at least about 80% amino acid sequence identity with the antibodies described herein, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. Optionally, a variant antibody will not have more than one conserved amino acid substitution compared to the antibody sequence provided herein, or will not have more than about 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions compared to the antibody sequence provided herein.

[0052] The term “immune cells” refers to a type of specialized cell that plays a vital role in the body’s defense against infection and foreign substances. They are part of the immune system and are responsible for identifying and eliminating harmful pathogens such as bacteria, viruses, and parasites, as well as abnormal or cancerous cells. As used herein, “immune cells” refers to all cells of the immune system, including but not limited to T cells, helper T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), granulocytes (basophils, eosinophils, neutrophils, etc.), mast cells, monocytes, and macrophages.

[0053] In the context of antibodies, the term “specifically” as used herein is intended to refer to an antibody that recognizes only a specific antigen, or an antibody that has a significantly higher binding affinity to a specific antigen compared to binding to a nonspecific antigen, for example, at least 5, 6, 7, 8, 9, or 10 times higher.

[0054] In the context of antibodies, the term “epitope” or “binding site” refers to the site (or portion) on an antigen to which an antibody paratope binds or recognizes. Epitopes can be formed from both consecutive amino acids (often called “linear epitopes”) or discontinuous amino acids formed by the tertiary folding of proteins (often called “concrete epitopes”). Epitopes formed from consecutive amino acids are typically retained even when exposed to denaturing solvents, while epitopes formed by folding are typically lost when treated with denaturing solvents. Epitopes have a unique spatial structure and typically contain at least three, more commonly, at least five to ten, amino acids. Epitopes usually consist of chemically active molecular surface groups such as amino acids or sugar side chains and typically have a specific three-dimensional structure and charge properties.

[0055] An antibody "class" refers to a type of constant domain or constant region held by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0056] The term "chimeric antibody" (or its antigen-binding fragment) refers to an antibody molecule (or its antigen-binding fragment) in which (a) the constant region (variable region) is modified, substituted, or exchanged so that the constant region or part thereof is linked to a different or modified class, effector function, and / or species of constant region, or to an entirely different molecule that gives the chimeric antibody new properties, such as an enzyme, toxin, hormone, growth factor, or drug; or (b) the variable region or part thereof is modified, substituted, or exchanged with a variable region having a different or modified antigen specificity. For example, a mouse antibody can be modified by substituting its constant region with the constant region of human immunoglobulin. By substituting with a human constant region, the chimeric antibody can be made less antigenic in humans compared to the original mouse antibody while retaining its specificity for recognizing the antigen.

[0057] The term "chimeric antigen receptor" refers to a receptor protein designed to give T cells a new ability to target specific antigens. The receptor is a chimeric entity, combining both antigen-binding and T-cell activation functions into a single receptor. CAR T-cell therapy uses T cells designed to possess CARs to treat cancer. In CAR T-cell immunotherapy, T cells are modified to recognize cancer cells in order to more effectively target and destroy them. CAR T cells can be obtained either from the patient's own T cells in their blood (autologous) or from T cells of another healthy donor (allogeneic). Once isolated from a human, these T cells are genetically engineered to express specific CARs and programmed to target antigens present on the tumor surface. For safety, CAR T cells are engineered to be specific to antigens expressed in tumors but not on healthy cells. CAR T cells destroy cells by increasing their toxicity to other living cells (cytotoxicity) through widespread cell proliferation, and by causing increased secretion of factors that can affect other cells, such as cytokines, interleukins, and growth factors. Two types of co-receptors, CD4 and CD8, can be present on the surface of CAR T cells, each having different and interacting cytotoxic effects.

[0058] As used herein, the terms “human antibody” or “humanized antibody” (or its antigen-binding fragment) are intended to include antibodies (or their antigen-binding fragments) having a variable region in which both the framework region and the CDR region are derived from human sequences. Based on the amino acid sequence of the constant region of its heavy chain, antibodies or immunoglobulins are classified into the following classes: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (subtypes), e.g., IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Therefore, when an antibody molecule is intended for therapeutic use and antibody effector function is required, the human IgG constant region domain, particularly of the IgG1 and IgG3 isotypes, may be used. Alternatively, when an antibody molecule is intended for therapeutic purposes and antibody effector function is not required, the IgG2 and IgG4 isotypes may be used. Furthermore, if an antibody contains a constant region, the constant region is also derived from such a human sequence. Humanized antibodies (or their antigen-binding fragments) retain the reactivity of non-human antibodies while exhibiting lower immunogenicity in humans. This can be achieved, for example, by retaining the non-human CDR region and substituting the rest of the antibody with the human-corresponding portion (i.e., the constant region as well as the framework portion of the variable region). Modifications to additional framework regions can be made within the human framework sequence as well as within the CDR sequence derived from the germline of another mammalian species. Humanized antibodies of this disclosure may contain amino acid residues not encoded by the human sequence (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro, or somatic mutations in vivo, or conservative substitutions to enhance stability or production). This definition of humanized antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries, transgenic animals modified to produce such antibodies in response to antigen challenge but with inactivated endogenous loci, such as administering antigens to immunized Xeno mice via human B-cell hybridoma technology.

[0059] 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 a transfectoma, from host cells transformed to express a humanized antibody, and antibodies prepared, expressed, created or isolated by any other means, including splicing all or part of a human immunoglobulin gene sequence with another DNA sequence.

[0060] Throughout this specification, the term “isolated” means that an antibody or polynucleotide is present in a physical environment different from what it would otherwise naturally be. The term “isolated” nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or that has been synthetically created. Isolated nucleic acids may include, for example, synthetic DNA, cDNA, genomic DNA, or combinations thereof produced by chemical processes. An isolated antibody refers to an antibody that is substantially free of other cellular material and / or chemical substances.

[0061] The term "complementarity-determining region" ("CDR") refers to an amino acid sequence with boundaries determined using one of several well-known schemes, including those described by Kabat (i.e., the "Kabat" numbering scheme); Al-Lazikani ("Chothia" numbering scheme); and ImMunoGenTics (IMGT) numbering ("IMGT" numbering scheme). The term "complementarity-determining region" ("CDR") refers to a hypervariable region containing a binding domain that interacts with an antigen. Antibodies typically contain six CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0062] As used herein, the term “sequence identity” refers to the percentage of sequence identity determined by an antibody sequence that has been maximally aligned according to the Kabat numbering convention. When, after alignment, the antibody region of interest (e.g., the entire maturation variable region of the heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the antibody region of interest and the reference antibody region is obtained by multiplying the number of positions occupied by the same amino acids in both the antibody region of interest and the reference antibody region by the total number of aligned positions in the two regions (excluding gaps), and then multiplying by 100 to convert it to a percentage. In some examples, antigen-binding proteins contain sequences that are at least 60% identical to any one of the sequences disclosed herein.For example, the antigen-binding protein is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least Approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical (for example, approximately 60%, or approximately 61%, or approximately 62%, or approximately 63%, or any one of the sequences disclosed herein). or approximately 64%, or approximately 65%, or approximately 66%, or approximately 67%, or approximately 68%, or approximately 69%, or approximately 70%, or approximately 71%, or approximately 72%, or approximately 73%, or approximately 74%, or approximately 75%, or approximately 76%, or approximately 77%, or approximately 78%, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or The sequences may include sequences with approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, approximately 99%, or approximately 100% sequence identity. In some examples, the antigen-binding protein includes a sequence or amino acid region that differs from the sequences disclosed herein by approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 or more amino acids or nucleic acid bases, or is encoded by a nucleotide region. In some examples, the antigen-binding protein includes an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein.In some examples, the antigen-binding protein comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, one or more amino acid mutations may be selected independently of substitutions, insertions, deletions, and truncations.

[0063] In some cases, amino acid mutations are amino acid substitutions, which may include conserved and / or non-conserved substitutions.

[0064] "Conservative substitutions" can be made, for example, based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the amino acid residues involved. The 20 naturally occurring amino acids can be classified into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, lIe; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0065] As used herein, “conservative substitution” is defined as the exchange of an amino acid by another amino acid listed within the same group as the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the thus modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0066] As used herein, “non-conservative substitution” is defined as the exchange of an amino acid by another amino acid listed in the six standard amino acid groups (1) through (6) shown above.

[0067] In some examples, substitutions may include non-classical amino acids. Examples of non-classical amino acids include selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylamino acids, α-methylamino acids, N This includes, but is not limited to, α-methyl amino acids and amino acid analogs in general.

[0068] In some cases, amino acid mutations may be present in the CDR (e.g., CDR1, CDR2, or CDR3 regions) of antigen-binding proteins. In other cases, amino acid changes may be present in the framework regions (FRs) of antigen-binding proteins (e.g., FR1, FR2, FR3, or FR4 regions).

[0069] In some cases, the mutation does not substantially reduce the ability of the antigen-binding protein to specifically bind to the target. In some cases, the mutation does not substantially reduce the ability of the antigen-binding protein to specifically bind to the target, and does not reduce the ability of the antigen-binding protein without functionally modifying the target (e.g., partially or completely neutralizing it).

[0070] Modification of amino acid sequences can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis.

[0071] The term "polynucleotide" refers to a linear polymer composed of many nucleotide units that make up part of a nucleic acid molecule. Polynucleotides are made up of long chains of nucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0072] The term "affinity" refers to the strength of all non-covalent interactions between an antibody and its target protein. Unless otherwise indicated, as used herein, the term "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule for its binding partner can generally be expressed as a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0073] As used herein, the "K d " term refers to the dissociation constant obtained from the ratio of K d to K a (i.e., K d / K a ) and expressed as molar concentration (M). K d and K a refer to the dissociation rate and association rate, respectively, of a specific antigen-antibody interaction. The K D value of an antibody can be determined using methods well established in the art. As used herein, the term "low affinity" refers to a K D of 100 nM or more.

[0074] As used herein, the term "moderate affinity" refers to a K D in the range of 10 nM to 100 nM.

[0075] As used herein, the term "high affinity" refers to a K D of 1 to 10 nM.

[0076] As used herein, the term "very high affinity" refers to a K D of 1 nM or less.

[0077] As used herein, the term "EC50" refers to the concentration of an antibody or antigen-binding protein / part thereof that induces a response that is 50% of the maximum response (i.e., midway between the maximum response and the baseline) in either an in vivo or in vitro assay.

[0078] As used herein, the terms “multispecific” or “multispecific antibody” refer to an antibody having at least two binding domains, i.e., two or more binding domains, for example, two or three binding domains, wherein at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. Multispecific antibodies are generally monovalent for each specificity (antigen). The multispecific antibodies described herein include monovalent and polyvalent, for example, bivalent, trivalent, and tetravalent multispecific antibodies.

[0079] As used herein, the terms “bispecific” or “bispecific antibody” refer to an antibody that has two antigen specificities, or an antibody that has the ability to bind to two target antigens / sites simultaneously.

[0080] As used herein, “bispecific T cell engagers (BiTEs)” refers to a class of artificially created bispecific monoclonal antibodies that direct the host’s immune system toward cytotoxic activity of T cells against target cells (such as cancer cells). A BiTE is a fusion protein consisting of two single-chain variable fragments (scFv) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of approximately 55 kDa. One scFv binds to an immune cell (such as a T cell via the CD3 receptor), and the other binds to a target of interest (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs form a link between the immune cell (e.g., a T cell) and the target cell (e.g., a tumor cell). This allows the immune cell (e.g., a T cell) to exert cytotoxic activity against the tumor cell. For example, if the immune cell is a T cell, the T cell exerts cytotoxic activity by producing proteins such as perforin and granzyme that enter the tumor cell and initiate cellular apoptosis.

[0081] In some cases, BiTE refers to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In some cases, BiTE may refer to a bispecific T-cell engager platform known in the art, which refers to a recombinant protein that has the ability to simultaneously bind two different antigens and attract immune cells (such as T cells).

[0082] As used herein, the term “nanobody” refers to a single-domain antibody (sdAb) having an antibody fragment consisting of a single monomeric variable antibody domain. In some examples, bispecific T-cell engagers (BiTEs) are nanobodies having only heavy chains (VHH). As used herein, “nanobody having only heavy chains” refers to a heavy-chain antibody based on a nanobody. A heavy-chain antibody is an antibody consisting of two heavy chains and lacking the two light chains typically found in antibodies.

[0083] As described herein, “vector” is any molecule or composition having the ability to deliver a nucleic acid sequence to a suitable host cell, for example, capable of synthesizing an encoded polypeptide. Typically, and preferably, a vector is a nucleic acid designed to incorporate a desired nucleic acid sequence (e.g., the nucleic acids of this disclosure) using recombinant DNA techniques known in the art. An expression vector typically comprises one or more components (if not already provided by a nucleic acid molecule): a promoter, one or more enhancer sequences, an origin of replication, a termination sequence, a complete intron sequence including donor and acceptor splice sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0084] Vectors are typically selected to be functional in the host cell in which they will be used (vectors are adapted to the host cell's mechanisms so that they can cause gene amplification and / or gene expression. Vectors described herein may be expression vectors and / or cloning vectors).

[0085] As used herein, the term “host cell” is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the progeny cells of such a cell. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but they are still included within the scope of the term “host cell” as used herein.

[0086] The terms “treating,” “treat,” and “therapy,” as well as their synonyms, refer to both therapeutic treatment and prophylactic or preventative measures aimed at preventing or delaying (mitigating) a medical condition, which includes, but is not limited to, diseases, symptoms, and disorders. A medical condition also includes the body’s response to a disease or disorder, such as inflammation. Those who need such treatment include not only those who already have a medical condition, but also those who are prone to developing a medical condition or who wish to prevent one.

[0087] As used herein, the term “subject” includes patients and non-patients. The term “patient” refers to an individual who has or is likely to have a medical condition, while “non-patient” refers to an individual who does not have or is unlikely to have a medical condition. “Non-patient” includes healthy individuals, non-disease individuals, and / or individuals who do not have a medical condition. The term “subject” includes humans and animals. Animals include, but are not limited to, mammals (e.g., non-human primates, dogs, mice, etc.). “Murine” refers to any mammal of the Muridae and / or Leporidae families, such as mice, rats, and rabbits.

[0088] As used herein, the terms “prevent” and / or “reduce the severity of symptoms” refer to a process that delays the onset of the disease, reduces the severity of symptoms, reduces and / or prevents weight loss, prevents death, inhibits exacerbation, inhibits further exacerbation, and / or improves at least one sign or symptom of the disease.

[0089] The term "and / or," for example, "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be taken as providing explicit support for both meanings or either meaning.

[0090] Furthermore, in this description, the term “substantially” is understood to include, but not be limited to, “entirely” or “completely” whenever it is used. In addition, terms such as “comprising” and “comprise” are intended to be non-restrictive descriptive language in that, whenever they are used, they broadly include elements / components described after such terms, in addition to other components not explicitly stated. For example, when “comprising” is used, a reference to “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting” and “consist” may be considered a subset of terms such as “comprising” and “comprise” in appropriate contexts. Thus, in embodiments disclosed herein using terms such as “comprising” and “comprise,” it will be understood that these embodiments provide teachings to corresponding embodiments using terms such as “consisting” and “consist.” Furthermore, whenever terms such as “approximately” or “about” are used, they typically mean a reasonable variation, for example, a variation of ±5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, or a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

[0091] Furthermore, in the description herein, certain values ​​may be disclosed within a range. Values ​​indicating the endpoints of a range are intended to describe a preferred range. Whenever a range is given, it is intended to cover and teach all possible partial ranges, as well as the individual numerical values ​​within that range. That is, the endpoints of a range should not be interpreted as inflexible limitations. For example, a statement of a range of 1% to 5% is intended to specifically disclose partial ranges such as 1% to 2%, 1% to 3%, 1% to 4%, and 2% to 3%, as well as individual values ​​within that range such as 1%, 2%, 3%, 4%, and 5%. Individual numerical values ​​within a range should be understood to include integers, fractions, and decimals. Furthermore, whenever a range is given, it is also intended that the range covers and teaches values ​​from the endpoints of the given numerical values ​​to two more decimal places or significant figures (where applicable). For example, a statement of a range of 1% to 5% is intended to specifically disclose the ranges of 1.00% to 5.00% and 1.0% to 5.0%, as well as all the intermediate values ​​within those ranges (such as 1.01%, 1.02%...4.98%, 4.99%, 5.00%, and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The above intent of specific disclosure is applicable to any depth / width range.

[0092] As used herein, "at least 95% identical" is intended to refer to an amino acid sequence that is 95% or more identical to a reference sequence over its entire length, such as 96%, 97%, 98%, or 99% identical. Software programs can be used to calculate the percentage of identity.

[0093] In addition, when describing certain embodiments, this disclosure may disclose methods and / or processes as a specific set of steps. However, unless specifically required, it will be understood that the methods or processes should not be limited to a specific set of steps disclosed. Other sets of steps may be possible. The specific order of the steps disclosed herein should not be construed as an unreasonable restriction. Unless specifically required, the methods and / or processes disclosed herein should not be limited to steps performed in the order written. The set of steps may change and still remain within the scope of this disclosure.

[0094] Furthermore, while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may be disregarded in other alternative embodiments, and it will be understood that this disclosure provides support for such disregards and these related alternative embodiments.

[0095] Description of the Embodiment Exemplary, non-limiting embodiments include cells expressing a chimeric antigen receptor targeting GPC3 (or GE3) and / or CD19, as well as a multispecific antigen-binding protein, variant, or binding fragment that binds to EpCAM and an immune cell marker. Also disclosed are designed cells expressing polynucleotides as described herein. Also disclosed are designed cells expressing (a) a chimeric antigen receptor targeting a first antigen, and (b) a multispecific antigen-binding protein that binds to EpCAM (an epithelial cell adhesion molecule), where the first target antigen in (a) is optionally a disease-related antigen. While we do not wish to be bound by any theory, designing cells so that EpCAM BiTE is secreted to a target site minimizes toxicity, reduces side effects, and / or reduces nonspecific cytotoxicity.

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

[0097] In some cases, chimeric antigen receptors target disease cells. In some cases, disease cells may include, but are not limited to, cells of proliferative disorders (tumors / cancer, inflammatory diseases, etc.). In some cases, disease cells may be cancer / tumor cells, and cancer cells may optionally be solid tumor cells.

[0098] In some cases, solid tumors may include epithelial tumors such as carcinomas. In some cases, carcinomas may include, but are not limited to, liver cancer (hepatocellular carcinoma, etc.), lung cancer (squamous cell carcinoma of the lung, etc.), stomach cancer (adenocarcinoma, etc.), breast cancer, skin cancer (melanoma, etc.), ovarian cancer (clear cell carcinoma of the ovary, etc.), kidney cancer, pancreatic cancer, head and neck cancer, prostate cancer, esophageal cancer, bladder cancer, colon cancer, and childhood cancers (hepatoblastoma, nephroblastoma, yolk sac tumor, etc.).

[0099] In some cases, tumor cells may include benign tumors, pre-malignant tumors, and malignant tumors. In some cases, tumor cells may include stem cells, progenitor cells, and so on.

[0100] In some cases, tumor cells may include human xenografts in animal models. In some cases, human xenografts may include Hep3B, HepG2, etc. In some cases, human xenografts are Hep3B xenografts and / or HepG2 xenografts in mouse models.

[0101] In some cases, tumor cells are derived from gastric adenocarcinoma cell lines (such as AGS), breast cancer cell lines (such as MDA-MB468), and hepatic adenocarcinoma cell lines (Hep3B(GPC3)). High EpCAM High ), HepG2(GPC3 High EpCAM High ), HT-29 (GPC3 lowEpCAM High ), HeyA8 (GPC3 -ve EpCAM -ve ), Huh7 (GPC3 High EpCAM High This may include, but is not limited to, tumor cell lines such as ) ).

[0102] In some cases, target antigens are epithelial markers and may include, but are not limited to, receptor tyrosine protein kinase erbB-2 (HER2), glypican 3 (GPC3), claudin 18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), delta-like canonical Notch ligand 3 (DLL3), carcinoembryonic antigen (CEA), mucin 1 (MUC1), mucin 16 (MUC16), CEA cell adhesion molecule 7 (CEACAM7), prominin-1 (CD133), cluster of differentiation antigens 147 (CD147), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), mesothelin (MSLN), mesenchymal epithelial transition factor (c-Met), folate receptor α (FRα), and others.

[0103] In some cases, the chimeric antigen receptor binds to GPC3.

[0104] While we do not wish to be bound by theory, GPC3 is a well-known carcinoembryonic protein that can be used 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 a wide variety of tumor types, including liver, lung, stomach, ovary, esophagus, and many others. At the same time, EpCAM is widely expressed in almost all carcinomas, but is also expressed at low levels in normal epithelium. Anti-EpCAM CAR T cells have been demonstrated to be highly toxic to normal tissues, and anti-EpCAM bimethoprim (such as solitomab and catumaxomab) have shown dose-limiting toxicity and have not received FDA approval.

[0105] Therefore, the inventors of this disclosure designed CAR-T cells to produce a second tumor target molecule, namely anti-EpCAM bispecific T cell engager (BiTE), via secretion. EpCAM is a well-known tumor-associated antigen that is frequently overexpressed in almost all solid tumors of epithelial origin; however, because EpCAM is also widely expressed, albeit at low levels, in normal epithelium, neither CAR T cells targeting EpCAM nor systemically administered BiTE have been successful due to their high systematic toxicity. Secretion of anti-EpCAM BiTE by CAR-T cells localizes anti-EpCAM BiTE to the tumor site or its vicinity, reducing tumor escape and significantly improving safety. Since anti-GPC3 CAR-T cells have previously demonstrated a clinically proven efficacy and safety profile, this disclosure applies GPC3-targeted CAR-T cells as carriers of anti-EpCAM BiTE. This disclosure demonstrates that GPC3-targeted CAR T cells secreting anti-EpCAM BiTE (named "GE CAR-BiTE T") exhibit superior efficacy in eradicating human xenograft tumors derived from hepatocellular carcinoma, both in vitro and in vivo.

[0106] Therefore, in one embodiment, (a) Chimeric antigen receptors targeting GPC3 and / or CD19, and (b) A multispecific antigen-binding protein, variant or binding fragment that binds to one or more targets, including a first antigen-binding protein that binds to EpCAM (epithelial cell adhesion molecule), a variant or binding fragment thereof, and a second antigen-binding protein that binds to an immune cell marker, a variant or binding fragment thereof. Cells expressing are provided. The first antigen-binding protein that binds to EpCAM, its variants, or binding fragments are: (i) GSIFSGND (sequence numbers 25-2C4, hu2C4, 1A5, 2B7, and 2D10), or ·GSSERFTS (Sequence ID 29-1B8) CDR-H1 · ITSGGST (Sequence IDs 26-2C4, hu2C4, 1A5, 2B7, and 2D10), or • ITNGGST (Sequence ID 30-1B8) Including CDR-H2, and ·TNGRWSGDTYYAHH (Sequence IDs 27-2C4, hu2C4, 1A5, 2C4, and 2D10), ·MAGTS (Sequence ID 31-1B8), or ·TNGRWSGDTYYAHL (Sequence ID 33-2B7) CDR-H3 Heavy chain variable regions including: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10) (ii) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6, 1C1, 1C11, 1D4, and 1H6) (iii) CDR-H1 containing GDSISSNSVA (Sequence ID 5), CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4) (iv) QSLLHSNGYNY (Sequence IDs 9-1B6, 1C1, 1C11 and 1H6), · QSLLHSNRYNY (Sequence ID 17-1D4), or • QSISDF (Sequence ID 19-1E4) CDR-L1 LGS (Sequence IDs 10-1B6, 1C1, 1C11, 1D4 and 1H6), or • AAS (Sequence ID 20-1E4) Including CDR-L2, and • MQALQTPYT (Sequence IDs 11-1B6, 1C1 and 1D4) · MQGLQSPWT (SEQ ID NO: 15-1C11), · QQSYIMPDT (SEQ ID NO: 21-1E4), or · MQGLQTPYT (SEQ ID NO: 23-1H6) A CDR-L3 comprising A light chain variable region comprising: (1B6, 1C1, 1C11, 1D4, 1E4 and 1H6); and A fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, A heavy chain variable region and / or a light chain variable region selected from the group consisting of.

[0107] In some examples, the first antigen-binding protein that binds to EpCAM, a variant or binding fragment thereof · GSIFSGND (SEQ ID NO: 25-2C4, hu2C4, 1A5, 2B7, and 2D10) or · GSSERFTS (SEQ ID NO: 29-1B8) A CDR-H1 comprising · ITSGGST (SEQ ID NO: 26-2C4, hu2C4, 1A5, 2B7, and 2D10), or · ITNGGST (SEQ ID NO: 30-1B8) A CDR-H2 comprising: and · TNGRWSGDTYYAHH (SEQ ID NO: 27-2C4, hu2C4, 1A5, 2D10), · MAGTS (SEQ ID NO: 31-1B8), or · TNGRWSGDTYYAHL (SEQ ID NO: 33-2B7) A CDR-H3 comprising A fragment or variant or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto A heavy chain variable region comprising: (2C4, hu2C4, 1A5, 1B8, 2B7, and 2D10).

[0108] In some examples, the first antigen-binding protein, variant or binding fragment that binds to EpCAM is: (i) CDR-H1 containing GSIFSGND (SEQ ID NO: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (SEQ ID NO: 27) Heavy chain variable region containing: (2C4-VHH, hu2C4-VHH, 1A5-VHH, and 2D10-VHH and) (ii) CDR-H1 containing GSSERFTS (SEQ ID NO: 29), CDR-H2 containing ITNGGST (SEQ ID NO: 30), and CDR-H3 containing MAGTS (SEQ ID NO: 31) Heavy chain variable region containing: (1B8-VHH); and (iii) CDR-H1 containing GSIFSGND (SEQ ID NO: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHL (SEQ ID NO: 33) Heavy chain variable region containing: (2B7-VHH) Or a fragment or variation or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto Comprising a heavy chain variable region selected from the group consisting of

[0109] In some examples, the first antigen-binding protein, variant or binding fragment that binds to EpCAM is: (i) CDR-H1 containing GSIFSGND (SEQ ID NO: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHH (SEQ ID NO: 27) Heavy chain variable region containing: (2C4, hu2C4, 1A5, and 2D10) (ii) CDR-H1 containing GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6, 1C1, 1C11, 1D4, 1H6) (iii) CDR-H1 containing GDSISSNSVA (Sequence ID 5), CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4) (iv) CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable regions including: (1B6 and 1C1) (v) CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) Light chain variable region including: (1C11) (vi) CDR-L1 containing QSLLHSNRYNY (Sequence ID 17), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable region including: (1D4) (vii) CDR-L1 containing QSISDF (sequence number 19), CDR-L2 containing AAS (Sequence ID 20), and CDR-L3 containing QQSYIMPDT (SEQ ID NO: 21) Light chain variable region including: (1E4) (viii) CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) Light chain variable region containing: (1H6) (ix) CDR-H1 containing GSSERFTS (SEQ ID NO: 29), CDR-H2 containing ITNGGST (SEQ ID NO: 30), and CDR-H3 containing MAGTS (SEQ ID NO: 31) Heavy chain variable region containing: (1B8-VHH); and (x) CDR-H1 containing GSIFSGND (SEQ ID NO: 25), CDR-H2 containing ITSGGST (SEQ ID NO: 26), and CDR-H3 containing TNGRWSGDTYYAHL (SEQ ID NO: 33) Heavy chain variable region containing: (2B7-VHH) or a fragment or variation or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto comprising a heavy chain variable region and / or a light chain variable region selected from the group consisting of

[0110] In some examples, the first antigen-binding protein that binds to EpCAM, a variant or binding fragment thereof is: (i) QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAVI TSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (SEQ ID NO: 35 - 2C4-VHH) comprising a heavy chain variable domain (ii) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH WGQGTL (SEQ ID NO: 37 - hu2C4-VHH) comprising a heavy chain variable domain (iii) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL (Sequence IDs 4-1B6, 1C1, 1C11, 1D4 and 1H6) Heavy chain variable domains including (iv)QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQGTM (Sequence ID 8-1E4), Heavy chain variable domains including (v)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK (Sequence IDs 12-1B6 and 1C1) Light chain variable domains including (vi)EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT FGQGTK(Sequence ID 16-1C11) Light chain variable domains including (vii)DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK(Sequence ID 18-1D4) Light chain variable domains including (viii)DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT FGQGTK(Sequence ID 22-1E4) Light chain variable domains including (ix)DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT FGQGTK (Sequence ID 24-1H6) Light chain variable domains including (x)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ(Sequence ID 28-1A5-VHH) Heavy chain variable domains including (xi)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ(Sequence ID 32-1B8-VHH) Heavy chain variable domains including (xii)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL WGQGTQ(Sequence ID 34-2B7-VHH) Heavy chain variable domains including (xiii)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ(Sequence ID 36-2D10-VHH) Heavy chain variable domains including; and Alternatively, a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. It includes heavy chain variable domains and / or light chain variable domains selected from the group consisting of the following.

[0111] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)QVQLQESGGGLVQAGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID 35-clone 2C4-VHH), or (ii) QVQLVESGGGLVQAGGSLRLSCAAS GSIFSGND MSWYRQAPGKGLELVAV ITSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TNGRWSGDTYYAHH WGQGTL (Sequence ID 37-hu2C4-VHH), or (iii)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID 28-Clone 1A5-VHH), or (iv)QVQLQESGGGLVQPGGSLRLSCAAS GSSERFTS VAWYRQAPGKERELVAF ITNGGST RYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC MAGTS WGQGTQ (Sequence ID 32-clone 1B8-VHH), or (v)QVQLQESGGGLVQPGGSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHL WGQGTQ (Sequence ID 34-Clone 2B7-VHH), or (vi)QVQLQESGGGLVQAGDSLRLSCADS GSIFSGND MAWYRRAPGVERELVAV ITSGGST HYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYC TNGRWSGDTYYAHH WGQGTQ (Sequence ID 36-Clone 2D10-VHH), Alternatively, a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. It includes a single-domain heavy chain variable domain having the following sequence.

[0112] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6, 1C1, 1C11, 1D4, and 1H6) (ii) CDR-H1 containing GDSISSNSVA (SEQ ID NO: 5) CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4) A heavy chain variable region selected from the group consisting of; and / or • QSLLHSNGYNY (Sequence IDs 9-1B6, 1C1, 1C11 and 1H6) · QSLLHSNRYNY (Sequence ID 17-1D4), or • QSISDF (Sequence ID 19-1E4) CDR-L1 LGS (Sequence IDs 10-1B6, 1C1, 1C11, 1D4 and 1H6), or • AAS (Sequence ID 20-1E4) Including CDR-L2, and • MQALQTPYT (Sequence IDs 11-1B6, 1C1 and 1D4) • MQGLQSPWT (Sequence ID 15-1C11) • QQSYIMPDT (Sequence ID 21-1E4), or • MQGLQTPYT(Sequence ID 23-1H6) CDR-L3 Light chain variable regions including: (1B6, 1C1, 1C11, 1D4, 1E4, and 1H6) Or, a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. Includes.

[0113] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable regions including: (1B6 and 1C1); and / or CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable region including (ii) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable region including: (1C11); and / or CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQSPWT (SEQ ID NO: 15) Light chain variable region including (iii) CDR-H1 containing GGTFSSYA (Sequence ID 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable region including: (1D4); and / or CDR-L1 containing QSLLHSNRYNY (Sequence ID 17), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQALQTPYT (SEQ ID NO: 11) Light chain variable region including (iv) CDR-H1 containing GGTFSSYA (SEQ ID NO: 1), CDR-H2 containing IIPIFGTA (SEQ ID NO: 2), and CDR-H3 containing ARSLGGRFRY (Sequence ID 3) Heavy chain variable region including: (1H6); and / or CDR-L1 containing QSLLHSNGYNY (Sequence ID 9), CDR-L2 containing LGS (Sequence ID 10), and CDR-L3 containing MQGLQTPYT (SEQ ID NO: 23) Light chain variable region including; and (v) CDR-H1 containing GDSISSNSVA (Sequence ID 5), CDR-H2 containing TYYRSKWYS (SEQ ID NO: 6), and CDR-H3 containing AREVEGSSYDAFDI (Sequence ID 7) Heavy chain variable region including: (1E4); and / or CDR-L1 containing QSISDF (SEQ ID NO: 19), CDR-L2 containing AAS (Sequence ID 20), and CDR-L3 containing QQSYIMPDT (SEQ ID NO: 21) Light chain variable region including: (1E4) Or a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. It includes a heavy chain variable region and / or a light chain variable region selected from the group consisting of the following.

[0114] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1B6 and 1C1), and / or DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK(SEQ ID NO: 12) Light chain variable domains including (ii)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1C11), and / or EIVLTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQGLQSPWT FGQGTK(SEQ ID NO: 16) Light chain variable domains including (iii)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1D4), and / or DVVMTQSPLSLPVTPGESASISCRSS QSLLHSNRYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC MQALQTPYT FGQGTK(Sequence ID 18) Light chain variable domains including (iv)QVQLQQSGPGLVKPSQTLSLTCAIS GDSISSNSVA WNWIRQSPSRGLEWLGR TYYRSKWYS DYAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYC AREVEGSSYDAFDI WGQGTM (Sequence ID 8) Heavy chain variable domains including: (1E4), and / or DIQLTQSPSSLSASVGDRVTITCRAS QSISDF LNWYQQKPGKAPKLLIY AAS SLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYC QQSYIMPDT FGQGTK(SEQ ID NO: 22) Light chain variable domains including, or (v)EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYC ARSLGGRFRY WGQGTL(Sequence ID 4) Heavy chain variable domains including: (1H6), and / or Light chain variable domain DVVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYC MQGLQTPYT FGQGTK (SEQ ID NO: 24), or Or fragments, variants, or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical and / or two or three amino acid substitutions. It includes heavy chain variable domains and / or light chain variable domains selected from the group consisting of the following.

[0115] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 13 - Clone 1B6 - Light Chain Constant Domain), or (ii)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFSRGEC (Sequence ID 14 clone 1C1 - light chain constant domain), Alternatively, a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. It contains a light chain constant domain having the sequence.

[0116] In one embodiment, the antigen-binding protein, its variant, or fragment is: (i) CDR-H1 containing GGAGGCACCTTCAGCAGCTATGCT (Sequence ID 38), CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID 40) Heavy chain variable regions including: (1B6 and 1C1); and / or CDR-L1 containing CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO: 46), CDR-L2 containing TTGGGTTCT (SEQ ID NO: 47), and CDR-L3 containing ATGCAAGCTCTACAAACTCCGTACACT (SEQ ID NO: 48) Light chain variable region including: (ii) CDR-H1 containing GGAGGCACCTTCAGCAGCTATGCT (Sequence ID 38), CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID 40) Heavy chain variable region including: (1C11); and / or CDR-L1 containing CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO: 46), CDR-L2 containing TTGGGTTCT (SEQ ID NO: 47), and CDR-L3 containing ATGCAAGGTCTACAAAGTCCCTGGACG (Sequence ID 52) Light chain variable region including (iii) CDR-H1 containing GGAGGCACCTTCAGCAGCTATGCT (Sequence ID 38), CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID 40) Heavy chain variable region including: (1D4); and / or CDR-L1 containing CAGAGCCTCCTGCATAGTAATAGATACAACTAT (SEQ ID NO: 54), CDR-L2 containing TTGGGTTCT (SEQ ID NO: 47), and CDR-L3 containing TGCAAGCTCTACAAACTCCGTACACT (SEQ ID NO: 48) Light chain variable region including (iv) CDR-H1 containing GGAGGCACCTTCAGCAGCTATGCT (Sequence ID 38), CDR-H2 containing ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and CDR-H3 containing GCGAGATCGTTGGGTGGGAGATTTCGCTAC (Sequence ID 40) Heavy chain variable region including: (1H6); and / or CDR-L1 containing CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO: 46), CDR-L2 containing TTGGGTTCT (SEQ ID NO: 47), and CDR-L3 containing ATGCAAGGTCTACAGACTCCGTACACT (SEQ ID NO: 60) Light chain variable region including; and (v) CDR-H1 containing GGGGACAGTATCTCTAGTAACAGTGTTGCT (Sequence ID 42), CDR-H2 containing ACATACTACAGGTCCAAGTGGTACAGT (SEQ ID NO: 43), and CDR-H3 containing GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC (Sequence ID 44) Heavy chain variable region including: (1E4); and / or CDR-L1 containing CAGAGTATTAGCGACTTT (SEQ ID NO: 56), CDR-L2 containing GCTGCATCG (SEQ ID NO: 57), and CDR-L3 containing TTACATTATGCCCGACACT (SEQ ID NO: 58) Light chain variable region including: (1E4) Alternatively, a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. It includes a heavy chain variable region and / or a light chain variable region encoded by a nucleic acid sequence selected from the group consisting of the following.

[0117] In one embodiment, the antigen-binding protein, its variant, or fragment is: (i) CDR-H1 containing GGAAGCATCTTCAGTGGCAATGAC (Sequence ID 62), CDR-H2 containing ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC (Sequence ID 64) Heavy chain variable regions including: (2C4-VHH, 1A5-VHH, 2D10-VHH, and hu2C4-VHH) (ii) CDR-H1 containing GGAAGCTCCGAAAGATTCACATCA (Sequence ID 66), CDR-H2 containing ATTACTAATGGTGGTAGCACA (SEQ ID NO: 67), and CDR-H3 containing ATGGCGGGTACGTCC (Sequence ID 68) Heavy chain variable region including: (1B8-VHH); and (iii) CDR-H1 containing GGAAGCATCTTCAGTGGCAATGAC (Sequence ID 62), CDR-H2 containing ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC (Sequence ID 70) Heavy chain variable region including: (2B7-VHH) Or a fragment, variation, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. It includes a heavy chain variable region encoded by a nucleic acid sequence selected from the group consisting of the following:

[0118] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 72 - Clone 2C4), or (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (SEQ ID NO: 74 - Clone hu2C4 - VHH); or (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (for sequence numbers 41-1B6, 1C1, 1C11, 1D4, 1H6 clones), or (iv)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG(Sequence ID 45-Clone 1E4); or (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID 65-Clone 1A5), or (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (Sequence ID 69-Clone 1B8), or (vii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (Sequence ID 71-Clone 2B7), or (viii)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID 73 - Clone 2D10) It includes a heavy chain variable domain encoded by a nucleotide sequence containing and / or The light chain variable domain is: (vi)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 49 - clones 1B6 and 1C1), or (vii)GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID 53-Clone 1C11), or (viii)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 55-Clone 1D4), or (ix)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence ID 59-Clone 1E4), or (x)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 61 - Clone 1H6), Alternatively, a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it, and / or a sequence having 10 to 20 nucleic acid substitutions. It is encoded by a nucleotide sequence containing [the specified character].

[0119] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)GAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (for sequence numbers 41-1B6, 1C1, 1C11, 1D4, 1H6 clones), or (ii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG(Sequence ID 45-Clone 1E4); or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID 65-Clone 1A5), or (iv)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (SEQ ID NO: 69 - Clone 1B8), or (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (SEQ ID NO: 71 - Clone 2B7), or (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 72 - Clone 2C4), or (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (Sequence ID 73-Clone 2D10), or (viii)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (Sequence ID 74 - Clone hu2C4-VHH) It includes a heavy chain variable domain encoded by a nucleotide sequence containing; and / or The light chain variable domain is: (xi)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 49 - clones 1B6 and 1C1), or (xii)GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID 53-Clone 1C11), or (xiii)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 55-Clone 1D4), or (xiv)GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence ID 59-Clone 1E4), or (xv)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 61 - Clone 1H6), Alternatively, a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it, and / or a sequence having 10 to 20 nucleic acid substitutions. It is encoded by a nucleotide sequence containing [the specified character].

[0120] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)GAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID 41) Heavy chain variable domains encoded by nucleotide sequences including: (1B6 and 1C1), and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence No. 49) Light chain variable domain encoded by a nucleotide sequence containing (ii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID 41) Heavy chain variable domain encoded by a nucleotide sequence containing: (1C11), and / or GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATAAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGGTCTACAAAGTCCCTGGACG TTCGGCCAAGGGACCAAG (Sequence ID 53) Light chain variable domain encoded by a nucleotide sequence containing (iii)GAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID 41) Heavy chain variable domain encoded by a nucleotide sequence containing: (1D4), and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATAGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 55) Light chain variable domain encoded by a nucleotide sequence containing (iv)GAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (Sequence ID 41) A heavy chain variable domain encoded by a nucleotide sequence containing: (1H6), and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGCAAATCAGCAGAGTGGAGGCTGAGGATGCTGGGGTTTATTACTGC ATGCAAGGTCTACAGACTCCGTACACT TTTGGCCAGGGGACCAAG (Sequence ID 61) Light chain variable domains encoded by nucleotide sequences including; and (v)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGT GATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC TGGGGCCAAGGGACAATG (Sequence No. 45) Heavy chain variable domain encoded by a nucleotide sequence containing: (1E4), and / or GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGTATTAGCGACTTT TTAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTAT GCTGCATCG AGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCATGGATCTGGGACAGAATTCACTCTCACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTACTGT CAACAGAGTTACATTATGCCCGACACT TTTGGCCAGGGGACGAAA (Sequence No. 59) Light chain variable domain encoded by a nucleotide sequence containing Alternatively, a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it, and / or a sequence having 10 to 20 nucleic acid substitutions. It includes heavy chain and / or light chain variable domains encoded by a nucleotide sequence selected from the group consisting of the following:

[0121] In one embodiment, an antigen-binding protein, its variant, or fragment is: CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCGCAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (Sequence ID 50 - Clone 1B6 - Light Chain Constant Domain), or CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAGCAGGGGAGAGTGT (Sequence ID 51 - Clone 1C1 - Light Chain Constant Domain), or A sequence that is at least 60% identical and / or has 10-20 nucleic acid substitutions. It contains a light chain constant domain encoded by a nucleotide sequence that includes [a specific nucleotide sequence].

[0122] In some examples, the first antigen-binding protein that binds to EpCAM, its variant or binding fragment, is: (i)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 72), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCATCTTCAGTGGCAATGAC ATGTCCTGGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCaAGAAcACCcTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCTG (SEQ ID NO: 74), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions; or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACTTGAAACCTGAGGACACGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (SEQ ID NO: 65), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (iv)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCT GGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTT ATTACTAATGGTGGTAGCACA AGATATACAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAAGCTGAGGACACGGCCGTCTATTATTGT ATGGCGGGTACGTCC TGGGGCCAGGGGACCCAG (Sequence ID 69), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (v)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCTC TGGGGCCAGGGGACCCAG (Sequence ID 71), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (vi)CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCT GGAAGCATCTTCAGTGGCAATGAC ATGGCCTGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTT ATTACTAGCGGTGGTAGTACA CACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCCAGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACACGGCCGTGTATTACTGC ACAAACGGAAGATGGTCAGGCGATACTTACTATGCCCATCAC TGGGGCCAGGGGACCCAG (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 a sequence having 10 to 20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT GGAGGCACCTTCAGCAGCTATGCT ATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGG ATCATCCCTATCTTTGGTACAGCA AACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT GCGAGATCGTTGGGTGGGAGATTTCGCTAC TGGGGCCAGGGAACCCTG (SEQ ID NO: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, (vii)CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC GGGGACAGTATCTCTAGTAACAGTGTTGCT TGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGG ACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGT GCAAGAGAAGTTGAGGGCAGCAGCTATGATGCTTTTGATATC Sequence TGGGGCCAAGGGACAATG (SEQ ID NO: 45), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. It contains a heavy chain variable domain encoded by a nucleotide sequence that includes [the specified nucleotide].

[0123] In some examples, a multispecific antigen-binding protein may comprise an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, the antigen-binding protein comprises an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, one or more amino acid mutations may be selected independently of substitutions, insertions, deletions, and truncations. In some examples, the amino acid mutations may be amino acid substitutions and may include conserved and / or non-conservative substitutions. In some examples, the mutations do not substantially reduce the ability of the antigen-binding protein to bind specifically to the target. In some examples, the mutations do not substantially reduce the ability of the antigen-binding protein to bind specifically to the target and do not reduce the ability of the antigen-binding protein without functionally modifying (e.g., partially or completely neutralizing) the target.

[0124] In some examples, a multispecific antigen-binding protein may be a bispecific, triplicate, or quadruplicate antigen-binding protein. In some examples, a multispecific antigen-binding protein is a bispecific antigen-binding protein. In some examples, a multispecific antigen-binding protein, its variant, or fragment is a bispecific antibody. In some examples, a multispecific antigen-binding protein may be provided as a nanobody. In some examples, a multispecific antigen-binding protein may be provided as an Fc region. In some examples, a multispecific antigen-binding protein is an inducible bispecific T cell engager containing a heavy-chain antibody variable region (i.e., VHH) and / or a single-chain variable fragment (scFv). In some embodiments, the bispecific T cell engager (BiTE) is a nanobody having only a heavy chain (VHH).

[0125] In some cases, multispecific antigen-binding proteins, their variants, or fragments are inducible bispecific immune cell engagers. In some cases, multispecific antigen-binding proteins are secreted by cells, optionally immune cells. In some cases, multispecific antigen-binding proteins, their variants, or fragments are bispecific T cell engagers (BiTEs).

[0126] As used herein, bispecific T cell engagers (BiTEs) refer to a class of artificially created bispecific monoclonal antibodies that direct the host immune system toward actions such as the cytotoxic activity of T cells against target cells (e.g., cancer cells). A BiTE is a fusion protein consisting of two single-chain variable fragments (scFv) on a single peptide chain of approximately 55 kDa, each being either a different antibody or the amino acid sequence of four different genes. One scFv binds to T cells via the CD3 receptor, while the other binds to the target of interest (e.g., tumor cells via tumor-specific molecules). Like other bispecific antibodies, BiTEs form a link between 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 such as perforin and granzyme that enter the tumor cells and initiate cellular apoptosis.

[0127] In some cases, BiTE may refer to the BiTE® immuno-oncology platform developed by Amgen® Oncology. In some cases, BiTE may also refer to the bispecific T-cell engager platform known in the art, which refers to a recombinant protein that has the ability to simultaneously bind to two different antigens and attract immune cells (such as T cells).

[0128] In some cases, a bispecific T cell engager (BiTE) binds to two antigens; the first antigen is EpCAM, and the second antigen is an immune cell marker. In some cases, the second antigen-binding protein, its variant, or binding fragment binds to an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16, and CD64.

[0129] In some cases, polyspecific antigen-binding proteins are inducible bispecific T cell engagers containing heavy-chain antibody variable regions (i.e., VHH) and / or single-chain variable fragments (scFv).

[0130] In some cases, multispecific antigen-binding proteins, their variants, or fragments are inducible bispecific T cell engagers. In some cases, multispecific antigen-binding proteins are secreted by cells.

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

[0132] In some cases, polyspecific antigen-binding proteins are inducible bispecific T cell engagers containing heavy-chain antibody variable regions (i.e., VHH) and / or single-chain variable fragments (scFv).

[0133] In some cases, bispecific T cell engagers (BiTEs) bind to two antigens; the first antigen is epithelial cell adhesion molecule (EpCAM); and the second antigen is CD3. In some cases, this may include CD3ζ, CD3ε, CD3γ, CD3δ, etc.

[0134] In some cases, the immune cells to be modified / designed may be, but are not limited to, macrophages, dendritic cells, T cells, B cells, eosinophils, basophils, neutrophils, mast cells, natural killer T cells (NKT cells), natural killer cells (NK cells), macrophages, monocytes, etc. In some cases, the immune cells to be modified / designed are T cells, NK cells, or macrophages. In some cases, the immune cells to be modified / designed are T cells.

[0135] In some cases, a cell may bind to more than one host cell antigen. Therefore, in some cases, a cell may bind to one, two, three, four, or more host cell antigens.

[0136] In some cases, the designed cells bind to a target antigen and secrete BiTE targeting EpCAM and CD3 (CAR-BITE T). In some cases, the cells i. bind to GPC3 and secrete an inducible bispecific T cell engager targeting EpCAM and CD3 (GE CAR-BiTE T); or ii. bind to CD19 and secrete an inducible bispecific T cell engager targeting EpCAM and CD3 (CD19 CAR-BiTE T). In some cases, the cells bind to GPC3 and secrete BiTE targeting EpCAM and CD3 (GE CAR-BiTE T). In some cases, the CAR T cells target GPC3 only (GE CAR-T).

[0137] While we don't want to be constrained by theory, CAR T cells directly kill target cells, whereas anti-EpCAM BiTE exerts cytotoxicity by recruiting nearby immune cells (such as T cells). This approach allows bystander T cells to be physically directed and activated in close proximity to the tumor, aiding in tumor cell clearance. Applying local secretion of anti-EpCAM BiTE by CAR T cells reduces the extratumor toxicity that can be brought about by systematic delivery, thus transforming a non-drug-capable target into a drug-capable target.

[0138] Since EpCAM is also defined as a cancer stem cell marker expressed in cancer progenitor cells and cancer stem cells, the secretion of anti-EpCAM BiTE by CAR T cells will likely contribute to the prevention of cancer relapse and recurrence by eliminating cancer stem cells and progenitor cells.

[0139] As described in the experimental data of this disclosure, GPC3-targeted CAR T cells secreting anti-EpCAM BiTE (GE CAR BiTE T) demonstrated superior efficacy in eradicating human xenograft tumors derived from hepatocellular carcinoma both in vitro and in vivo.

[0140] From the perspective of in vitro data, GE CAR-BiTE T cells showed a slightly faster rate of Hep3B (GPC3) than anti-GPC3 CAR alone (GE CAR T). High , EpCAM High ) Target cells were killed (Figure 2C), suggesting that BiTE plays a crucial role in promoting target cell killing. Culture supernatant recovered from GE CAR-BiTE T cells was shown to efficiently direct T cell-mediated target cell killing (Figure 2D).

[0141] From an in vivo data perspective, in Hep3B xenografts, both CAR T and GE CAR-BiTE T therapy reduced tumor growth, but only mice treated with GE CAR-BiTE achieved complete tumor regression (Figure 3C, Figure 3D) and showed a favorable safety profile without causing weight loss in the mice (Figure 3B). Figures 3F and 3G further demonstrate the superior efficacy of using GE CAR-BiTE T cells grown in culture medium containing IL-7 and IL-15 in tumor control and long-term survival of mice. In HepG2 tumor xenografts, a more aggressive mouse model, unlike the inefficiency of the CAR T monotherapy group, the tumors of all three GE CAR-BiTE T cell-treated mice were successfully cured (Figure 4D, Figure 4E), and all mice survived without significant weight loss (Figure 4F) (Figure 4G).

[0142] Polypeptides containing multispecific antigen-binding proteins are also disclosed. In some examples, the multispecific antigen-binding protein is a bispecific antibody. In some examples, the polypeptide contains multispecific antigen-binding proteins that bind to EpCAM (epithelial cell adhesion molecule) and immune cells.

[0143] In some cases, multispecific antigen-binding proteins are bispecific immune cell engagers that have the ability to attract both antigens and immune cells.

[0144] In some examples, the polypeptide contains an anti-EpCAM antigen-binding protein. In some examples, the polypeptide contains a single-domain anti-EpCAM antibody, optionally an anti-EpCAM H-chain antibody variable region (i.e., VHH).

[0145] In some cases, polypeptides contain anti-immune cell antigen-binding proteins. In some cases, polypeptides contain anti-immune cell antigen-binding proteins that bind to immune cell activation markers. In some cases, immune cell activation markers include CD3, NKG2D, CD4, CD8, CD16, and CD64. In some cases, polypeptides bind to CD3. In some cases, CD3 may include CD3ζ, CD3ε, CD3γ, and CD3δ.

[0146] In some examples, the polypeptide contains an anti-CD3 antigen-binding protein. In other examples, the polypeptide contains a single-chain variable fragment (anti-CD3 scFv) of an anti-CD3 antibody.

[0147] In some cases, immune cells are T cells, NK cells, macrophages, or monocytes. In some cases, immune cells are T cells.

[0148] In some cases, the polypeptide is a bispecific antibody / antigen-binding protein. In some cases, the bispecific antibody / antigen-binding protein is a bispecific T cell engager (BiTE), such as an inducible BiTE, a non-inducible BiTE, or a constitutively expressed BiTE. In some cases, the bispecific T cell engager (BiTE) binds to two antigens, the first being EpCAM and the second being an immune cell marker. In some cases, the second antigen is an immune cell marker involved in the activation of immune cells. In some cases, the second antigen targeted by the bispecific T cell engager (BiTE) may include, but is not limited to, CD3, NKG2D, CD28, CD16, CD64, etc. In some cases, the polypeptide contains a BiTE that bispecifically binds to EpCAM and T cells. In some cases, the polypeptide contains a BiTE that bispecifically binds to EpCAM and CD3. In some cases, the EpCAM targeted by the BiTE is modified with anti-EpCAM VHH paired with anti-CD3 scFv.

[0149] In some cases, anti-EpCAM VHH may be paired with anti-CD3 scFv which may include clone Okt3 (Nb01-013A) or anti-CD3 clone (MT110) (Nb01-013B) used by another anti-EpCAM in the art.

[0150] In another embodiment, polynucleotides encoding cell and / or polypeptides and / or polyspecific antigen-binding proteins are provided as described herein.

[0151] Also disclosed are polynucleotides comprising sequences encoding an immune cell engager and a chimeric antigen receptor (CAR), wherein the immune cell engager has the ability to bispecifically bind to EpCAM (epithelial cell adhesion molecule) and immune cells, and the CAR has the ability to bind to a first antigen.

[0152] In some examples, polynucleotides are provided that contain sequences encoding chimeric antigen receptors capable of recognizing, binding to, and attracting GPC3-positive cells. In some examples, the primary antigen is GPC3. Thus, in some examples, the CAR has the ability to bind to GPC3.

[0153] In some examples, polynucleotides further include sequences encoding one or more costimulatory domains, signal peptides, hinges, and / or signaling domains.

[0154] In some examples, the co-stimulatory domains may include, but are not limited to, 4-1BB, CD28, CD27, OX-40, etc.

[0155] In some examples, the signal peptide may be, but is not limited to, an IgH signal peptide, an IgK signal peptide, or a CD8 signal peptide.

[0156] In some examples, 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), CD28, CD8, 4-1 BB, etc.

[0157] In some cases, the sequence encoding the CAR encodes an antigen-binding protein capable of binding to GPC3 (or anti-GPC3 antigen-binding protein), or a fragment or variant thereof. In some cases, the sequence encoding the CAR encodes a single-chain variable fragment. In some cases, the sequence encoding the CAR encodes anti-GPC3 scFv.

[0158] In some cases, the sequence encoding the CAR further encodes an immune cell signaling domain. In some cases, the signaling domain may include, but is not limited to, the intracellular domains of CD3, TCFζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3ε, CD3η, CD3ζ, CD22, CD79a, CD79b, CD66d, etc. In some cases, the intracellular domain of CD3 may be one or more of CD3ζ, CD3ε, CD3γ, CD3δ, etc. In some cases, the sequence encoding the CAR further encodes the intracellular domain of CD3 (signaling domain).

[0159] In some examples, the sequence encoding the CAR encodes the anti-GPC3 scFv, the CD28 costimulatory domain, the 4-1BB costimulatory domain, the IgH signaling peptide, the IgH hinge, and the CD3 signaling domain.

[0160] In some cases, the sequence encoding the immune cell engager encodes a multispecific antigen-binding protein. In some cases, the multispecific antigen-binding protein is a bispecific antibody. In some cases, the sequence encoding the immune cell engager encodes an antigen-binding protein (anti-EpCAM antigen-binding protein), or a fragment thereof, or a variant thereof, that is capable of binding EpCAM, and an anti-immune cell antigen-binding protein.

[0161] In some examples, sequences encoding immune cell engagers encode single-chain variable fragments (scFv) or single-chain variable domains located on heavy chains (VHH).

[0162] In some cases, sequences encoding immune cell engagers encode anti-EpCAM scFv or anti-EpCAM VHH.

[0163] In some cases, sequences encoding immune cell engagers encode anti-immune cell antigen-binding proteins that bind to immune cell activation markers. In some cases, immune cell activation markers may include, but are not limited to, CD3, NKG2D, CD4, CD8, CD16, CD64, etc.

[0164] In some cases, the immune activation marker may be of the VHH or scFv type. In some cases, the sequence encoding the immune cell engager encodes an antigen-binding protein (anti-CD3 antigen-binding protein) or fragment, or a variant thereof, that has the ability to bind CD3. In some cases, the sequence encoding the immune cell engager encodes a single-chain variable fragment (scFv) or a VHH type. In some cases, the sequence encoding the immune cell engager encodes anti-CD3 scFv or anti-CD3 single-domain VHH.

[0165] In some cases, immune cell engagers may include the His tag.

[0166] In some examples, immune cell engagers include anti-EpCAM antigen-binding proteins, linkers, anti-CD3 scFv or anti-CD3 single-domain VHH, and His tags. In some examples, the linker is a cleavable linker and may include, but is not limited to, P2A, T2A, F2A, etc.

[0167] In some cases, immune cells are T cells, NK cells, macrophages, or monocytes. In some cases, immune cells are T cells.

[0168] In some examples, the polynucleotide containing the sequence encoding the immune cell engager is the sequence encoding the bispecific T cell engager (BiTE), such as inducible BiTE, non-inducible BiTE, or constitutively expressed BiTE. In some examples, the polynucleotide contains the sequence encoding the BiTE that bispecifically binds to EpCAM and T cells. In some examples, the polynucleotide contains the sequence encoding the anti-GPC3 scFv CAR with an intracellular CD3 domain and the sequence encoding the BiTE that binds to EpCAM and T cells.

[0169] Also disclosed herein are vectors expressing polynucleotides or polypeptides. In some examples, the vectors are selected from the group consisting of plasmids, viral particles, phages, baculoviruses, yeast plasmids, lipid-based vehicles, polymer microspheres, liposomes, and cell-based vehicles, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, viral vehicles, adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, foam viruses, cytomegaloviruses, Semryki forest viruses, poxviruses, pseudorabies viruses, RNA virus vectors, DNA virus vectors, and vectors derived from combinations of plasmids and phage DNA. Further optionally, the polynucleotides are operably ligated to expression control sequences to direct peptide synthesis. Even more optionally, the vectors include one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0170] In some cases, the vector is a lentiviral vector.

[0171] Also disclosed herein are host cells comprising vectors disclosed herein, or polynucleotides or polypeptides disclosed herein. In some examples, the host cells comprise nucleic acid sequences encoding the cloning or expression vectors described herein, and / or antigen-binding proteins, antibodies and their binding fragments described herein.

[0172] A 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 its binding fragment / protein encoded by it. A host cell containing a nucleic acid or vector can be used to produce an antigen-binding protein or its binding fragment / protein, or a portion thereof (e.g., a heavy chain sequence or light chain sequence encoded by the nucleic acid or vector). After introducing the nucleic acid or vector into the cell, the cell is cultured under conditions suitable for the expression of the encoded sequence. The antibody, antigen-binding protein, or fragment, or portion of the antibody, can then be isolated from the cell.

[0173] The host cell may be a prokaryotic host cell (such as E. coli) or a eukaryotic host cell (such as yeast cells, insect cells, or vertebrate cells). When cultured under appropriate conditions, the host cell will express the antibody or its conjugated fragment, which can then be recovered from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it does not secrete it). The selection of a suitable host cell will depend on various factors, such as the desired expression level, polypeptide modifications desirable or required for activity, such as glycosylation or phosphorylation, and the ease of folding into a biologically active molecule. The selection of the host cell will also depend in part on whether the antibody or its conjugated fragment is post-transcriptionally modified (e.g., glycosylation and / or phosphorylation). Host cells may include bacterial cells, yeast cells, animal cells, e.g., mammalian cells and / or plant cells.

[0174] Suitable mammalian host cells include CHO cells, myeloma cells, or hybridoma cells. Many are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples of mammalian cells include Chinese hamster ovary cells (CHO) (ATCC No. CCL61), human fetal kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), 3T3 cells (ATCC No. CCL92), or PER.C6 cells. Other cell types that can be used to express antibodies include lymphocyte cell lines, such as NSO myeloma cells and SP2 cells, and COS cells.

[0175] In one embodiment, a host cell expresses / secretes an antigen-binding protein, a variant thereof, or a fragment thereof as disclosed herein.

[0176] In one embodiment, cells that express / secrete EpCAM-specific immune cell engagers are provided.

[0177] In one embodiment, the cells are stem cells selected from a group that includes, for example, mesenchymal stem cells, neural stem cells, and pluripotent stem cells such as induced pluripotent stem cells (iPSCs). Therefore, in one embodiment, the stem cells are mesenchymal stem cells. In one embodiment, the stem cells are neural stem cells. In one embodiment, the stem cells are pluripotent stem cells such as iPSCs.

[0178] In one embodiment, the cells are immune cells.

[0179] In one embodiment, immune cells express / secrete EpCAM-specific immune cell engagers. In one embodiment, the immune cell engagers are selected from a group including T cell engagers, NK cell engagers, monocyte engagers, and macrophage engagers.

[0180] In one embodiment, immune cells express / secrete bispecific T cell engagers (BiTEs) that are specific to EpCAM.

[0181] In one embodiment, immune cells express / secrete bispecific T cell engagers (BiTEs), such as inducible, non-inducible, or constitutively expressed BiTEs, which include antigen-binding proteins, variants, or fragments thereof, as disclosed herein.

[0182] Surprisingly, the inventors have demonstrated that immune cells, such as CAR T cells, can secrete EpCAM BiTE. This was previously unknown. Advantageously, by designing immune cells to express the EpCAM immune engager, the EpCAM immune engager can be secreted to a target site (e.g., a solid tumor site), thereby minimizing toxicity and / or side effects.

[0183] In some examples, 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, and the like. In one embodiment, the immune cells are NK cells. In one embodiment, the immune cells are macrophages. In one embodiment, the immune cells are dendritic cells. In one embodiment, the immune cells are monocytes.

[0184] In one embodiment, the immune cells are T cells. In one embodiment, the immune cells are CAR T cells such as anti-GPC3, anti-HER2, or anti-CD19 CAR T cells. Therefore, in one embodiment, the CAR T cells are anti-GPC3 CAR T cells. In one embodiment, the CAR T cells are anti-HER2 CAR T cells. In one embodiment, the CAR T cells are anti-CD19 CAR T cells. In one embodiment, the immune cells are CAR T cells, CAR NK cells, CAR macrophages, or CAR monocytes.

[0185] In some cases, immune cells can bind to more than one host cell antigen. Therefore, in some cases, immune cells can bind to one host cell antigen, two host cell antigens, three host cell antigens, four host cell antigens, and so on.

[0186] In yet another embodiment, a method for producing / generating cells as described herein is provided, comprising introducing the polynucleotides described herein into cells.

[0187] Also disclosed are methods for producing / generating the cells described herein, in which one or more nucleic acids encoding host cell target antigens are fused in a vector via a linker (such as a P2A cleavable linker); and the vector is transformed or transfected into host cells. In some examples, the culture supernatant of the host cells transformed or transfected with the vector is collected.

[0188] In some examples, the method involves introducing the vector described herein into cells. In some examples, the vector is introduced via viral transduction.

[0189] Furthermore, compositions comprising cells or cell supernatants described herein are also disclosed.

[0190] Also disclosed herein are cells or cell supernatants described (designed) herein, and pharmaceutical compositions comprising such appropriate pharmaceutical compositions.

[0191] In some examples, the compositions are prophylactic and / or therapeutic compositions.

[0192] The pharmaceutically acceptable agents for use in the pharmaceutical compositions of the present invention include carriers, excipients, diluents, antioxidants, preservatives, colorants, fragrances, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonic agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobial agents, and surfactants.

[0193] Furthermore, compositions described herein or pharmaceutical compositions for use in therapeutics / pharmaceuticals / vaccines are also disclosed, and optionally, the compositions may further comprise excipients and / or stabilizers.

[0194] In yet another embodiment, a method is provided for treating a disease in a subject requiring such treatment, the method comprising administering cells described herein to a subject, optionally, the disease being a proliferative disorder.

[0195] Furthermore, methods for treating diseases in subjects requiring such treatment are disclosed, the methods comprising administering the designed cells or compositions described herein to a subject.

[0196] Furthermore, methods for preventing or reducing the severity of symptoms caused by a disease in subjects requiring such prevention or reduction are also disclosed, the methods comprising administering the designed cells or compositions described herein to a subject.

[0197] In some cases, cells or compositions or pharmaceutical compositions are administered to a subject through one or more routes of administration, including but not limited to topical, intravascular, intravenous, oral, subcutaneous, intra-arterial, intrathecal, intraperitoneal, intranasal, intradermal, and intramuscular administration.

[0198] Furthermore, the use of cells described herein in the manufacture of agents for the prevention and / or treatment of diseases is also disclosed.

[0199] Also disclosed herein are polynucleotides, antibodies, bispecific T cell engagers, designed cells, designed immune cells, methods, compositions, or pharmaceutical compositions described herein.

[0200] In some cases, the disease is a proliferative disorder. In some cases, the disease is a tumor or cancer. In some cases, the disease is a carcinoma and may include, but is not limited to, liver cancer (hepatocellular carcinoma, etc.), lung cancer (squamous cell carcinoma of the lung, etc.), stomach cancer (adenocarcinoma of the stomach, etc.), breast cancer, skin cancer (melanoma, etc.), ovarian cancer (clear cell carcinoma of the ovary, etc.), kidney cancer, pancreatic cancer, head and neck cancer, prostate cancer, esophageal cancer, bladder cancer, colon cancer, and childhood cancers (hepatoblastoma, nephroblastoma, yolk sac tumor, etc.).

[0201] In addition, when describing certain embodiments, this disclosure may disclose methods and / or processes as a specific set of steps. However, unless specifically required, it will be understood that the methods or processes should not be limited to a specific set of steps disclosed. Other sets of steps may be possible. The specific order of the steps disclosed herein should not be construed as an unreasonable limitation. Unless specifically required, the methods and / or processes disclosed herein should not be limited to steps performed in the order written. The order of the steps may change and still remain within the scope of this disclosure.

[0202] Furthermore, while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may be disregarded in other alternative embodiments, and it will be understood that this disclosure provides support for such disregards and these related alternative embodiments.

[0203] array CDR refers to bold (CDR1); bold and italics (CDR2); or bold, italics and underlined ( CDR3 ) The highlighted residues / bases show the difference between the 1B6 and 1C1 light chain constant domains.

[0204] 1. Amino acid sequence

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[0209] JPEG2026521452000007.jpg104145

[0210] 2. Nucleotide sequence

[0211] JPEG2026521452000008.jpg56145 JPEG2026521452000009.jpg44145

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[0214] JPEG2026521452000014.jpg58145 [Brief explanation of the drawing]

[0215] The embodiments of this disclosure will be well understood and readily apparent to those skilled in the art from the following discussion and, where applicable, in combination with the drawings. It should be understood that other modifications may be made without departing from the scope of the invention. Some of the embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new exemplary embodiments. The embodiments should not be construed as limiting the scope of this disclosure.

[0216] [Figure 1]Figures 1A-1H show that Hep3B xenografts in NSG mice can be suppressed by anti-GPC3 CAR T(5C4) cells, but complete eradication is not possible because the surviving tumor cells lose GPC3 expression. Figure 1A shows FACS analysis of tumor markers (GPC3 and EpCAM) expressed on Hep3B cells. Figures 1B-1D show the results of subcutaneous injection of 2 million Hep3B cells into the right flank of NSG mice (-26 days). On day 0, mice were grouped according to tumor size, and these mice were intravenously injected with 10 million anti-GPC3 CAR T(5C4) cells or Mock T cells via the tail vein (day 0). Figure 1B shows the percentage of CAR expression on anti-GPC3 CAR T(5C4) cells detected by flow cytometry analysis. Figure 1C shows tumor size measured and recorded every 3-7 days. Figure 1D shows FACS analysis of tumor markers (GPC3 and EpCAM) expressed in tumors that reappeared in anti-GPC3 CAR T(5C4) treated mice 70 days after CAR T cell injection. Figures 1E-1H show the results of subcutaneous injection of 2 million Hep3B cells into the right flank of NSG mice (-21 days). On day 0, the mice were grouped according to tumor size, and 4 million anti-GPC3 CAR T(5C4) cells or mock T cells, sorted from the enriched CAR-positive population, were intravenously injected into these mice via the tail vein (day 0). Figure 1E shows that after lentiviral transduction, T cells expressing anti-GPC3 CAR T(5C4) cells were enriched by flow sorting, and the percentage of CAR expression on the sorted anti-GPC3 CAR T(5C4) cells was detected by flow cytometry analysis. The tumor size (Figure 1F) and body weight (Figure 1G) of each mouse were measured and recorded every 3–7 days. Figure 1H shows the FACS analysis of tumor markers expressed in tumors that reappeared in three anti-GPC3 CAR T (5C4) treated mice 71 days after CAR T injection.

[0217] [Figure 2]Figures 2A-2D show the structure and in vitro characterization of GE CAR-BiTE T cells. Figure 2A shows the structural map of GE CAR-BiTE. Figure 2B shows the percentage of CAR expression on anti-GPC3 CAR T (5C4) and GE CAR-BiTE T (the sequence "MT110" was used as BiTE) cells detected by flow cytometry. Figure 2C shows the percentage of Hep3B cell lysis mediated by human GE CAR-BiTE T cells, measured over time using xCelligence impedance assay (E:T ratio = 1:1). Figure 2D shows the percentage of Hep3B cell lysis mediated by anti-EpCAM BiTE activated naive T cells. Anti-EpCAM BiTE was secreted by human GE CAR-BiTE T cells, and naive human T cells were isolated from PBMCs of healthy donors. Time-course measurements were performed using xCelligence impedance assay (E:T ratio = 6:1).

[0218] [Figure 3]Figures 3A–3G show that human GE CAR-BiTE T cells completely eradicated large Hep3B xenografts in vivo. Figures 3A–3E show the results after lentiviral transduction, where anti-GPC3 CAR T(5C4) cells, GE CAR-BiTE T cells, or mock T cells were cultured and grown in T cell growth medium containing IL-2 (50 units per ml). Two million Hep3B cells were subcutaneously injected into the right flank of NSG mice (-27 days). 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 mock T cells were intravenously injected into these mice via the tail vein. Figure 3A shows the percentage of CAR expression on anti-GPC3 CAR T(5C4) and GE CAR-BiTE T (the sequence "MT110" was used as BiTE) cells as detected by flow cytometry analysis. Body weight (Figure 3B) and tumor size (Figure 3C) of each mouse were measured and recorded every 3–7 days. Figure 3D shows the tumor size of individual mice in each treatment group. Figure 3E shows that tumors were excised from selected mice and the expression of tumor markers GPC3 and EpCAM was analyzed. Figures 3F and 3G show the results after lentiviral transduction, in which anti-GPC3 CAR T(5C4) cells, GE CAR-BiTE T cells, or mock T cells were cultured and grown 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 flank of NSG mice (-21 days). 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 mock T cells were intravenously injected into these mice via the tail vein. Figure 3F shows that the tumor size of each mouse was measured and recorded every 3-4 days.Figure 3G shows the survival curves for mice in the "tumor only" group (gray dotted line), the "Mock T" group (gray solid line), the "CAR T(5C4)" group (black dotted line), and the "GE CAR-BiTE T" group (black solid line). Death was defined as either autonomous death of the mouse or the size of the mouse's tumor exceeding 2700 mm3.

[0219] [Figure 4] Figures 4A–4G show that human GE CAR-BiTE T cells completely eradicated HepG2 xenografts in vivo. Figures 4A and 4B show the results after subcutaneous injection of 1 million HepG2 cells into the right flank of NSG mice (-9 days). On day 0, mice were regrouped according to measurable tumor size, and these mice were intravenously injected with 10 million CAR T(5C4) cells or mock T cells via the tail vein. Figure 4A shows the percentage of CAR expression on anti-GPC3 CAR T(5C4) cells detected by flow cytometry analysis. Figure 4B shows the tumor size of each mouse measured and recorded every 3–7 days. Figures 4C–4G show the results after subcutaneous injection of 1 million HepG2 cells into the right flank of NSG mice (-4 days). On day 0, mice were regrouped according to measurable tumor size, and these mice were intravenously injected via the tail vein with 3 million GE CAR-BiTE T cells, CAR T(5C4) cells, or mock T cells. Figure 4C shows the percentage of CAR expression on anti-GPC3 CAR T(5C4) cells and GE CAR-BiTE T (the sequence "MT110" was used as BiTE) cells as detected by flow cytometry analysis. Figure 4D shows that the tumor size of each mouse was recorded and measured every 3–7 days. Figure 4E shows the tumor size of individual mice in each treatment group. Figure 4F shows that the body weight of each mouse was measured and recorded every 3–7 days. Figure 4G shows the survival curve of the mice. Death was defined as either autonomous death of the mouse or a tumor size exceeding 1700 mm3.

[0220] [Figure 5] Figures 5A-5H demonstrate that GE CAR-BiTE T cells using anti-EpCAM VHH constructed BiTE possess superior characteristics in terms of killing ability and specificity. Figure 5A shows the structural forms of MT110 BiTE, Nb01-013A BiTE, or Nb01-013B BiTE antibodies secreted by GE CAR-BiTE cells. Figure 5B shows FACS analysis of tumor markers (GPC3 and EpCAM) expressed in HepG2, HT-29, and HeyA8 cells. Killing assays of HepG2 (Figure 5C), HT-29 (Figure 5D), and HeyA8 (Figure 5E) cells by CAR T (5C4) or GE CAR-BiTE T cells (MT110, Nb01-013A, or Nb01-013B) were performed using xCelligence cell impedance assays and are shown over 72 hours after co-culture of target cells and effector cells (E:T ratio = 2:1). Interferon-r was measured using ELISA obtained 48 hours after co-culturing HepG2 (Figure 5F), HT-29 (Figure 5G), and HeyA8 (Figure 5H) cells with effector CAR T cells.

[0221] [Figure 6] Figures 6A and 6B demonstrate that human GE CAR-BiTE T cells using anti-EpCAM VHH constructed BiTE are superior tumor killers in in vivo HepG2 xenografts. Thirty male NSG mice were subcutaneously injected with 1 million HepG2 cells into the right flank (days 1-4). On day 0, the mice were regrouped according to measurable tumor size, and 3 million CAR T(5C4) cells, GE CAR-BiTE T cells (using "MT110", "Nb01-013A", or "Nb01-013B" as BiTE), or Mock T cells were intravenously injected into the tail vein. Figure 6A shows the tumor size of individual mice in each treatment group, measured and recorded every 3–7 days. Figure 6B shows the body weight of mice in different groups, measured and recorded every 3–7 days.

[0222] [Figure 7] Figures 7A to 7D show that GE CAR-BiTE T cells using anti-EpCAM VHH constructed BiTE exhibited superior killing ability, demonstrating that anti-EpCAM BiTE secreted by GE CAR-BiTE T cells could induce target cell killing independently of high GPC3 expression. Figure 7A shows the structural forms of anti-GPC3 CAR T (5C4) or GE CAR-BiTE T cells (anti-GPC3 CAR T cells secreting anti-EpCAM BiTE, "MT110", "Nb01-013A", or "Nb01-013B" used as BiTE) or 19E CAR-BiTE T cells (anti-CD19 CAR T cells secreting anti-EpCAM BiTE, "Nb01-013A" or "Nb01-013B" used as BiTE). In vitro killing assays of (Figure 7B) Hep3B (GPC3High, EpCAMHigh), (Figure 7C) HT-29 (GPC3Low, EpCAMHigh), and (Figure 7D) HeyA8 (GPC3-ve, EpCAM-ve) using anti-GPC3 CAR T cells, GE CAR-BiTE T cells, or 19E CAR-BiTE T cells were performed using xCelligence cell impedance assays and were shown over 72 hours after co-culture of target cells and effector cells (E:T ratio = 1:1).

[0223] [Figure 8]Figures 8A-D show that human GE CAR-BiTE T cells using anti-EpCAM VHH-constituted BiTE demonstrated superior tumor killing in in vivo Hep3B xenografts. Figure 8A shows a schematic timeline of the in vivo Hep3B xenograft model. 2 million Hep3B cells were subcutaneously injected into the right flank of 50 male NSG mice (-21 days). On day 0, mice were regrouped according to the size of their measurable tumors, and 8 million CAR T(5C4) cells, GE CAR-BiTE T cells (using anti-EpCAM BiTE, "MT110", "Nb01-013A", or "Nb01-013B" as BiTE, which secretes anti-GPC3 CAR T cells), 19E CAR-BiTE T cells (using anti-EpCAM BiTE, "Nb01-013A" or "Nb01-013B" as BiTE, which secretes anti-CD19 CAR T cells), or Mock T cells were intravenously injected into these mice via the tail vein. Two nights after CAR T or CAR-BiTE T cell therapy, three surviving mice from the GE CAR-BiTE T (Nb01-013A) group, along with five naive male NSG mice ("tumor only") as controls, were rechallenged with 1 million Hep3B cells (subcutaneous injection into the left flank). Figure 8B shows the tumor size of each mouse, measured and recorded every 3–7 days. The curve shows the tumor size up to day 92. Figure 8C shows the tumor size of each mouse in each treatment group up to day 92. Figure 8D shows the tumor size of the rechallenged tumor in the left flank (from day 92 onwards).

[0224] [Figure 9]Figures 9A–9F show that human GE CAR-BiTE T cells using anti-EpCAM VHH-constituted BiTE demonstrated superior in vivo T cell proliferation in a Hep3B mouse xenograft model. In the same experiment as in Figure 8, cheek bleeding was performed at weekly intervals for flow cytometry analysis to monitor the number of T cells in peripheral blood and for Luminex® Multiplex Assays regarding cytokine levels. Figure 9A shows the number of CD3-positive human T cells in the peripheral blood of selected mouse groups after 5 weeks of T cell infusion. Figure 9B shows the time-course analysis of CAR-positive human T cells in the peripheral blood of mice in the CAR T(5C4) treatment group. Figure 9C shows the time-course analysis of CAR-positive human T cells in the peripheral blood of mice in the GE CAR-BiTE T(Nb01-013A) treatment group. After 5 weeks of T-cell infusion, the levels of interferon-γ (Figure 9D), GM-CSF (Figure 9E), and perforin (Figure 9F) in the peripheral blood of selected mouse groups were measured using Luminex® Multiplex Assays.

[0225] [Figure 10] Figures 10A–10C show that human GE CAR-BiTE cells using anti-EpCAM VHH-constituted BiTE cells exhibited superior localized T cell proliferation within tumors in a Hep3B mouse xenograft model. In the same experiment as in Figure 8, selected mice were sacrificed after 5 weeks of T cell infusion, and the xenograft tumors were excised and dissected for flow cytometry analysis to analyze the number of human T cells within the tumors. Figure 10A shows the number of CD3-positive human T cells in the mouse tumors of selected mice, expressed as cells per gram of tumor. Figure 10B shows the number of CAR-positive human T cells in the mouse tumors of selected mice, expressed as cells per gram of tumor. Figure 10C shows the percentage of CAR-positive T cells within the human T cell population in the mouse tumors of selected mice. Mice showing effective tumor control are marked with an asterisk (*).

[0226] [Figure 11]Figure 11 shows a comparison of CAR T cells concentrated in tumors and CAR T cells dispersed in peripheral blood. In the same experiment as in Figure 8, selected mice (M18, M49, M38, and M40) that showed tumor control after 5 weeks of T cell infusion were analyzed by flow cytometry for both CAR T cells in peripheral blood and CAR T cells from excised tumors. The total number of CAR-positive T cells in the tumor was calculated by multiplying it by the total weight of the tumor, and the total number of CAR-positive T cells in peripheral blood was calculated by multiplying it by the average blood volume (~2 ml) of the experimental mice.

[0227] [Figure 12]Figure 12 shows the detection of the anti-EpCAM BiTE molecule Nb01-013A secreted from GE CAR-BiTE T cells by ELISA. Figure 12A shows the detection of the anti-EpCAM BiTE molecule 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 starting cell density was 500,000 / ml, and the percentage of CAR expression in both cultures was approximately 60%. To detect the amount of secreted anti-EpCAM BiTE molecule Nb01-013A, cell culture supernatant was collected daily (24 hours, 48 ​​hours, 72 hours, 96 hours) for ELISA. Figure 12B shows the detection of anti-EpCAM BiTE molecules when GE CAR-BiTE T (Nb01-013A) cells were co-cultured with Hep3B (GPC3High, EpCAMHigh), HT-29 (GPC3Low, EpCAMHigh), or HeyA8 (GPC3-ve, EpCAM-ve) cells in an E:T ratio of 2:1. The percentage of CAR expression in GE CAR-BiTE T (Nb01-013A) cells used in all co-cultures was 8.5%. After co-culturing GE CAR-BiTE T (Nb01-013A) cells with different target cells, the supernatant was collected daily (24 hours, 48 ​​hours, 72 hours) for ELISA to detect the amount of secreted anti-EpCAM BiTE molecule Nb01-013A. Briefly, human EpCAM-Fc protein was used to coat ELISA plates overnight, and after blocking with casein for 2 hours, the culture supernatant containing the 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 with an HRP-conjugated secondary antibody against the His tag. [Examples]

[0228] GPC3 single-targeting CAR T cells resulted in antigen loss in an in vivo xenograft mouse model. The in vivo therapeutic efficacy of 5C4 CAR T cells was demonstrated in two independent experiments using a human hepatocellular carcinoma cell Hep3B engrafted NSG mouse model (Figure 1). In vitro cultured Hep3B cells were shown to express high levels of GPC3 and EpCAM (Figure 1A). In the first experiment, 5C4 CAR T cells were successfully produced with 25.9% CAR expression (Figure 1B). The CAR T cells showed excellent tumor control and effectively suppressed the growth of engrafted Hep3B tumors (Figure 1C). However, after a tumor-free period, tumors reappeared (Figure 1C), and while EpCAM expression remained high, the antigen target GPC3 appeared to be lost (Figure 1D). In the second experiment, sorted anti-GPC3 CAR T (5C4) cells with a highly enriched CAR-positive population (94.1%, Figure 1E) were used to treat the same Hep3B xenografts. Despite treatment with these enriched CAR-expressing cells, which showed enhanced tumor control compared to the initial experiment (Figure 1F), tumor regrowth could not be prevented. These surviving tumor cells were also GPC3 -ve and EpCAM +ve This further confirmed the concept of a multi-target approach in the treatment of solid tumors. Moreover, GPC3-targeted CAR T-cell therapy did not cause weight loss in these mice, suggesting that it is low-toxicity or non-toxic (Figure 1G).

[0229] Construction and in vitro functional characterization of GE CAR-BiTE T constructs The GE CAR-BiTE T construct was designed by fusing the anti-EpCAM BiTE gene (the "MT110" sequence was used) to the anti-GPC3 CAR (clone 5C4) lentiviral construct via a P2A cleavable linker (Figure 2A). After lentiviral transduction and proliferation (Figure 2B), CAR T cells were tested for in vitro killing efficacy. Interestingly, GE CAR-BiTE T cells performed better than anti-GPC3 CAR alone when combined with Hep3B(GPC3) High , EpCAM High) killed target cells at a slightly faster rate (Figure 2C), suggesting a crucial role for BiTE in promoting target cell killing. The cytotoxic function of GE CAR-BiTE was further tested in an assay that showed that only the culture supernatant recovered from GE CAR-BiTE T cells efficiently directed T cell (naive T cells isolated from donor PBMCs)-mediated target cell killing (Figure 2D).

[0230] Human GE CAR-BiTE T cells completely eradicated large Hep3B xenografts in vivo. The efficacy of GE CAR-BiTE T cell therapy in tumor control was evaluated using Hep3B xenografts of NSG mice, assessed by transduction rates determined by flow cytometry (Figure 3A). Both CAR T(5C4) and GE-CAR-BiTE T therapy suppressed tumor growth, while only GE-CAR-BiTE-treated mice achieved complete tumor regression (Figures 3C, 3D) and exhibited a favorable safety profile without causing weight loss in the mice (Figure 3B). Further analysis revealed that tumor cells surviving anti-GPC3 CAR T(5C4) therapy lost GPC3 while still expressing EpCAM (Figure 3E). Nevertheless, when the inventors analyzed cells isolated from residual tissue at tumor engraftment sites from representative GE CAR-BiTE T cell-treated mice, they were unable to detect any cells expressing either GPC3 or EpCAM (Figure 3E). The double-negative cells were mouse stromal cells present at the engraftment site, suggesting that all human Hep3B tumor cells were likely eradicated by GE CAR-BiTE T cell therapy. Next, we slightly modified the T cell culture protocol by replacing IL-2 in the culture medium with a combination of IL-7 and IL-15, which is a widely accepted standard protocol for CAR T cell production in a clinical setting. Repeated experiments using GE CAR-BiTE T cells grown in a culture medium containing IL-7 and IL-15 reproduced the excellent efficacy results in tumor control (Figure 3F) and long-term survival of mice (Figure 3G).

[0231] Human GE CAR-BiTE T cells completely eradicated HepG2 xenografts in vivo. Similar to Hep3B, another HCC cell line, HepG2, also expressed high levels of GPC3 and EpCAM on its cell surface. However, compared to the Hep3B model, anti-GPC3 targeted CAR T cells alone were relatively ineffective in treating HepG2-derived xenografts, even when injected much earlier after tumor inoculation (9 days in the HepG2 model compared to 21-27 days in the Hep3B model) (Figures 4A and 4B). We suspected this was because HepG2 xenografts proliferated considerably faster in mice than Hep3B xenografts. We considered whether GE CAR-BiTE T cell therapy could be successful in this more aggressive mouse model. After lentiviral transduction (Figure 4C), mice were intravenously injected with either CAR T(5C4) alone or GE CAR-BiTE T cells 4 days after HepG2 tumor cell inoculation. Unlike the inefficiency of the CAR T (5C4) treatment group, all three GE CAR-BiTE T cell-treated mice successfully cured their tumors (Figure 4D, Figure 4E), and all mice survived without significant weight loss until voluntarily discontinuing the experiment on day 80 (Figure 4F) (Figure 4G).

[0232] Human GE CAR-BiTE cells using the novel BiTE demonstrated superior characteristics in terms of killing ability and specificity. The inventors noticed that GE CAR-BiTE T cells using "MT110" generally had lower T cell viability and poorer T cell proliferation compared to CAR T(5C4) cells. Therefore, "MT110" was replaced with a different version of BiTE, the anti-EpCAM arm was replaced with anti-EpCAM VHH (clone 2C4) identified by us, 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 the cytotoxic effects of GE CAR-BiTE T cells using "MT110," three representative cell lines (Figure 5B) were selected, including HepG2 (expressing both GPC3 and EpCAM at high levels), HT-29 (expressing GPC3 at very low levels but EpCAM at high levels), and HeyA8 (expressing neither GPC3 nor EpCAM). Using three cells expressing different levels of surface antigens, GE CAR-BiTE cells equipped with the novel BiTE (Nb01-013A or Nb01-013B) showed superior properties in killing HepG2 and HT-29 cells with comparable ability to MT110 BiTE. Interestingly, T cells secreting MT110 induced strong cytotoxicity against HeyA8 cells, while GE CAR-BiTE T cells using a new BiTE (Nb01-013A or Nb01-013B) missed these target-negative cells (Figures 5C, 5D, and 5E). This may be due to the nonspecific binding of MT110's anti-EpCAM arms to different, unknown cell surface proteins, or to specific binding to the EpCAM protein, which is expressed at extremely low levels outside the detection limit by flow cytometry. This may be due to the high affinity of MT110 for EpCAM. T cell activation-mediated target cell killing can also be reflected by parallel interferon-γ ELISA measurements (Figures 5F, 5G, and 5H).

[0233] In summary, to overcome the challenge of tumor heterogeneity, the inventors have developed a novel CAR T-cell therapy targeting GPC3-positive solid tumors by providing single-antigen-targeting CAR T cells through local secretion of anti-EpCAM BiTE. By using CAR T cells that secrete anti-EpCAM BiTE, the efficacy of the CAR T-cell therapy can be maximized by recruiting nearby bystander T cells (a population of T cells that do not express CAR) to contribute to tumor clearance and simultaneously avoid systemic toxicity that may be caused by other means of delivering anti-EpCAM BiTE. This disclosure presents the development of a prototype (GE CAR-BiTE), which also serves as a proof of concept. While GE CAR-BiTE has been demonstrated to function normally, this technology is not limited to CARs targeting GPC3 but can be extended to many other tumor antigens that would be applicable as CAR targets for various types of cancer occurring in multiple organs.

[0234] Human GE CAR-BiTE cells using the VHH BiTE molecule demonstrated superior tumor-killing capabilities in a HepG2 mouse xenograft model. After constructing GE CAR-BiTE T cells using different anti-EpCAM BiTE molecules ("MT110", "Nb01-013A", and "Nb01-013B"), the inventors proceeded to test their tumor control ability using an in vivo xenograft model. First, the HepG2 xenograft model was tested (Figure 6). While CAR T (5C4) failed to control tumor growth, all three groups of GE CAR-BiTE T cell therapy showed some in vivo tumor control efficacy without significant weight loss (Figure 6B) (Figure 6A). Tumor reduction was observed in 40% (2 / 5 mice), 60% (3 / 5 mice), and 80% (4 / 5 mice) of mice in the "MT110", "Nb01-013A", and "Nb01-013B" groups, respectively (Figure 6A).

[0235] Human GE CAR-BiTE cells using the VHH BiTE molecule demonstrated superior CAR antigen-independent in vitro cytotoxicity in killing EpCAM-expressing cells. Next, GE CAR-BiTE T cells using different anti-EpCAM BiTE ("MT110", "Nb01-013A", "Nb01-013B") were compared with anti-CD19 CAR T cells that secrete anti-EpCAM BiTE ("Nb01-013A" or "Nb01-013B"), which were named "19E CAR-BiTE T" cells (Figure 7A), along with Hep3B (GPC3) cells. High , EpCAM High ), HT-29 (GPC3 Low , EpCAM High ), and HeyA8(GPC3 -ve, EpCAM -ve The in vitro killing efficacy was tested using ) cells.

[0236] In an in vitro killing assay using the xCelligence RTCA system, an E:T ratio of 1:1 (adjusted to 10% CAR expression level) was applied. All GE CAR-BiTE T cells, including GE CAR-BiTE T (MT110) cells, did not kill HeyA8 (GPC3-ve, EpCAM-ve) cells at low E:T ratios (Figure 7D), however, strong cytotoxicity was observed in killing EpCAM-positive cells Hep3B (Figure 7B) and HT-29 (Figure 7C). 19E CAR-BiTE T cells (using "Nb01-013A" as BiTE) killed EpCAM-positive cells Hep3B (GPC3 High , EpCAM High )(Figure 7B) and HT-29(GPC3 Low , EpCAM High Both (Figure 7C) showed partial killing, indicating that, in the current in vitro setting, the amount of anti-EpCAM BiTEs secreted by 19E CAR-BiTE T (Nb01-013A) cells exceeded the threshold for BiTE-mediated cell killing and was independent of CAR-specific antigen expression levels. On the contrary, 19E CAR-BiTE T cells ("Nb01-013B" used as BiTE) were HT-29 (GPC3 Low , EpCAM HighSince it showed only minimal cell killing, it was suggested that the “Nb01-013B” BiTE is considerably less capable of mediating T cell killing than the “Nb01-013A” BiTE (Figure 7C).

[0237] Human GE CAR-BiTE cells using the VHH BiTE molecule demonstrated excellent CAR antigen-dependent tumor-killing ability in a Hep3B mouse xenograft model. This observation allows us to hypothesize whether in vivo tumor control depends on or does not depend on CAR-specific antigen expression (Figure 8). Interestingly, Hep3B xenografts from mice in two 19E CAR-BiTE T cell therapy groups showed similar proliferation rates to the control group ("tumor only" group and "Mock T" group), suggesting that, unlike in vitro environments, the effectiveness of in vivo tumor control by CAR-BiTE T cells depends on CAR-specific antigen expression. In this experiment, we did not observe the effect of GE CAR-BiTE T(MT110) cells on Hep3B xenograft control, as we had consistently observed in previous experiments. This is because the in vivo proliferation of GE CAR-BiTE T(MT110) cells reached a threshold point (tumor size 3000 mm) before achieving a significant tumor control effect. 3This is because the mice were spontaneously disposed of when the threshold was exceeded. This may be due to variations in the donor of the T cells used to generate CAR T cells in different experiments. However, GE CAR-BiTE T cells (either "Nb01-013A" or "Nb01-013B" used as BiTE) generated using the same T cell donor showed much stronger tumor control efficacy, suggesting that replacing the anti-EpCAM arm with our anti-EpCAM 2C4-VHH significantly improved both the in vivo killing capacity and in vivo proliferation of GE CAR-BiTE T cells. Mice in the GE CAR-BiTE T (Nb01-013A) group achieved efficient tumor control in all nine mice tested. In addition, three mice survived in a healthy state for more than 90 days after treatment (Figure 8B, Figure 8C). 92 days after GE CAR-BiTE T(Nb01-013A) cell therapy, a rechallenge study was conducted by subcutaneously injecting 1 million Hep3B cells into the left flank of the remaining three surviving mice. Five naive NSG mice were also included as a "tumor-only" control. Approximately 20 days later, tumors began to grow in all naive mice injected with 1 million Hep3B cells, averaging 445.3 mm at day 133 (41 days after subcutaneous injection of 1 million Hep3B cells). 3 This was reached. However, no tumor growth was observed in the left flank of any of the three re-challenge mice in the GE CAR-BiTE T(Nb01-013A) group (Figure 8D).

[0238] Human GE CAR-BiTE cells using the VHH BiTE molecule showed excellent in vivo T cell proliferation in a Hep3B mouse xenograft model. To monitor the in vivo proliferation and cytotoxic function of CAR T cells, we performed weekly cheek bleeding for flow cytometry analysis of T cell counts and Luminex® Multiplex Assays for cytokine levels (Figure 9). From week 4 onward (day 28 after T cell therapy), significant proliferation of human T cells was detected in mouse blood, and their numbers continued to increase until reaching a peak level, after which they subsided (Figures 9B, 9C). As expected, when comparing the levels of CD3-positive T cells in each group after 5 weeks of T cell infusion, mice in the GE CAR-BiTE T(Nb01-013A) and GE CAR-BiTE T(Nb01-013B) groups showed much deeper T cell proliferation than mice in the GE CAR-BiTE T(MT110) group (Figure 9A). In addition, the levels of serum cytokines such as human interferon-γ (Figure 9D), GM-CSF (Figure 9E), and perforin (Figure 9F) secreted by human T cells in the first two mouse groups were comparable to those in the CAR T(5C4) group and significantly higher than those in the three control groups, including GE CAR-BiTE T(MT110) and Mock T, 19E CAR-BiTE T(Nb01-013A), and 19E CAR-BITE T(Nb01-013B). As a result of efficient T cell proliferation in mice, all tumors in the CAR T(5C4) group and GE CAR-BiTE T(Nb01-013A) group, as well as five tumors in the GE CAR-BiTE T(Nb01-013B) group, were found to be 3000 mm 3 It was observed that the sample contracted before reaching this point (Figure 8C).

[0239] Human GE CAR-BiTE cells using the VHH BiTE molecule showed superior localized T cell proliferation within tumors in a Hep3B mouse xenograft model. On days 33–35, all mice in the "tumor only," "Mock T," "GE CAR-BiTE T (MT110)," "19E CAR-BiTE T (Nb01-013A)," and "19E CAR-BiTE T (Nb01-013B)" groups were voluntarily euthanized because most of the tumor size was close to the humane point. The inventors selected 1–2 mice from each group to analyze tumor-infiltrating human T cells from the excised tumors. In the CAR T (5C4) and GE CAR-BiTE T (Nb01-013A) groups, all treated mice showed good control of tumor growth; therefore, the inventors randomly selected one mouse from each of these two groups for FACS analysis, while leaving the remaining 8 mice for continued monitoring. However, in the GE CAR-BiTE T (Nb01-013B) group, not all mice showed tumor reduction on the day of analysis. Therefore, the inventors included a total of five mice in the FACS analysis. Effective tumor control was observed in mice M38 and M40, but not in mice M34, M37, and M39. Interestingly, the inventors observed abundant accumulation of human T cells (CD3-positive cells, Figure 10A) and CAR-positive T cells (Figure 10B) in mice M18, M49, M38, and M40, suggesting that effective proliferation of CAR T cells within the tumor is necessary for effective tumor control activity. In addition, moderate to high percentages of anti-GPC3 CAR T cells could be detected in all M18 (CAR T group) and GE CAR-BiTE T group (M29, M49, M34, M37, M38, M39, and M40) mice, but not in the 19E CAR-BiTE T group (M27, M22, and M23) (a population of less than 10% CAR-positive T cells) (Figure 10C). This suggests that CAR-positive T cells are enriched in vivo only when CAR T cells encounter CAR target antigens.

[0240] Even more interestingly, when the inventors compared the total number of CAR-positive T cells in the excised tumors with the total number of CAR-positive T cells in the peripheral blood of four mice that showed effective tumor control (Figure 11), they observed that the amount of CAR-positive T cells present in the tumors was significantly greater than the amount of T cells circulating in the peripheral blood.

[0241] Anti-EpCAM rama VHH BiTE secreted by GE CAR-BiTE T cells can be detected in cell cultures. Both anti-GPC3 CAR T(5C4) cells 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 / ml. Cell culture supernatant was collected daily for four consecutive days, and the amount of secreted anti-EpCAM BiTE molecule Nb01-013A was detected by ELISA. The results clearly showed that the amount of secreted Nb01-013A molecule increased over time, suggesting that anti-EpCAM VHH BiTE is continuously secreted and accumulated from GE CAR-BiTE T(Nb01-013A) cell cultures (Figure 12A).

[0242] Interestingly, in another experiment, GE CAR-BiTE T(Nb01-013A) cells were treated with Hep3B(GPC3 High ,EpCAM High ), HT-29 (GPC3 Low ,EpCAM High ), and HeyA8(GPC3 -ve ,EpCAM -ve The cells were co-cultured with different target cells, including GE CAR-BiTE T(Nb01-013A). The percentage of CAR expression in GE CAR-BiTE T(Nb01-013A) cells used in all co-cultures was 8.5%. The culture supernatant from the co-cultures was also collected daily for 3 days, and ELISA showed that Nb01-013A secretion increased over time only after co-culture with Hep3B cells, and the proliferation of the CAR-positive T cell population was observed when GE CAR-BiTE T(Nb01-013A) cells were GPC High This was suggested to occur only when the target cell is encountered (Figure 12B).

[0243] In summary, this disclosure demonstrates that the use of VHH BiTE molecules ("Nb01-013A" and "Nb01-013B") constructed from anti-EpCAM VHH clone 2C4 in GE CAR-BiTE T cells achieves more efficient and potent tumor killing than GE CAR-BiTE T cells using "MT110," with the highest efficacy observed when "Nb01-013A" is used as the BiTE molecule.

[0244] Applicable Embodiments of modified cells disclosed herein utilize the following factors: 1) tumor-targeted T cell proliferation, and 2) tumor-specific anti-EpCAM BiTE secretion, the latter of which cannot be achieved by conventional systematic delivery approaches. 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.

[0245] The technology described in this TDF may have the following applications:

[0246] 1. Anti-GPC3 CAR T cells that secrete anti-EpCAM BiTE can be used to treat GPC3-expressing cancers such as hepatocellular carcinoma, ovarian clear cell carcinoma (OCCC), lung squamous cell carcinoma, melanoma, and several childhood cancers (hepatoblastoma, nephroblastoma, and yolk sac tumors), as well as certain gastric cancers.

[0247] 2. Anti-EpCAM BiTE-secreting CAR T cells can be configured to target other tumor-specific antigens, including but not limited to HER2, claudin 18.2, 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 a variety of solid cancers, including liver cancer, breast cancer, head and neck cancer, gastric cancer, pancreatic cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, esophageal cancer, bladder cancer, and colon cancer.

[0248] This disclosure provides an improvement in the therapeutic efficacy of current single-target CAR T-cell therapy against solid tumors by advantageously overcoming tumor heterogeneity and tumor escape associated with treatment using anti-EpCAM BiTE. Furthermore, by applying local secretion of anti-EpCAM BiTE by CAR T cells, non-drug-unavailable targets are transformed into drug-available targets by reducing the extratumor toxicity of the target that may be caused by systematic delivery. In addition, targeting cancer progenitor cells and cancer stem cells that express EpCAM further contributes to complete tumor eradication and prevention of tumor relapse.

[0249] As is broadly described, it will be understood by those skilled in the art that other variations and / or modifications can be made to the embodiments disclosed herein without departing from the spirit or scope of this disclosure. For example, features of different exemplary embodiments may be mixed, combined, swapped, incorporated, adopted, modified, included, etc., across different exemplary embodiments in this description. Thus, these embodiments are intended to be illustrative and not limiting in all respects.

Claims

1. (a) Chimeric antigen receptors targeting GPC3 or CD19, and (b) A multispecific antigen-binding protein, variant or binding fragment that binds to one or more targets, comprising a first antigen-binding protein that binds to EpCAM (epithelial cell adhesion molecule), a variant or binding fragment thereof, and a second antigen-binding protein that binds to an immune cell marker, a variant or binding fragment thereof. Modified cells that express, A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (Sequence ID 25), or GSSERFTS (Sequence ID 29): CDR-H1, or - ITSGST (Sequence ID 26), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, - ITNGGST (Sequence ID 30) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it: Including CDR-H2, and - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHH (Sequence ID 27), - MAGTS (Sequence ID 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, - Sequence TNGRWSGDTYYAHL (SEQ ID NO: 33), or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it: Includes, CDR-H3: Heavy chain variable region including, (ii) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GGTFSSYA (Sequence ID 1), CDR-H2, which includes IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and CDR-H3: Contains a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ARSLGGRFRY (SEQIDNO:3) or therewith. Heavy chain variable region including, (iii) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GDSISSNSVA (Sequence ID 5), CDR-H2, which includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TYYRSKWYS (Sequence ID 6), and CDR-H3 containing AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity: Heavy chain variable region including, (iv) - QSLLHSNGYNY (Sequence ID 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. - QSLLHSNRYNY (Sequence ID 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, - QSISDF (Sequence ID 19) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it: CDR-L1 - LGS (Sequence ID 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, - AAS (Sequence ID 20-1E4) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it: Including CDR-L2, and - MQALQTPYT (Sequence ID 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it. - Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to MQGLQSPWT (Sequence ID 15). - QQSYIMPDT (Sequence ID 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, - MQGLQTPYT (SEQ ID NO: 23) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it: Includes, CDR-L3: Light chain variable region including: Modified cells comprising heavy chain variable regions and / or light chain variable regions selected from the group consisting of the following.

2. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (SEQ ID NO: 25), CDR-H2, which includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (Sequence ID 26), and CDR-H3 contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHH (Sequence ID 27): Heavy chain variable region including, (ii) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GGTFSSYA (Sequence ID 1), CDR-H2, which includes IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and CDR-H3, which contains ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith: Heavy chain variable region including, (iii) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GDSISSNSVA (Sequence ID 5), CDR-H2, which includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TYYRSKWYS (Sequence ID 6), and CDR-H3 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith: Heavy chain variable region including, (iv) CDR-L1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNGYNY (Sequence ID 9), CDR-L2, which includes LGS (Sequence ID 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, and CDR-L3 contains MQALQTPYT (Sequence ID 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto: Light chain variable region including (v) CDR-L1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNGYNY (Sequence ID 9), CDR-L2, which includes LGS (Sequence ID 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, and CDR-L3 includes MQGLQSPWT (Sequence ID 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto: Light chain variable region including, (vi) CDR-L1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNRYNY (Sequence ID 17), CDR-L2, which includes LGS (Sequence ID 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, and CDR-L3 contains MQALQTPYT (Sequence ID 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto: Light chain variable region including, (vii) CDR-L1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSISDF (Sequence ID 19), CDR-L2, which includes AAS (Sequence ID 20) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, and CDR-L3 contains a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QQSYIMPDT (SEQ ID NO: 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith: Light chain variable region including, (viiii) CDR-L1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNGYNY (Sequence ID 9), CDR-L2, which includes LGS (Sequence ID 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, and CDR-L3 includes MQGLQTPYT (Sequence ID 23) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto: Light chain variable region including (ix) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSSERFTS (SEQ ID NO: 29), CDR-H2, which includes ITNGGST (SEQ ID NO: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, and CDR-H3 containing MAGTS (SEQ ID NO: 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto: Heavy chain variable region including; and (x) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (SEQ ID NO: 25), CDR-H2, which includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (Sequence ID 26), and CDR-H3 containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHL (SEQ ID NO: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith: Heavy chain variable region including The cell according to claim 1, comprising a heavy chain variable region and / or a light chain variable region selected from the group consisting of the following.

3. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) CDR-H1, comprising 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 therewith. CDR-H2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with IIPIFGTA (SEQ ID NO: 2), CDR-H3 comprising 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 therewith: Heavy chain variable region including; and / or CDR-L1, comprising a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNGYNY (SEQ ID NO: 9) CDR-L2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (SEQ ID NO: 10), 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 therewith: Light chain variable region including (ii) CDR-H1, comprising 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 therewith. CDR-H2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with IIPIFGTA (SEQ ID NO: 2), CDR-H3 comprising 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 therewith: Heavy chain variable region including; and / or CDR-L1, comprising a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNGYNY (SEQ ID NO: 9) CDR-L2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (SEQ ID NO: 10), 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 therewith: Light chain variable region including (iii) CDR-H1, comprising 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 therewith. CDR-H2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with IIPIFGTA (SEQ ID NO: 2), CDR-H3 comprising 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 therewith: Heavy chain variable region including; and / or CDR-L1, comprising a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNRYNY (SEQ ID NO: 17), CDR-L2 containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (SEQ ID NO: 10), and 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 therewith: Light chain variable region including (iv) CDR-H1 containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GGTFSSYA (SEQ ID NO: 1) CDR-H2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with IIPIFGTA (SEQ ID NO: 2), CDR-H3 comprising 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 therewith: Heavy chain variable region including; and / or CDR-L1, comprising a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSLLHSNGYNY (SEQ ID NO: 9) CDR-L2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with LGS (SEQ ID NO: 10), 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 therewith: Light chain variable region including; Furthermore (v) CDR-H1, comprising 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 therewith. CDR-H2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TYYRSKWYS (SEQ ID NO: 6), CDR-H3 comprising 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 therewith: Heavy chain variable region including; and / or CDR-L1, comprising a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QSISDF (SEQ ID NO: 19), CDR-L2, and containing a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with AAS (SEQ ID NO: 20), CDR-L3 comprises a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with QQSYIMPDT (SEQ ID NO: 21) or therewith. Light chain variable region including A cell according to any one of the above claims, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of the above.

4. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (Sequence ID 25), or - Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSSERFTS (Sequence ID 29): Including CDR-H1, - ITSGST (Sequence ID 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it, - ITNGGST (Sequence ID 30) or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it: Including CDR-H2; and - A sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHH (Sequence ID 27), - MAGTS (Sequence ID 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, - Sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHL (SEQ ID NO: 33): CDR-H3 A cell according to any one of the above claims, comprising a heavy chain variable region.

5. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSIFSGND (SEQ ID NO: 25), CDR-H2, which includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (Sequence ID 26), and CDR-H3 contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TNGRWSGDTYYAHH (Sequence ID 27): Heavy chain variable region including, (ii) CDR-H1, containing a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with GSSERFTS (SEQ ID NO: 29), CDR-H2, which includes ITNGGST (SEQ ID NO: 30), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity therewith, and CDR-H3 containing MAGTS (SEQ ID NO: 31), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto: Heavy chain variable region including; and (iii) CDR-H1, containing a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSIFSGND (SEQ ID NO: 25), CDR-H2, which includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with ITSGST (Sequence ID 26), and CDR-H3 includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with TNGRWSGDTYYAHL (SEQ ID NO: 33), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it: Heavy chain variable region including A cell according to any one of the above claims, comprising a heavy chain variable region selected from the group consisting of the above.

6. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAAHHWGQGTL (SEQ ID NO: 37), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains (ii) 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 thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains (iii)EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID 4), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains (iv) QVQLQQSGPGLVKPSQTLSLTCCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions, Heavy chain variable domains (v) DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Light chain variable domains (vi) EIVLTQSPLSLLPPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Light chain variable domains (vii) DVVMTQSPLSLPVTPPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLLIYLGSNRASGVPDRFSGSGSGTDFTTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Light chain variable domains (viiii)DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAAPKLLIYAASSLQTGVPSRFGGGSGSGTEFFTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Light chain variable domains (ix) DVVMTQSPLSLPVTPPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (SEQ ID NO: 24), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Light chain variable domains (x) 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 thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains (xi)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 thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains (xi)QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAAHLWGQGTQ (SEQ ID NO: 34), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains (xiiii)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 thereto, and / or having two or three amino acid substitutions. Heavy chain variable domains A cell according to any one of the above claims, comprising a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of the above.

7. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID 4), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions: Heavy chain variable domains including, and / or DVVMTQSPLSLPVTPPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 12), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Light chain variable domains including (ii) EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (SEQ ID NO: 4), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions: Heavy chain variable domains including, and / or EIVLTQSPLSLPPVTPPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFFTLKISRVEAEDVGVYYCMQGLQSPWTFGQGTK (SEQ ID NO: 16), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions. Light chain variable domains including (iii) EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (Sequence ID 4), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions: Heavy chain variable domains including, and / or DVVMTQSPLSLPVTPPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTK (SEQ ID NO: 18), or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions. Light chain variable domains including (iv) QVQLQQSGPGLVKPSQTLSLTCCAISGDSISSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYSDYAAISVKGRLDINPDTSKNQFSLQLNSVTPEDTAVYYCAREVEGSSYDAFDIWGQGTM (SEQ ID NO: 8), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions: Heavy chain variable domains including, and / or DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAAPKLLIYAASSLQTGVPSRFGGGSGSGTEFLTLTISSLQPEDLGTYYCQQSYIMPDTFGQGTK (SEQ ID NO: 22), or a fragment, variant, or sequence identical thereto by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and / or having two or three amino acid substitutions. Light chain variable domains including, or (v) EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQNFQGRVTTMTAADTSISTAYMELSSLLRSEDTAVYYCARSLGGGRFRYWGQGTL (SEQ ID NO: 4), or any fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or having two or three amino acid substitutions: Heavy chain variable domains including, and / or Light chain variable domain DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQTPYTFGQGTK (Sequence ID 24, or a fragment, variant, or sequence thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or has two or three amino acid substitutions) A cell according to any one of the above claims, comprising a heavy chain variable domain and / or a light chain variable domain selected from the group consisting of the above.

8. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (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 thereto, and / or having two or three amino acid substitutions, or (ii) QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLELVAVITSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTNGRWSGDTYYAAHHWGQGTL (SEQ ID NO: 37), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, 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 thereto, 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 thereto, and / or having two or three amino acid substitutions, or (v) QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAAHLWGQGTQ (SEQ ID NO: 34), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions, or (vi) QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRRAPGVERELVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYYCTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36), or a fragment, variant, or sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and / or having two or three amino acid substitutions. A cell according to any one of the above claims, comprising a single-domain heavy chain variable domain having a sequence.

9. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) CDR-H1, containing GGAAGCATCTTCAGTGGCAATATGAC (Sequence ID 62), CDR-H2, which includes ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAC (Sequence ID 64): Heavy chain variable region including (ii) CDR-H1 containing GGAAGTCCCGAAAAGATTCACATCA (Sequence ID 66), CDR-H2, which includes ATTACTAATGGGGTAGCCACA (SEQ ID NO: 67), and CDR-H3 containing ATGGCGGGTAACGTCC (Sequence ID 68): Heavy chain variable region including; and (iii) CDR-H1, containing GGAAGCATCTTCAGTGGCAATATGAC (Sequence ID 62), CDR-H2, which includes ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 63), and CDR-H3 containing ACAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATTC (Sequence ID 70): Heavy chain variable region including Alternatively, a fragment, variant, or sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it. A cell according to any one of the above claims, comprising a heavy chain variable domain encoded by a nucleotide sequence containing

10. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTG GTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACCTGAAACAGGCCCGTATATTACTACTGCCAAAAGAGGTCAAGAGGCGAATACTATACTATGCCCCATACACTGGGGGCCAAGGGGCAGAG (Sequence ID 72), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. or (ii) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCT GGTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCaAGAACACCcTAATATCTGCAAATGAACAGCCCTGAGAGCCTGAGGACACGGCCGTGTATATTAACTGCCAACAAACGGGAAGATGGGCGAATACTATACTATGCCCCATCAACTGGGGGCCAGGGGACCCTG (SEQ ID NO: 74), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions; or (iii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCA GCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGAC CGCAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCACAGCCCTGAGAGTGAACAGGCCCGTGTATATTACTGTGCGCAGATCGTTGGGGGGAGAGATTTCGCTACTGGGGGCCAGGGGAGACCTG (SEQ ID NO: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. or (iv) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGA ACTGGATCAGGCAGTCCCCATCGAGAGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCCAAACC CAGACACATCCCAAGAACCACAGTTTCCCCTGCACAGCTGAACTCTGTGACTCCCCGAGGAACAGGCTGTGTTATTATTTGTGCCAAGACAGAGTTGAGGGCCAAGCTATGAATGCCTTTTTGAATCTGGGGGCCAAGAGGAATG (Sequence ID 45), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions; or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTG GTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACTTTGAAAACCCTGAGGACACGGCCGTGTATATTACTGCCAAAACGGGAAGAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGCCCAGGGGACCCAG (Sequence ID 65), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions, or (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCAC CACTTCCAGAGACAACGCCCAAGAACACGGGTGTATCTGCCAAAAATGAACAGCCCTGAAAAAGCTGAGGCAACAGGCCCGTCTATTTATTTGTTATTGGCGGTACGTCCCTGGGGCCCAGGGGACCACAG (Sequence ID 69), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions, or (vii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCC TGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAC AATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACCTGAAACAGGCCCGTATATTACTGCCAAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCTCTGGGGGCCAGGGGACCCAG (Sequence ID 71), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. or (viii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCC TGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAC AATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACCTGAAACAGGCCCGTGTATATTACTGCCAAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGGCCAGGGGACCCAG (Sequence ID 73), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. It includes a heavy chain variable domain encoded by a nucleotide sequence containing and / or The aforementioned light chain variable domain is: (xvi) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATG GATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGG CAGTGGATCAGGCAACAGATTTTTACACTGAAAAATCAGCAGAGGAGGCTGAGGATGTTTGGGGTTTTATTATTACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGCCCAGGGGACCAAG (Sequence ID 49), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions, or (xvii) GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAAT GGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTG GCAGTGGATCAGGTACAGATTTTTAACACTGAAAAATAAGCAGAGGGAGAGCTGAGGATGTGGGGTTTTATTATTACTGCATGCCAAGGTCTCAAAAGTTCCCCTGGACGTTCCGGCCAAGGGACCAAG (SEQ ID NO: 53), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. or (xviii) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAAT AGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTG GCAGTGGATCAGGGCACAGATTTTTACACTGAAAAATCAGCAGAGGGAGCTGAGGATGTGGGGTTTTATTTACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGGCCAGGGGACCAAG (SEQ ID NO: 55), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. or (xix) GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTT TTAAAATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGA TCTGGGACAGAATTCTCTCACCATAAGCAGTCTACACACCTGAAGATTTGGGAACTTATTACTGTCAACAGAGTTTAACATTTATGCCCCGACACTTTTGGCCCAGGGGGAAAA (Sequence ID 59), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions, or (xx) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGG ATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGG CAGTGGATCAGGGCACAGATTTTTACACTGCCAAAATCAGCCAGAGTGGAGCTGAGGATGCTGGGGTTTTATACTGCATGCCAAGGTTCTACAGAACTCCCGTAACACTTTTTGGGCCAGGGGACCAAG (SEQ ID NO: 61), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. Encoded by a nucleotide sequence containing, The cell according to any one of the above claims.

11. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (vi) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTG GGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCG CAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCCAGCCTGAGAATCTGAGGACACGGCCCGGTGTATATTACTGTGCCGAGATCGTTGGGGGGAGAGATTTTCCGCTACTGGGGGCCAGGGGAGACCCTG (SEQ ID NO: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: A heavy chain variable domain encoded by a nucleotide sequence containing, and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGCACAGATTTTTACACTGAAAAATCAGCAGAGGCCTGAGGATGTGGGGGTTTATACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGCCCAGGGGACCAAG (SEQ ID NO: 49), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: Light chain variable domain encoded by a nucleotide sequence containing (vii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTG GGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCG CAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCCAGCCTGAGAATCTGAGGACACGGCCCGGTGTATATTACTGTGCCGAGATCGTTGGGGGGAGAGATTTTCCGCTACTGGGGGCCAGGGGAGACCCTG (SEQ ID NO: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: A heavy chain variable domain encoded by a nucleotide sequence containing, and / or GAAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGTACAGATTTTTACACTGAAAAATAAGCAGAGTGGAGCTGAGGATGTGGGGGTTTATTATTACTGCATGCCAAGGTTCCTACAAAGTTCCCCTGGACGTTCCGGCCAAGGGACCAAG (SEQ ID NO: 53), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: Light chain variable domain encoded by a nucleotide sequence containing (viiii) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTG GGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCG CAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCCAGCCTGAGAATCTGAGGACACGGCCCGGTGTATATTACTGTGCCGAGATCGTTGGGGGGAGAGATTTTCCGCTACTGGGGGCCAGGGGAGACCCTG (SEQ ID NO: 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: A heavy chain variable domain encoded by a nucleotide sequence containing, and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGTCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATAGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGCACAGATTTTTACACTGAAAAATCAGCAGAGGCCTGAGGATGTGGGGGTTTTTATACTGCATGCCAAGCTCTACAAACTCCGTACACTTTTTGGCCCAGGGGACCAAG (SEQ ID NO: 55), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: Light chain variable domain encoded by a nucleotide sequence containing (ix) GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTG GGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCG CAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCCAGCCTGAGAATCTGAGGACACGGCCCGGTGTATATACTGTGCCGAGATCGTTGGGGGGAGATTTTCCGCTACTGGGGGCCAGGGCAGACCCTG (Sequence ID 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. A heavy chain variable domain encoded by a nucleotide sequence containing, and / or GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATA CAACTATTTGGATTGGTACCTGCAGAAGCCAGGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCA GTGGATCAGGCACAGATTTTTACACTGCCAAAATCAGCCAGAGTGGAGAGCTGAGGATGCTGGGGGTTTATTTACTGCATGCCAAGGTTCTACAGAACTCCCGTAACTTTTGGCCCAAGGGGGACCAAG (SEQ ID NO: 61), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: Light chain variable domain encoded by a nucleotide sequence containing; Furthermore (x) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGGAACT GGATCAGGCAGTCCCCATCGAGAGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCCAACCCA GACACATCCCAAGAACCAGTTCTCCCCTGCCAGCTGAACTCTGTGACTCCCCGAGGACACGGCTGTGTGTATTTATGTGCCAAGAGAAGTTGAGGGCCAGCAAGCTATGAATGCTTTTGAATCTGGGGGCCAAGGGAATG (Sequence ID 45), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions: A heavy chain variable domain encoded by a nucleotide sequence containing, and / or GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTAGCGACTTTTTAA ATTGGTACCAGCAGAAACCAGGTAAAGCCCCGAAGCTCCTGATCTATGCTGCATCGAGTTTACAAACTGGGGTCCCCTCAAGATTCGGTGGCAGTGGATCT GGGACAGAATTCCAACTTCTCCAACCATAAGCAGTCTACAACCTGAAGATTTGGGAACTTATTTACTGTCAACAGAGTTTAACATTTAGCCCCGAACACTTTTTGGCCCAGGGGGAAAA (SEQ ID NO: 59), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions: Light chain variable domain encoded by a nucleotide sequence containing A cell according to any one of the above claims, comprising a heavy chain and / or light chain variable domain encoded by a nucleotide sequence selected from the group consisting of the above.

12. A first antigen-binding protein, a variant thereof, or a binding fragment that binds to the aforementioned EpCAM is: (i) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCTCTGGAAGCATCTTCAGTGGCAATGACATGTCCTG GTACCGCCAGGCTCCAGGGAAGGGACTCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCaAGAACACCcTAATATCTGCAAATGAACAGCCCTGAGAGCCTGAGGACACGGCCGTGTATATTAACTGCCAACAAACGGGAAGATGGGCGAATACTATACTATGCCCCATCAACTGGGGGCCAGGGGACCCTG (SEQ ID NO: 74), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions; or (ii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCT GGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACCTGAAACAGGCCCGTATATTACTACTGCCAAAAGAGGTCAAGAGGCGAATACTATACTATGCCCCATACACTGGGGGCCAAGGGGCAGAG (Sequence ID 72), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. or (iii) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCC TGGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAC AATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACTTTGAAAACCCTGAGGACACGGCCGTGTATATTTACTGCCAAAACGGGAAGATGGTCCAGGGCGAATACTTACTATGCCCCATCAACTGGGGCCCAGGGGACCCAG (Sequence ID 65), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions. or (iv) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCTCCGAAAGATTCACATCA GTGGCCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTGGTCGCATTTATTACTAATGGTGGTAGCACAAGATATACAGACCCCGTGAAGGGCCGATTCAC CACTTCCAGAGACAACGCCCAAGAACACGGGTGTATCTGCCAAAAATGAACAGCCCTGAAAAAGCTGAGGCAACAGGCCCGTCTATTTATTTGTTATTGGCGGTACGTCCCTGGGGCCCAGGGGACCACAG (Sequence ID 69), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions, or (v) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCTG GTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACCTGAAACAGGCCCGTATATTACTACTGCCAAAAGAGGTCAAGAG (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 a sequence having 10 to 20 nucleic acid substitutions, or (vi) CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGACTCTGGAAGCATCTTCAGTGGCAATGACATGGCCT GGTACCGCCGGGCTCCAGGGGTGGAGCGCGAGTTGGTCGCGGTTATTACTAGCGGTGGTAGTACACACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATGCCCCAGAAGACCGTATATCTGCCAAAACGAACGACCTGAAACAGGCCCGTATATTACTACTGCCAAAAGAGGTCAAGAGGCGAATACTATACTATGCCCCATCAACTGGGGGCCAAGGGGCAGAG (Sequence ID 73), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions, GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTG GGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAAACTACGCACAGAACTTCCAGGGCAGAGTCACCATGACCG CAGACACCTCCATAAGCACAGCCTACATGGAGCTGAGCCAGCCTGAGAGTGAACAGGCCCGTGTATATTACTGTGCGAGAATCGTTGGGTGGGAGAGATTTCCGCTACTGGGGGCCAGGGAACCCTG (Sequence ID 41), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and / or a sequence having 10 to 20 nucleic acid substitutions, or (vii) CAGGTACAGCTGCAGCAGTCAGGTCCAGGGCTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTATCTCTAGTAACAGTGTTGCTTGG AACTGGATCAGGCAGTCCCCATCGAGAGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTACAGTGATTATGCAATATCTGTGAAAGGTCGATTAGACATCCAAC CCAGACACATCCCAAGAACCACAGTTTCCCCTGCACAGCTGAACTCTGTGACTCCCCGAGGACACGGCTGTGTTATTATTTGTGCCAAGAGACAGTAAGTTGAGGGCCAAGCTATGAATGCCTTTTTGAATCTGGGGGCCAAGAGGAATG (Sequence ID 45), or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or a sequence having 10 to 20 nucleic acid substitutions. A cell according to any one of the above claims, comprising a heavy chain variable domain encoded by a nucleotide sequence containing

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

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

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

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

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

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

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

20. The aforementioned cells, i. Secrete an inducible bispecific T cell engager that binds to GPC3 and targets EpCAM and CD3 (GECAR-BiTE T); or ii. It binds to CD19 and secretes inducible bispecific T cell engagers that target EpCAM and CD3 (CD19 CAR-BiTE T). The cell according to any one of the above claims.

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

22. A vector expressing the polynucleotide described in claim 21.

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

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

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

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

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