WO2024253595A1 - Protéine de liaison à un antigène multispécifique contre epcam - Google Patents

Protéine de liaison à un antigène multispécifique contre epcam Download PDF

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WO2024253595A1
WO2024253595A1 PCT/SG2024/050383 SG2024050383W WO2024253595A1 WO 2024253595 A1 WO2024253595 A1 WO 2024253595A1 SG 2024050383 W SG2024050383 W SG 2024050383W WO 2024253595 A1 WO2024253595 A1 WO 2024253595A1
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sequence
seq
identity
cdr
chain variable
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Cheng-I Wang
Bei Wang
Zi Xian Eve NGOH
Siti Nazihah MOHD SALLEH
Yee Chin Yvonne YEAP
Mun Kuen SOH
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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Priority to KR1020267000266A priority Critical patent/KR20260032536A/ko
Priority to AU2024284754A priority patent/AU2024284754A1/en
Priority to CN202480051122.5A priority patent/CN121666399A/zh
Publication of WO2024253595A1 publication Critical patent/WO2024253595A1/fr
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    • A61K40/41—Vertebrate antigens
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    • A61K40/42—Cancer antigens
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    • A61K40/421—Immunoglobulin superfamily
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    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates broadly to antigen binding proteins, variants and fragments thereof specific to Epithelial Cellular Adhesion Molecule (EpCAM).
  • EpCAM Epithelial Cellular Adhesion Molecule
  • the present disclosure encompasses the nucleotide and amino-acid sequences of 6 human antibodies, 5 llama antibodies and 1 humanized llama antibody, or the antigen-binding portions thereof, that specifically target human EpCAM, both in solution and on the surface of cells.
  • multispecific antigen binding proteins such as bispecific T cell engagers (BiTEs), which bind to EpCAM and also other targets.
  • Advanced therapeutics such as antibody-based or cell-based immunotherapies, offers a new paradigm in oncology, and hold immense potential for more effective treatments for cancers compared to conventional treatment strategies.
  • antibody and cell-based therapies are increasingly gaining immense attention and investments for its development as a potential more promising standard of care for the treatment of cancer.
  • BiTE bi-specific T-cell engager
  • ADC antibody-drug conjugates
  • cancer targeted therapies rarely cure patients with solid tumors.
  • Cell-based therapy such as chimeric antigen receptor (CAR) T achieves great success in haematological malignancy but shows limited efficacy in treating solid tumors.
  • EpCAM Epithelial cell adhesion molecule
  • the presently disclosed fully human anti-EpCAM antibodies (1 B5, 1C1 , t d f , 1 D4, 1 E4 and 1 H6). despite their relatively low binding affinity, exhibited potent cytotoxic activity when used in a BiTE format to mediate target specific cell killing. Applications using the BiTE format can therefore overcome issues of immunogenicity and high toxicity/low tolerability brought by high-affinity non-human anti-EpCAM antibodies as shown by most prior arts.
  • the nanobody format of BiTE have superior advantages: i) high stability; ii) large production yield; and iii) small gene size to be packaged in the lentivirus system to achieve high T cell transduction efficiency
  • the present inventors have also generated anti-EpCAM nanobodies of llama-derived single-domain antibody fragments (VHH) against EpCAM, which can be fused to many different anti-CD3 T cell agonist antibodies via a flexible linker.
  • VHH llama-derived single-domain antibody fragments
  • These BiTE antibodies can be used for targeting EpCAM locally.
  • CAR- T cells secreting anti-EpCAM BiTE the present inventors could achieve not only the specific tumor antigen directed T cell expansion but also tumor restricted expression of BiTE targeting a more widely expressed tumor antigen EpCAM to avoid toxicity brought by the traditional systemic delivery.
  • a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a heavy chain variable region comprising: a CDR-H1 comprising GDSISSNSVA (SEQ ID NO: 5) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising TYYRSKWYS (SEQ ID NO: 6) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a light chain variable region comprising: a CDR-L1 comprising:
  • QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto,
  • QSLLHSNRYNY (SEQ ID NO: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or
  • CDR-L2 • QSISDF (SEQ ID NO: 19) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto a CDR-L2 comprising:
  • LGS SEQ ID NO: 10
  • LGS SEQ ID NO: 10
  • AAS SEQ ID NO: 20
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto and a CDR-L3 comprising:
  • MQALQTPYT SEQ ID NO: 11
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto SEQ ID NO: 11
  • MQGLQSPWT SEQ ID NO: 15
  • SEQ ID NO: 15 a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • QQSYIMPDT SEQ ID NO: 21 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or
  • MQGLQTPYT SEQ ID NO: 23
  • a heavy chain variable region comprising: a CDR-H1 comprising:
  • GSIFSGND SEQ ID NO: 25
  • GSIFSGND SEQ ID NO: 25
  • GSSERFTS SEQ ID NO: 29
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto a CDR-H2 comprising:
  • ITSGGST SEQ ID NO: 26
  • ITSGGST SEQ ID NO: 26
  • ITNGGST SEQ ID NO: 30
  • TNGRWSGDTYYAHH SEQ ID NO: 27
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto SEQ ID NO: 27
  • MAGTS SEQ ID NO: 31
  • MAGTS SEQ ID NO: 31
  • TNGRWSGDTYYAHL SEQ ID NO: 33
  • the antigen binding protein, variant or fragment thereof of comprises a heavy chain variable region selected from the group consisting of:
  • a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a heavy chain variable region comprising: a CDR-H1 comprising GDSISSNSVA (SEQ ID NO: 5) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising TYYRSKWYS (SEQ ID NO: 6) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and comprises a light chain variable region comprising: a CDR-L1 comprising:
  • QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto,
  • QSLLHSNRYNY (SEQ ID NO: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or
  • CDR-L2 • QSISDF (SEQ ID NO: 19) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto a CDR-L2 comprising:
  • LGS SEQ ID NO: 10
  • LGS SEQ ID NO: 10
  • AAS SEQ ID NO: 20
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto and a CDR-L3 comprising:
  • MQALQTPYT SEQ ID NO: 11
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto SEQ ID NO: 11
  • MQGLQSPWT SEQ ID NO: 15
  • SEQ ID NO: 15 a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • QQSYIMPDT SEQ ID NO: 21 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or
  • MQGLQTPYT SEQ ID NO: 23
  • the antigen binding protein, variant or binding fragment thereof comprises a heavy chain variable region comprising: a CDR-H1 comprising:
  • GSIFSGND SEQ ID NO: 25
  • GSIFSGND SEQ ID NO: 25
  • GSSERFTS SEQ ID NO: 29
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto a CDR-H2 comprising:
  • ITSGGST SEQ ID NO: 26
  • ITSGGST SEQ ID NO: 26
  • ITNGGST SEQ ID NO: 30
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto and a CDR-H3 comprising:
  • TNGRWSGDTYYAHH SEQ ID NO: 27
  • a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto SEQ ID NO: 27
  • MAGTS SEQ ID NO: 31
  • MAGTS SEQ ID NO: 31
  • TNGRWSGDTYYAHL SEQ ID NO: 33
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable region and/or a light chain variable region selected from the group consisting of:
  • a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a heavy chain variable region comprising: a CDR-H1 comprising GDSISSNSVA (SEQ ID NO: 5) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising TYYRSKWYS (SEQ ID NO: 6) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-L3 comprising MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-L3 comprising MQGLQSPWT (SEQ ID NO: 15) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a light chain variable region comprising: a CDR-L1 comprising QSLLHSNRYNY (SEQ ID NO: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-L3 comprising MQALQTPYT (SEQ ID NO: 11) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a light chain variable region comprising: a CDR-L1 comprising QSISDF (SEQ ID NO: 19) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-L2 comprising AAS (SEQ ID NO: 20) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-L3 comprising QQSYIMPDT (SEQ ID NO: 21) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-L2 comprising LGS (SEQ ID NO: 10) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-L3 comprising MQGLQTPYT (SEQ ID NO: 23) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a heavy chain variable region comprising: a CDR-H1 comprising GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a heavy chain variable region comprising: a CDR-H1 comprising GSSERFTS (SEQ ID NO: 29) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising ITNGGST (SEQ ID NO: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising MAGTS (SEQ ID NO: 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and
  • a heavy chain variable region comprising: a CDR-H1 comprising GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising TNGRWSGDTYYAHL (SEQ ID NO: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto,
  • a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto,
  • a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and a light chain variable region comprising: a CDR-L1 comprising QSLLHSNRYNY (SEQ ID NO: 17) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a heavy chain variable region comprising: a CDR-H1 comprising GGTFSSYA (SEQ ID NO: 1) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising IIPIFGTA (SEQ ID NO: 2) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising ARSLGGRFRY (SEQ ID NO: 3) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and a light chain variable region comprising: a CDR-L1 comprising QSLLHSNGYNY (SEQ ID NO: 9) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto,
  • a heavy chain variable region comprising: a CDR-H1 comprising GDSISSNSVA (SEQ ID NO: 5) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising TYYRSKWYS (SEQ ID NO: 6) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising AREVEGSSYDAFDI (SEQ ID NO: 7) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and a light chain variable region comprising: a CDR-L1 comprising QSISDF (SEQ ID NO: 19) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity there
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable region selected from the group consisting of:
  • a heavy chain variable region comprising: a CDR-H1 comprising GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising TNGRWSGDTYYAHH (SEQ ID NO: 27) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto
  • a heavy chain variable region comprising: a CDR-H1 comprising GSSERFTS (SEQ ID NO: 29) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising ITNGGST (SEQ ID NO: 30) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising MAGTS (SEQ ID NO: 31) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; and
  • a heavy chain variable region comprising: a CDR-H1 comprising GSIFSGND (SEQ ID NO: 25) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, a CDR-H2 comprising ITSGGST (SEQ ID NO: 26) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a CDR-H3 comprising TNGRWSGDTYYAHL (SEQ ID NO: 33) or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable domain and/or a light chain variable domain selected from the group consisting of:
  • a light chain variable domain comprising DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQ SPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM QALQTPYTFGQGTK (SEQ ID NO: 12) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • a light chain variable domain comprising EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQS PQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ GLQSPWTFGQGTK (SEQ ID NO: 16) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • a light chain variable domain comprising DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQ SPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM QALQTPYTFGQGTK (SEQ ID NO: 18) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIY AASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDT FGQGTK (SEQ ID NO: 22) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • a light chain variable domain comprising DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQ SPQLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCM
  • QGLQTPYTFGQGTK (SEQ ID NO: 24 ) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • a heavy chain variable domain comprising QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERE LVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYY CTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 28) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • a heavy chain variable domain comprising QVQLQESGGGLVQPGGSLRLSGAASGSSERFTSVAWYRQAPGKERE LVAFITNGGSTRYTDPVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYY CMAGTSWGQGTQ (SEQ ID NO: 32) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • (x) a heavy chain variable domain comprising QVQLQESGGGLVQPGGSLRLSCADSGSIFSGNDMAWYRRAPGVERE LVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYY CTNGRWSGDTYYAHLWGQGTQ (SEQ ID NO: 34) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • a heavy chain variable domain comprising QVQLQESGGGLVQAGGSLRLSCADSGSIFSGNDMAWYRRAPGVERE LVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYY CTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • a heavy chain variable domain comprising QVQLQESGGGLVQAGDSLRLSCADSGSIFSGNDMAWYRRAPGVERE LVAVITSGGSTHYADSVKGRFTISRDNAQKTVYLQTNDLKPEDTAVYY CTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 36) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions; and a heavy chain variable domain comprising QVQLVESGGGLVQAGGSLRLSCAASGSIFSGNDMSWYRQAPGKGLE LVAVITSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CTNGRWSGDTYYAHHWGQGTL (SEQ ID NO: 37 ) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable domain and a light chain variable domain selected from the group consisting of:
  • EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARS LGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions, and a light chain variable domain comprising DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSP QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQT PYTFGQGTK (SEQ ID NO: 12) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino
  • a heavy chain variable domain comprising: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARS LGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions, and a light chain variable domain comprising
  • EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQSP WTFGQGTK (SEQ ID NO: 16) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions
  • DVVMTQSPLSLPVTPGESASISCRSSQSLLHSNRYNYLDWYLQKPGQSP QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQT PYTFGQGTK (SEQ ID NO: 18) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions (iv) a heavy chain variable domain comprising:
  • DIQLTQSPSSLSASVGDRVTITCRASQSISDFLNWYQQKPGKAPKLLIYA ASSLQTGVPSRFGGSGSGTEFTLTISSLQPEDLGTYYCQQSYIMPDTFG QGTK (SEQ ID NO: 22) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions, or
  • a heavy chain variable domain comprising: EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTANYAQNFQGRVTMTADTSISTAYMELSSLRSEDTAVYYCARS LGGRFRYWGQGTL (SEQ ID NO: 4) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions, and a light chain variable domain
  • DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSP QLLIYLGSNRASGVPDRFSGSGSGTDFTLQISRVEAEDAGVYYCMQGLQT PYTFGQGTK (SEQ ID NO: 24) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions.
  • the antigen binding protein, variant or fragment thereof comprises a single domain heavy chain variable domain having a sequence:
  • CTNGRWSGDTYYAHHWGQGTQ (SEQ ID NO: 35) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions, or
  • the antigen binding protein, variant or fragment thereof comprises a light chain constant domain having a sequence:
  • RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVAEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO: 13) or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions, or
  • RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFSRGEC SEQ ID NO: 14 or a fragment, variant or sequence thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having two or three amino acids substitutions.
  • the antigen binding protein, variant or fragment thereof is an IgG antibody, in particular an lgG1 antibody.
  • the antigen binding protein, variant or fragment thereof is a multi-specific antigen binding protein, variant or fragment thereof, such as a bispecific antibody.
  • the multi-specific antigen binding protein, variant or fragment thereof binds to an immune marker selected from the group consisting of CD3, NKG2D, CD4, CD8, CD16 and CD64.
  • the antigen binding protein, variant or fragment thereof is a bispecific T cell engager (BiTE).
  • the bispecific T cell engager comprises an anti- EpCAM Heavy chain antibody variable region (i.e. VHH) or a single chain variable fragment (scFv).
  • VHH EpCAM Heavy chain antibody variable region
  • scFv single chain variable fragment
  • the antigen binding protein, variant or fragment thereof comprises a Fc region.
  • the heavy chain variable domain is encoded by a nucleotide sequence comprising:
  • CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCC AGAAGACCGTATATCTGCAAACGAACGACCTGAAACCTGAGGACA CGGCCGTGTATTACTGCACAAACGGAAGATGGTCAGGCGATACTT ACTATGCCCATCTCTGGGGCCAGGGGACCCAG (SEQ ID NO: 71) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having 10-20 nucleic acid substitutions,
  • TACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGG ACCAAG SEQ ID NO: 49 or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having 10-20 nucleic acid substitutions, or
  • TACTGCATGCAAGCTCTACAAACTCCGTACACTTTTGGCCAGGGG ACCAAG SEQ ID NO: 55 or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having 10-20 nucleic acid substitutions, or
  • the heavy chain and light chain variable domains are encoded by nucleotide sequences selected from the group consisting of:
  • CTACTGGGGCCAGGGAACCCTG (SEQ ID NO: 41 ) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having 10-20 nucleic acid substitutions, and a light chain variable domain encoded by the nucleotide sequence comprising:
  • nucleotide sequence comprising:
  • GACCAAG (SEQ ID NO: 61) or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto and/or having I D- 20 nucleic acid substitutions;
  • nucleotide sequence comprising:
  • the single domain heavy chain variable domain is encoded by a nucleotide sequence comprising
  • the light chain constant domain is encoded by a nucleotide sequence comprising
  • a vector expressing the polynucleotide as described above.
  • a host cell comprising the vector as described above.
  • a cell expressing/secreting the antigen binding protein, variant of fragment thereof as described above.
  • a cell expressing/secreting an immune cell engager which is specific to EpCAM which is specific to EpCAM.
  • the immune cell engager is selected from the group comprising a T cell engager, an NK cell engager, a monocyte engager and a macrophage engager.
  • the immune cell engager is a bispecific T cell engager (BITE), such as an inducible BiTE, non-inducible BiTE or a constitutive expression BiTE comprising the antigen binding protein, variant of fragment thereof as described above.
  • BITE bispecific T cell engager
  • the cell is an immune cell, for example selected from the group comprising a T cell, a macrophage, a monocyte and an NK cell.
  • the immune cell is a T-cell, in particular a CAR T-cell.
  • the cell is a stem cell, for example selected from the group comprising a mesenchymal stem cell, a neural stem cell and a pluripotent stem cell, such as an induced pluripotent stem cell (iPSC).
  • a stem cell for example selected from the group comprising a mesenchymal stem cell, a neural stem cell and a pluripotent stem cell, such as an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • composition comprising the antigen binding protein, variant or fragment thereof and/or cell as described above.
  • an antigen binding protein, variant or fragment thereof, cell or a composition as described above for use in treatment of a disease is provided.
  • a method of treating a disease in a subject in need thereof comprising administering to the subject an antigen binding protein, variant or fragment thereof, cell or a composition as described above.
  • the antigen binding protein, variant or fragment thereof, cell or the composition as described above in the manufacture of a medicament for preventing and/or treating a disease.
  • the disease is a proliferative disease such as a tumor or cancer.
  • a method of diagnosis / determining the prognosis or presence of a solid tumor originating from epithelium comprising detecting the expression of high EpCAM on the solid tumor using the antigen binding protein, variant or fragment thereof or a composition as described above.
  • antigen binding protein herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies as long as they exhibit the desired antigen-binding activity.
  • antibody as used herein relates to whole (i.e., full length) antibodies (i.e., comprising the elements of two heavy chains and two light chains) and functionally active fragments thereof (i.e., molecules that contain an antigen binding domain that specifically binds an antigen, also termed antibody fragments or antigen-binding fragments).
  • functionally active fragments thereof i.e., molecules that contain an antigen binding domain that specifically binds an antigen, also termed antibody fragments or antigen-binding fragments.
  • antibody encompasses monovalent, i.e., antibodies comprising only one antigen binding domain (e.g., one- armed antibodies comprising a full-length heavy chain and a full-length light chain interconnected, also termed “half-antibody”), and multivalent antibodies, i.e., antibodies comprising more than one antigen binding domain, e.g., bivalent.
  • antigen binding fragment refers to functionally active antibody binding fragments including but not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies, scFv, Fv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above.
  • binding fragment refers to a fragment capable of binding a target peptide or antigen with sufficient affinity to characterize the fragment as specific for the peptide or antigen.
  • mAb refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. each individual of a monoclonal antibody preparation are identical except for possible mutations (e.g., naturally occurring mutations), that may be present in minor amounts. Certain differences in the protein sequences linked to post-translational modifications (for example, cleavage of the heavy chain C-terminal lysine, deamidation of asparagine residues and/or isomerization of aspartate residues) may nevertheless exist between the various different antibody molecules present in the composition. Contrary to polyclonal antibody preparations, each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • diabody refers to two Fv pairs, a first VH/VL pair and a further VH/VL pair which have two inter-Fv linkers, such that the VH of a first Fv is linked to the VL of the second Fv and the VL of the first Fv is linked to the VH of the second Fv.
  • trimer (also referred to a Fab(scFv)2) as employed herein refers to a Fab fragment with a first scFv appended to the C-terminal of the light chain and a second scFv appended to the C-terminal of the heavy chain.
  • tetrabody refers to a format similar to the diabody comprising fours Fvs and four inter-Fv linkers.
  • multivalent antibody refers to an antibody comprising more than one antigen binding domain e.g., bivalent.
  • Fv refers to two variable domains of full-length antibodies, for example co-operative variable domains, such as a cognate pair or affinity matured variable domains, i.e., a VH and VL pair.
  • scFv refers to single chain variable fragment which is a fusion protein of the variable regions of the heavy and light chains of the immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.
  • bis-scFv as described herein refers to a bispecific scFv.
  • dsscFv or “disulphide-stabilised single chain variable fragment” as employed herein refers to a single chain variable fragment which is stabilised by a peptide linker between the VH and VL variable domain and also includes an inter-domain disulphide bond between VH and VL.
  • DVD-lg also known as dual V domain IgG refers to a full-length antibody with 4 additional variable domains, one on the N-terminus of each heavy and each light chain.
  • Fab refers to as used herein refers to an antibody fragment comprising a light chain fragment comprising a VL (variable light) domain and a constant domain of a light chain (CL), and a VH (variable heavy) domain and a first constant domain (CHI) of a heavy chain.
  • Dimers of a Fab’ according to the present disclosure create a F(ab’)2 where, for example, dimerization may be through the hinge.
  • F(ab’) refers to a monovalent fragment of a single light chain homodimer, which is obtained by pepsin digestion of IgG, followed by reduction of the light chain disulfide bond.
  • F(ab')2 refers to a fragment of IgG that is prepared by pepsin digestion of IgG.
  • the F(ab’)2 fragment is a disulfide-linked homodimer of the two light chain dimers, so it retains bivalent epitope binding like whole IgG, but as it lacks the heavy chains, it is smaller in size compared to a whole IgG.
  • F(ab’)2 and F(ab’) fragments do not bind to immunoglobulin receptors on cells, which can be useful for achieving specific staining of the primary antibody target.
  • DiFab refers to two Fab molecules linked via their C-terminus of the heavy chains or two Fab’ molecules linked via one or more disulfide bonds in the hinge region thereof.
  • an antibody refers to a single domain antibody (sdAb), with an antibody fragment consisting of a single monomeric variable antibody domain.
  • antibody binding variant refers to a polypeptide, for example, an antibody possessing the desired characteristics described herein and comprising a VH and/or a VL that has at least about 80% amino acid sequence identity with a VH and/or a VL of the reference antibody.
  • antibody variants include, for instance, antibodies wherein one or more amino acid residues are added to or deleted from the VH and/or a VL domain.
  • an antibody variant will have at least about 80% amino acid sequence identity, alternatively at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to an antibody described herein.
  • variant antibodies will have no more than one conservative amino acid substitution as compared to an antibody sequence provided herein, alternatively no more than about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to an antibody sequence provided herein.
  • epitopes or “binding site” in the context of antibodies refers to a site (or a part) on an antigen to which the paratope of an antibody binds or recognizes.
  • Epitopes can be formed both from contiguous amino acids (also often called “linear epitopes”) or noncontiguous amino acids formed by tertiary folding of a protein (often called “conformational epitopes”). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least
  • Epitopes usually consist of chemically active surface groups of molecules such as amino acids, sugar side chains and usually have specific 3D structural and charge characteristics.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
  • chimeric antibody is an antibody molecule (or antigen-binding fragment thereof) in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e. g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a mouse antibody can be modified by replacing its constant region with the constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in human as compared to the original mouse antibody.
  • chimeric antigen receptors refers to receptor protein that has been engineered to give T cells the new ability to target a specific antigen.
  • the receptors are chimeric in that they combine both antigen-binding and T cell activating functions into a single receptor.
  • CAR T cell therapy uses T cells engineered with CARs to treat cancer.
  • T cells in CAR T immunotherapy are modified to recognize cancer cells in order to more effectively target and destroy them.
  • CAR T cells can be derived either from T cells in a patient's own blood (autologously) or from the T cells of another, healthy, donor (allogeneically). Once isolated from a person, these T cells are genetically engineered to express a specific CAR, which programs them to target an antigen that is present on the surface of tumors.
  • CAR T cells are engineered to be specific to an antigen that is expressed on a tumor but is not expressed on healthy cells.
  • CAR T cells destroy cells through extensive stimulated cell proliferation, increasing the degree to which they are toxic to other living cells (cytotoxicity) and by causing the increased secretion of factors that can affect other cells such as cytokines, interleukins and growth factors.
  • the surface of CAR T cells can bear either two types of co-receptors, CD4 and CD8, each with different and interacting cytotoxic effects.
  • human antibody or “humanized antibody” (or antigen-binding fragment thereof), as used herein, is intended to include antibodies (and antigen-binding fragments thereof) having variable regions in which both the framework and CDR regions are derived from sequences of human origin.
  • antibodies or immunoglobulins are divided into the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (subtypes), e.g. lgG1 , lgG2, lgG3, and lgG4, lgA1 , and lgA2.
  • human IgG constant region domains may be used, especially of the lgG1 and lgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required.
  • lgG2 and lgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required.
  • the constant region also is derived from such human sequences.
  • a humanized antibody (or antigen-binding fragment thereof) retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts (i.
  • the humanized antibodies of the present disclosure may include amino acid residues not encoded by human sequences (e. g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing). This definition of a humanized antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries, administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e. g., immunized xenomice via a human B-cell hybridoma technology.
  • recombinant humanized antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell transformed to express the humanized antibody, e. g., from a transfectoma, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences.
  • isolated means, throughout this specification, that the antibody, or polynucleotide, as the case may be, exists in a physical milieu distinct from that in which it may occur in nature.
  • isolated nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or that has been synthetically created.
  • An isolated nucleic acid may comprise synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof.
  • An isolated antibody refers to an antibody that is substantially free of other cellular material and/or chemicals.
  • CDRs Complementarity Determining Regions
  • Kabat i.e., "Kabat” numbering scheme
  • Al-Lazikani Chothia
  • ImMunoGenTics IMGT numbering scheme
  • CDRs Complementarity Determining Regions
  • Antibodies typically comprise six CDRs: three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
  • sequence identity refers to the percentage sequence identities that are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
  • “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1 ) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • non-conservative substitutions are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1 ) to (6) shown above.
  • affinity refers to the strength of all noncovalent interactions between an antibody thereof and the target protein.
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule for its binding partner can be generally represented by the dissociation constant (Ko). Affinity can be measured by common methods known in the art, including those described herein.
  • KD refers to the constant of dissociation which is obtained from the ratio of K rf to K a (i.e. K rf / K a ) and is expressed as a molar concentration (M).
  • Kd and K a refer to the dissociation rate and association rate, respectively, of a particular antigen-antibody interaction.
  • K D values for antibodies can be determined using methods well established in the art.
  • the term “low affinity” refers to KD of 100 nM or more.
  • moderate affinity refers to KD ranging from 10nM to 100nM.
  • high affinity refers to KD of 1 to 10nM.
  • very high affinity refers to KD of 1 nM or less.
  • EC50 refers to the concentration of an antibody or an antigen-binding protein/portion thereof, which induces a response, either in an in vivo or an in vitro assay, which is 50% of the maximal response (i.e., halfway between the maximal response and the baseline).
  • multi-specific antibody refers to an antibody as described herein which has at least two binding domains, i.e. two or more binding domains, for example two or three binding domains, wherein the at least two binding domains independently bind two different antigens or too different epitopes on the same antigen.
  • Multi-specific antibodies are generally monovalent for each specificity (antigen).
  • Multi-specific antibodies described herein encompass monovalent and multivalent, e.g. bivalent, tri valent, tetravalent multi-specific antibodies.
  • bispecific or “bispecific antibody” as employed herein refers to an antibody with two antigen specificities or an antibody that has the ability to simultaneously bind to two target antigens/sites.
  • a “bispecific T cell engager” refers to a class of artificial bispecific monoclonal antibodies that direct a host’s immune system, such as the T cells’ cytotoxic activity against target cells (such as cancer cells).
  • BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kDa.
  • One of the scFvs binds to an immune cell (such as a T cell via the CD3 receptor), and the other to target of interest (e.g., a tumor cell via a tumor specific molecule).
  • BiTEs form a link between an immune cell (e.g., a T cell) and a target cell (such as a tumor cell). This causes the immune cell (e.g., T cell) to exert cytotoxic activity on tumor cells. For example, if the immune cell is a T cell, the T cell would exert cytotoxic activity by producing proteins like perforin and granzymes that enter tumor cells and initiate the cell’s apoptosis.
  • immune cell refers to a type of specialized cell that plays a crucial role in the body's defense against infections and foreign substances. They are a part of the immune system, which is responsible for identifying and eliminating harmful pathogens, such as bacteria, viruses, and parasites, as well as abnormal or cancerous cells.
  • an ‘immune cell’ refers to any cell of the immune system, including but not limited to T-cells, helper T-cells, B-cells, natural killer (NK) cells, dendritic cells (DC), granulocytes (such as basophils, eosinophils, neutrophils), mast cells, monocytes, and macrophages.
  • a “nanobody with a heavy chain only” refers to nanobody-based heavy chain antibody.
  • a heavy-chain antibody is an antibody which consists of two heavy chains and lacks the two light chains usually found in antibodies.
  • a "vector” is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where e.g., synthesis of the encoded polypeptide can take place.
  • a vector is a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g., a nucleic acid of the present disclosure).
  • Expression vectors typically contain one or more of the following components (if they are not already provided by the nucleic acid molecules): a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • Vectors are typically selected to be functional in the host cell in which the vector will be used (the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur.
  • the vector as described herein may be an expression vector and/or a cloning vector.
  • host cell is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • treating refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition, which includes but is not limited to diseases, symptoms and disorders.
  • a medical condition also includes a body’s response to a disease or disorder, e.g. inflammation.
  • Those in need of such treatment include those already with a medical condition as well as those prone to getting the medical condition or those in whom a medical condition is to be prevented.
  • subject as used herein includes patients and non-patients.
  • patient refers to individuals suffering or are likely to suffer from a medical condition
  • non-patients refer to individuals not suffering and are likely to not suffer from the medical condition.
  • Non-patients include healthy individuals, non-diseased individuals and/or an individual free from the medical condition.
  • subject includes humans and animals. Animals may include, but is not limited to, mammals (for example nonhuman primates, canine, murine and the like), and the like.
  • “Murine” refers to any mammal from the family Muridae and / or Leporidae, such as mouse, rat, rabbit, and the like.
  • preventing and/or “reducing the severity of symptoms” as used herein refers to process of delaying the onset, reducing the severity of symptoms, reducing and/or preventing weight loss, preventing death, inhibiting deterioration, inhibiting further deterioration, and/or ameliorating at least one sign or symptom of a disease.
  • the word “substantially” whenever used is understood to include, but not restricted to, “entirely” or “completely” and the like.
  • terms such as “comprising”, “comprise”, and the like whenever used are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited.
  • reference to a “one” feature is also intended to be a reference to “at least one” of that feature.
  • Terms such as “consisting”, “consist”, and the like may in the appropriate context, be considered as a subset of terms such as “comprising”, “comprise”, and the like.
  • the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1 .00% to 5.00% and also 1 .0% to 5.0% and all their intermediate values (such as 1 .01%, 1 .02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1 .2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth/breadth of a range.
  • At least 95% identical as employed herein is intended to refer to an amino acid sequence which over its full length is 95% identical or more to a reference sequence, such as 96, 97, 98 or 99% identical. Software programmes can be employed to calculate percentage identity.
  • the disclosure may have disclosed a method and/or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.
  • an antigen binding protein an antigen binding variant, and/or an antigen binding fragment thereof that binds specifically to Epithelial Cellular Adhesion Molecule (EpCAM).
  • EpCAM Epithelial Cellular Adhesion Molecule
  • the antigen binding protein, variant or fragment thereof is a monoclonal antibody.
  • the antigen binding protein or variant thereof is a full length antibody.
  • the antigen binding fragment thereof is selected from the group comprising: an Fab, a modified Fab, an Fab', a modified Fab', an F(ab')2, an Fv, a single domain antibody, a VHH, an scFv, an Fv, an bivalent antibody, a trivalent antibody, a tetra-valent antibody, a Bis-scFv, a diabody, a triabody, a tetrabody, an epitope-binding fragment, and the like.
  • the antigen binding protein, variant or fragment thereof is an IgG, IgA, IgD, IgE, or IgM antibody.
  • the antigen binding protein, variant or fragment thereof is a chimeric antibody, a human antibody / humanized antibody, recombinant humanized antibody, an animal-derived antibody (such as llama antibody) or the like.
  • the antigen binding protein, variant or fragment thereof is a human antibody. In one embodiment, the antigen binding protein, variant or fragment thereof is a llama antibody. In one embodiment, the antigen binding protein, variant or fragment thereof is a humanized llama antibody.
  • the antigen binding protein, variant or fragment thereof comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 37.
  • the antigen binding protein, variant or fragment thereof is encoded by one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 38 to 74.
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable region and a light chain variable region encoded by nucleic acid sequences selected from the group consisting of:
  • a heavy chain variable region comprising: (1 B6 and 1 C1 ) a CDR-H1 comprising GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and a CDR-H3 comprising GCGAGATCGTTGGGTGGGAGATTTCGCTAC (SEQ ID NO: 40); and a light chain variable region comprising: a CDR-L1 comprising
  • CAGAGCCTGCTGCATAGTAATGGATACAACTAT (SEQ ID NO: 46)
  • CDR-L2 comprising TTGGGTTCT
  • CDR-L3 comprising ATGCAAGCTCTACAAACTCCGTACACT (SEQ ID NO: 48)
  • a heavy chain variable region comprising: (1 C11 ) a CDR-H1 comprising GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 39), and a CDR-H3 comprising GCGAGATCGTTGGGTGGGAGATTTCGCTAC (SEQ ID NO: 40); and a light chain variable region comprising: a CDR-L1 comprising
  • CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO: 46)
  • CDR-L2 comprising TTGGGTTCT
  • CDR-L3 comprising ATGCAAGGTCTACAAAGTCCCTGGACG (SEQ ID NO: 52)
  • a heavy chain variable region comprising: (1 D4) a CDR-H1 comprising GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 38), a CDR-H2 comprising ATCATCCCTATCTTTGGTACAGCA (SEQ ID NO: 38), a CDR-H2 comprising
  • CAGAGCCTCCTGCATAGTAATAGATACAACTAT (SEQ ID NO: 54), a CDR-L2 comprising TTGGGTTCT (SEQ ID NO: 47), and a CDR-L3 comprising ATGCAAGCTCTACAAACTCGGTACACT (SEQ ID NO: 54), and a CDR-L3 comprising ATGCAAGCTCTACAAACTCGGTACACT (SEQ ID NO: 54).
  • a heavy chain variable region comprising: (1 H6) a CDR-H1 comprising GGAGGCACCTTCAGCAGCTATGCT (SEQ ID NO:
  • CAGAGCCTCCTGCATAGTAATGGATACAACTAT (SEQ ID NO: 46)
  • CDR-L2 comprising TTGGGTTCT
  • CDR-L3 comprising ATGCAAGGTCTACAGACTCCGTACACT
  • a heavy chain variable region comprising: (1 E4) a CDR-H1 comprising GGGGACAGTATCTCTAGTAACAGTGTTGCT
  • a light chain variable region comprising: (1 E4) a CDR-L1 comprising CAGAGTATTAGCGACTTT (SEQ ID NO: 56), a CDR-L2 comprising GCTGCATCG (SEQ ID NO: 57), and a CDR-L3 comprising TTACATTATGCCCGACACT (SEQ ID NO: 58) or fragment or variation or sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable region and a light chain variable region encoded by nucleic acid sequences selected from the group consisting of:
  • a heavy chain variable region comprising: (1 A5-VHH, 2C4-VHH, 2D10-VHH and hu2C4-VHH) a CDR-H1 comprising GGAAGCATCTTCAGTGGCAATGAC (SEQ ID NO:
  • a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO:
  • a heavy chain variable region comprising: (1 B8-VHH) a CDR-H1 comprising GGAAGCTCCGAAAGATTCACATCA (SEQ ID NO:
  • a CDR-H2 comprising ATTACTAATGGTGGTAGCACA (SEQ ID NO: 66), a CDR-H2 comprising ATTACTAATGGTGGTAGCACA (SEQ ID NO: 66), a CDR-H2 comprising ATTACTAATGGTGGTAGCACA (SEQ ID NO: 66), a CDR-H2 comprising ATTACTAATGGTGGTAGCACA (SEQ ID NO: 66), a CDR-H2 comprising ATTACTAATGGTGGTAGCACA (SEQ ID NO:
  • a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO: 62), a CDR-H2 comprising ATTACTAGCGGTGGTAGTACA (SEQ ID NO:
  • ID NO: 70 or fragment or variation or sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.
  • the antigen binding protein, variant or fragment thereof comprises a heavy chain variable domain encoded by the nucleotide sequence comprising
  • the antigen binding protein, variant or fragment thereof comprises a light chain variable domain encoded by the nucleotide sequence comprising
  • AGGCTGAGGATGCTGGGGTTTATTACTGCATGCAAGGTCTACAGACTCCG TACACTTTTGGCCAGGGGACCAAG (SEQ ID NO: 61 - clone 1 H6), or a sequence at least 60% identical thereto and/or having 10-20 nucleic acid substitutions.
  • the antigen binding protein, variant or fragment thereof comprises a single domain heavy chain variable domain encoded by the nucleotide sequence comprising:
  • the antigen binding protein, variant or fragment thereof comprises a light chain constant domain encoded by the nucleotide sequence comprising
  • the antigen binding protein is an antibody. In some embodiments, the antigen binding protein may include an isolated antibody.
  • the antigen binding protein comprises a sequence that is at least 60% identical to any one of the sequences disclosed herein.
  • the antigen binding protein may comprise a sequence that is at least about 60%, at least about 61 %, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71 %, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 9
  • the antigen binding protein comprises a sequence or an amino acid region or is encoded by a nucleotide region that differs by about one, about two, about three, about four, about five, about six, about seven, about eight, about nine, about ten or more amino acids or nucleobase with the sequence as disclosed herein.
  • the antigen binding protein comprises an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, the antigen binding protein comprises an amino acid sequence having one, or two, or three, or four, or five, or six, or seen, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to any one of the sequences disclosed herein. In some examples, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
  • the substitution replaces the amino acids with natural amino acids, such as, but is not limited to, alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the substitution does not include replacement with cysteine.
  • the substitution replaces the amino acids with natural amino acids, such as, but is not limited to, alanine, arginine, asparagine, aspartate, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • natural amino acids such as, but is not limited to, alanine, arginine, asparagine, aspartate, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the substitution may be a conservative substitution that substitute one for another of similar properties. For example, substitution of one amino acid with another from the same group. In various embodiments, the substitution may include substitution with an amino acid with different properties. In various embodiments, the substitution is made without affecting the biological activity of the antigen binding protein, variant or fragment thereof as described herein. In various embodiments, the substitution increases binding affinity to EpCAM.
  • the substitutions may also include non-classical amino acids.
  • non-classical amino acids include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine p-alanine, GABA and 5-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4- diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalan
  • the amino acid mutation may be in the CDRs of the antigen binding protein (e.g., the CDR1 , CDR2 or CDR3 regions).
  • amino acid alteration may be in the framework regions (FRs) of the antigen binding protein (e.g., the FR1 , FR2, FR3, or FR4 regions).
  • Modification of the amino acid sequences may be achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis.
  • the mutations do not substantially reduce the antigen binding protein’s capability to specifically bind to a target. In some examples, the mutations do not substantially reduce the antigen binding protein’s capability to specifically bind to a target and without functionally modulating (e.g., partially or fully neutralizing) the target.
  • the antigen binding protein, variant or fragment thereof binds to EpCAM with low affinity, moderate affinity or high affinity.
  • the antigen binding protein, variant or fragment thereof binds to EpCAM with low to moderate affinity (KD ranging from 1 -100nM).
  • the antigen binding protein, variant or fragment thereof binds with moderate affinity KD ranging from 10-100nM.
  • the antigen binding protein, variant or fragment thereof binds to EpCAM with low affinity.
  • the binding affinity of the antigen binding protein of the disclosure for the full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or monomeric and/or dimeric forms and/or any other naturally occurring or synthetic analogs, variants, or mutants (including monomeric and/or dimer forms) of antigen binding protein may be described by the equilibrium dissociation constant (K D ).
  • the antigen binding protein binds to the full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or any other naturally occurring or synthetic analogs, variants, or mutants (including monomeric and/or dimeric forms) of antigen binding protein with a K D of less than about 1
  • binding affinities of the presently disclosed antigen binding proteins/fragments thereof were measured via Bio-layer Interferometry (BLI) analysis (results shown in Example 4).
  • BLI Bio-layer Interferometry
  • the antigen binding protein may be a nanobody.
  • the bispecific T cell engager (BiTE) is a nanobody with a heavy chain only (VHH).
  • the second antigen targeted by the bispecific T cell engager is selected from the group comprising CD3, NKG2D, CD28, CD16, and the like.
  • the CD3 may include CD3 , CD3s, CD3y, CD36, and the like.
  • 1 C1 , 1 C11 , 1 D4 and 1 H6) were able to kill 70-100% of HT-29 cells within 72 hours at a concentration as low as 0.1 nM (FIG. 4B).
  • 1 C1 and 1 H6 BiTE antibodies showed the lowest EC50 values of 18.5 pM and 12.02 pM, respectively; while 1 B6, 1C1 1 , 1 D4, 1 E4 showed low to moderate cell killing efficacy (FIG. 4C).
  • the five disclosed llama anti-EpCAM BiTE clones (1A5-VHH, 1 B8-VHH, 2B7-VHH, 2C4-VHH and 2D10- VHH) show comparable or even lower EC50 values as the benchmark known in the art anti-EpCAM BiTE “MT110” in binding to both mobilized biotinylated EpCAM antigen (FIG. 6B) and EpCAM expressing cells (FIG. 6F), suggesting high binding affinities to the antigen.
  • the four clones (1A5-VHH, 2B7-VHH, 2C4-VHH and 2D10-VHH) all bound strongly to EpCAM expressing cell HT-29 as well as human T cells (CD3 positive) (FIG. 6E).
  • the binding EC50 to HT-29 cells range from 10-20 pM and binding specificity was superior as none of them showed binding to EpCAM negative, CD negative HeyA8 cells even at the highest concentration of 10nM (FIG. 6D).
  • the four anti-EpCAM VHH BiTE antibodies (1A5-VHH, 2B7-VHH, 2C4-VHH and 2D10-VHH) were able to kill 90-100% of EpCAM positive HT-29 (FIG. 7A), HepG2 (FIG. 7B), and Hep3B (FIG. 7C) within 96 hours at concentrations as low as 10 pM but spared EpCAM negative cell HeyA8.
  • the antigen binding fragments of VHH antibodies are advantageously much smaller and have a higher tissue penetration than conventional antibodies, and possess superior properties including high solubility, stability and resistance to heat-denaturation.
  • the immunoglobulin fragment is an IgG Fc fragment.
  • the sequence encoding the immune cell engager encodes for a multi-specific antigen binding protein.
  • the multi-specific antigen binding protein is a bi-specific antibody.
  • the sequence encoding the immune cell engager encodes for an antigen binding protein capable of binding EpCAM (anti-EpCAM antigen binding protein), or fragment, or variant thereof, and an anti- immune cell antigen binding protein.
  • the sequence encoding the immune cell engager encodes for a single chain variable fragment (scFv) or a single variable domain located on a heavy chain (VHH).
  • the sequence encoding the immune cell engager encodes for an anti-EpCAM scFv or an anti-EpCAM VHH.
  • the sequence encoding the immune cell engager encodes for an anti-immune cell antigen binding protein that binds to an immune cell activation marker.
  • the immune cell activation marker may include but is not limited to CD3, NKG2D, CD4, CD8, CD16, CD64, and the like.
  • the immune activation marker may be in VHH form or scFv form.
  • the sequence encoding the immune cell engager encodes for an antigen binding protein capable of binding CD3 (anti-CD3 antigen binding protein), or fragment, or variant thereof.
  • the sequence encoding the immune cell engager encodes for a single chain variable fragment (scFv) or a VHH form.
  • the sequence encoding the immune cell engager encodes for an anti-CD3 scFv or an anti-CD3 single domain VHH.
  • the immune cell engager may include a His-tag.
  • the immune cell engager comprises an anti-EpCAM antigen binding protein, a linker, an anti-CD3 scFv or an anti-CD3 single domain VHH, and a His- tag.
  • the linker is a cleavable linker, which may include but is not limited to, P2A, T2A, F2A, and the like.
  • the polynucleotide comprises the sequence encoding an immune cell engager is a sequence encoding a bispecific T cell engager (BiTE). In some examples, the polynucleotide comprises the sequence encoding BiTE that bi-specifically binds to EpCAM and a T cell. In some examples, the polynucleotide comprises the sequence encoding for an anti-GPC3 scFv CAR with CD3 intracellular domain, and the sequence encoding for a BiTE that binds to EpCAM and a T cell.
  • the polynucleotide comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 38 to 74.
  • the polynucleotide sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences as disclosed herein.
  • the vector is an expression vector.
  • the vector is selected from the group consisting of a plasmid, a viral particle, a phage, a baculovirus, a yeast plasmid, a lipid based vehicle, a polymer microsphere, a liposome, and a cell based vehicle, a colloidal gold particle, lipopolysaccharide, polypeptide, polysaccharide, a viral vehicle, an adenovirus, a retrovirus, a lentivirus, an adeno-associated viruses, a herpesvirus, a vaccinia virus, a foamy virus, a cytomegalovirus, a Semliki forest virus, a poxvirus, a pseudorabies virus, an RNA virus vector, a DNA virus vector and a vector derived from a combination of a plasmid and a phage DNA, further optionally wherein said polynucleotide is operatively linked to an expression control sequence(s) to direct peptid
  • the vector is a lentiviral vector.
  • the host cell comprises cloning or expression vectors as described above and/or nucleic acid sequences encoding for the antigen binding protein, antibodies and binding fragments thereof as described above.
  • the host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell, or a vertebrate cell).
  • the host cell when cultured under appropriate conditions, expresses an antibody or binding fragment thereof which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). Selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity, such as glycosylation or phosphorylation, and ease of folding into a biologically active molecule.
  • the host cell may comprise a bacterial cell, a yeast cell, an animal cell e.g., a mammalian cell and/or a plant cell.
  • Suitable mammalian host cells include CHO, myeloma or hybridoma cells. Many are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61 ), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), 3T3 cells (ATCC No. CCL92), or PER.C6 cells. Other cell types of use in expressing antibodies include lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells.
  • lymphocytic cell lines e.g., NSO myeloma cells and SP2 cells, COS cells.
  • the host cell expresses/secretes the antigen binding protein, variant or fragment thereof as disclosed herein.
  • a cell that expresses/secretes an immune cell engager that is specific to EpCAM.
  • the cell is a stem cell, for example selected from the group comprising a mesenchymal stem cell, neural stem cell and a pluripotent stem cell, such as an induced pluripotent stem cell (IPSC).
  • a mesenchymal stem cell a mesenchymal stem cell.
  • the stem cell is a neural stem cell.
  • the stem cell is a pluripotent stem cell, such as an iPSC.
  • the cell is an immune cell.
  • the immune cell expresses/secretes an immune cell engager which is specific to EpCAM.
  • the immune cell engager is selected from the group comprising a T cell engager, an NK cell engager, a monocyte engager and a macrophage engager.
  • the immune cell expresses/secretes a bispecific T cell engager (BITE) which is specific to EpCAM.
  • BITE bispecific T cell engager
  • the immune cell expresses/secretes a bispecific T cell engager (BiTE), such as an inducible, non-inducible or constitutive expression BiTE comprising the antigen binding protein, variant or fragment thereof as disclosed herein.
  • BiTE bispecific T cell engager
  • the present inventors have established that immune cells, such as CAR T-cells, are able to secrete EpCAM BiTEs. See Examples 5 to 7. It was not previously known that this was possible.
  • immune cells such as CAR T-cells
  • this allows the EpCAM immune engagers to be secreted at the target site (for example at the site of a solid tumour), thereby minimising toxicity and/or side effects.
  • the immune cell may include but is not limited to a macrophage, a dendritic cell, a T cell, a B cell, an eosinophil, a basophil, a neutrophil, a mast cell, a natural killer T cell (NKT cell), natural killer cell (NK cell), a macrophage, a monocyte, and the like.
  • the immune cell is a NK cell.
  • the immune cell is a macrophage.
  • the immune cell is a dendritic cell.
  • the immune cell is a monocyte.
  • the immune cell is a T-cell.
  • the immune cell is a CAR T-cell, such as an anti-GPC3, anti-HER2 or anti-CD19 CAR T-cell.
  • the CAR T-cell is an anti-GPC3 CAR T-cell.
  • the CAR T-cell is an anti-HER2 CAR T-cell.
  • the CAR T-cell is an anti- CD19 CAR T-cell.
  • the immune cell is a CAR T-, CAR NK-, CAR macrophage-, or CAR monocyte-cell,
  • the immune cell may bind to more than one host cell antigen. Therefore, in some examples, the immune cell may further bind to one host cell antigen, two host cell antigens, three host cell antigens, four host cell antigens, and the like.
  • the anti-EpCAM BiTEs exert their cytotoxicity by recruiting the immune cells (such as T cells) nearby.
  • immune cells such as T cells
  • bystander T cells are physically directed to the close proximity of tumors and at the same time being activated and help the clearance of tumor cells.
  • CAR T cells By applying local secretion of anti-EpCAM BiTE by CAR T cells, it converts a non-druggable target into a druggable target as it diminishes the on-target, off-tumor toxicity that might be brought by systematic delivery.
  • EpCAM is also defined as a cancer stem cell marker expressed on cancer progenitor cells and cancer stem cells
  • secretion of anti-EpCAM BiTE by CAR T cells will concomitantly contribute to the prevention of cancer relapse and recurrence by eliminating cancer stem cells and progenitor cells.
  • the immune cell expresses an inducible bispecific T cell engager (BITE) comprising a heavy chain variable region that comprises the heavy chain complementarity determining region:
  • BITE bispecific T cell engager
  • the immune cell expresses an inducible bispecific T cell engager (BiTE) that further binds to an immune cell activator.
  • BiTE bispecific T cell engager
  • the immune cell expresses an inducible bispecific T cell engager (BiTE) that further binds to CD3.
  • BiTE bispecific T cell engager
  • polypeptide comprising a multispecific antigen binding protein.
  • the multispecific antigen binding protein is a bi-specific antibody.
  • the polypeptide comprises a multispecific antigen binding protein that binds to EpCAM (epithelial cell adhesion molecule) and an immune cell.
  • the multispecific antigen binding protein is a bispecific immune cell engager that is capable of engaging both an antigen and an immune cell.
  • the polypeptide comprises an anti-EpCAM antigen binding protein. In some examples, the polypeptide comprises a single domain anti-EpCAM antibody, optionally an anti-EpCAM H-chain antibody variable region (i.e., VHH).
  • VHH anti-EpCAM H-chain antibody variable region
  • the polypeptide comprises an anti-immune cell antigen binding protein. In some examples, the polypeptide comprises an anti-immune cell antigen binding protein that binds to an immune cell activation marker. In some examples, the immune cell activation marker is CD3, NKG2D, CD4, CD8, CD16, CD64, and the like. In some examples, the polypeptide binds to CD3. In some examples, the CD3 may include CD3 , CD3E, CD3y, CD36, and the like.
  • the polypeptide comprises an anti-CD3 antigen binding protein. In some examples, the polypeptide comprises a single-chain variable fragment of an anti-CD3 antibody (anti-CD3 scFv).
  • the polypeptide is a bispecific antibody/antigen binding protein.
  • the bispecific antibody/antigen binding protein is a bispecific T cell engager (BITE).
  • the bispecific T cell engager (BiTE) binds to two antigens, wherein the first antigen is EpCAM and wherein the second antigen is an immune cell marker.
  • the second antigen is an immune cell marker that is involved in the activation of the immune cell.
  • the second antigen targeted by the bispecific T cell engager (BiTE) may include but is not limited to CD3, NKG2D, CD28, CD16, CD64, and the like.
  • the polypeptide comprises a BiTE that bi-specifically binds to EpCAM and a T cell. In some examples, the polypeptide comprises a BiTE that bi-specifically binds to EpCAM and CD3. In some examples, the EpCAM targeted by the BiTE is modified with an anti-EpCAM VHH paired with an anti-CD3 scFv.
  • the anti-EpCAM VHH pairs with an anti-CD3 scFv that may include a clone Okt3 (NbO1 -O13A), or an anti-CD3 clone used by another anti-EpCAM in the art (MT110)(Nb01-013B).
  • an anti-CD3 scFv that may include a clone Okt3 (NbO1 -O13A), or an anti-CD3 clone used by another anti-EpCAM in the art (MT110)(Nb01-013B).
  • 21 clones showing the positive Fab supernatant binding signals to biotinylated hEpCAM-His protein and specific binding to EpCAM positive HT-29 cells were sequence and 6 unique sequences were identified: 1 B6, 1C1 , 1 C11 , 1 D4, 1 E4 and 1 H6.
  • Clones 1 B6, 1 C1 , 1C11 , 1 D4 and 1 H6 share the same sequence in the heavy chain variable region but differ in the light chain sequences of the variable region, except for 1 B6 and 1 C1 that share the same sequences in the light chain variable region but with three amino acid differences in the kappa light chain constant region.
  • the disclosed anti-EpCAM human Fab I VHH clones are used to construct anti-EpCAM BiTE antibodies.
  • a method of producing / generating the antigen binding protein, variant or fragment thereof as disclosed herein comprising expressing the polynucleotide as described herein in a host cell.
  • CDRs are in bold (CDR1 ) ;in bold and in italics (CDR2). or in bold, in italics and underlined (CDR3 ⁇ . Highlighted residues/bases show differences between the 1 B6 and 1 C1 light chain constant domains.
  • Figure 1 shows the flow cytometry analysis of the surface expression of human EpCAM protein on multiple cells lines with epithelial morphology (HT-29, AGS, HepG2, Hep3B, MCF-7) and non-epithelial morphology (HeyA8, A172, U-87) and human T cells isolated from healthy donor blood tissue.
  • epithelial morphology HT-29, AGS, HepG2, Hep3B, MCF-7
  • HeyA8, A172, U-87 human T cells isolated from healthy donor blood tissue.
  • a commercially available mouse anti-human EpCAM monoclonal antibody directly conjugated to BV421 fluorescent dye was used.
  • Figure 2A shows the binding ELISA assay results of 6 Fab clones to the antigen protein.
  • the 6 Fab clones were tested using crude culture supernatants in a binding ELISA assay to assess antigen binding to biotinylated human EpCAM with a poly-His tag, detected by goat-anti-human Fab-HRP.
  • Figure 2B shows the flow cytometry analysis of the 6 Fab clones in their binding to cell surface expressed EpCAM. Using crude culture supernatants, the 6 Fab clones were tested on their binding to EpCAM positive cell HT-29 and EpCAM negative human T cells. In both assays, supernatant from a culture medium alone well (“Blank”) was used as background control.
  • Figure 2C shows the binding affinity results of the lgG1 antibodies.
  • Six anti- EpCAM Fab clones were cloned and expressed as lgG1 antibodies and their binding affinity to the biotinylated human EpCAM protein antigen were tested using affinity binding ELISA and compared to the benchmark IgG (the anti-EpCAM arm of “MT110”).
  • Antigen binding EC50 values of anti-EpCAM IgG 1 antibodies were calculated by PRISM.
  • Figure 3A shows the structural format of anti-EpCAM BiTE antibodies.
  • Figure 3B shows the binding kinetics of anti-EpCAM BiTE antibodies for biotinylated human EpCAM protein antigen using binding ELISA.
  • Figure 3C shows the results of the flow cytometry analysis at different concentrations for the anti-EpCAM BiTE antibodies to EpCAM positive HT-29 cells
  • Figure 3D shows the results of the flow cytometry analysis at different concentrations for the anti-EpCAM BiTE antibodies to EpCAM negative HeyA8 cells
  • Figure 3E shows the results of the flow cytometry analysis at different concentrations for the anti-
  • EpCAM BiTE antibodies to CD3 positive human T cells The percentages of cells positively stained by anti-EpCAM BiTE antibodies are plotted.
  • Figure 3F shows the binding of Benchmark BiTE antibody (“MT1 10”) to HT-29 and HeyA8 cells.
  • Figure 3G shows the binding EC50 values of anti-EpCAM BiTE antibodies to HT- 29 cells as calculated by PRISM, with top and bottom constraints set at 100% and 0% respectively.
  • Figure 4A shows the xCelligence impedance assay results for the anti-EpCAM BiTE antibodies. Cytolysis of EpCAM positive HT-29 cells was mediated by activated human T cells with time-course measurement using xCelligence impedance assay, upon treatment with 6 different anti-EpCAM BiTE antibodies (clones 1 B6, 1C1 , 1C11 , 1 D4, 1 E4 and 1 H6).
  • Figure 4B shows the Mean % cytolysis ⁇ SD in duplicate wells at different concentrations at 72 hours.
  • Figure 4C shows the EC50 values of anti-EpCAM BiTE antibodies in killing of HT-29 cells as calculated by PRISM, with top and bottom constraints set at 100% and 0% respectively.
  • Figure 5A shows the binding ELISA results of 5 llama VHH clones to the antigen protein.
  • the 5 llama VHH clones were tested using crude culture supernatants in a binding ELISA assay to assess their antigen binding to biotinylated human EpCAM with a poly-His tag, and detected by HRP-conjugated anti-HA.1 1 epitope tag antibody.
  • Figure 5B shows the flow cytometry analysis of the 5 llama VHH clones in their binding to cell surface expressed EpCAM. Using crude culture supernatants, the 5 VHH clones were tested for binding to EpCAM positive cell AGS. In both assays, supernatant from a culture medium alone well (“Blank”) was used as background control.
  • Figure 6A shows the structural format of anti-EpCAM VHH BiTE antibodies.
  • Figure 6B binding kinetics of anti-EpCAM VHH BiTE antibodies for biotinylated human EpCAM protein antigen using binding ELISA.
  • Figure 6C shows the results of the flow cytometry analysis at different concentrations for the anti-EpCAM VHH BiTE antibodies to EpCAM positive HT-29 cells
  • Figure 6D shows the results of the flow cytometry analysis at different concentrations for the anti-EpCAM VHH BiTE antibodies to EpCAM negative HeyA8 cells
  • Figure 6E shows the results of the flow cytometry analysis at different concentrations for the anti-EpCAM VHH BiTE antibodies to CD3 positive human T cells.
  • the percentages of cells positively stained by anti-EpCAM VHH BiTE antibodies were plotted using PRISM software.
  • Figure 6F shows the binding EC50 values of anti-EpCAM VHH BiTE antibodies to HT-29 cells as calculated by PRISM, with top and bottom constraints set at 100% and 0% respectively. All anti-EpCAM VHH BiTE antibodies used the same anti-CD3 clone as used in the benchmark antibody (“MT 110”).
  • FIG. 7A shows the cytolysis of EpCAM positive HT-29 cells
  • Figure 7B shows the cytolysis of EpCAM positive HepG2 cells
  • Figure 7C shows the cytolysis of EpCAM positive Hep3B cells
  • Figure 7D shows the cytolysis of EpCAM negative HeyA8 cells.
  • Cytolysis of the cells was mediated by activated human T cells with time-course measurement using xCelligence impedance assay, upon treatment with 5 different anti- EpCAM VHH BiTE antibodies (clones 1 A5-VHH, 1 B8-VHH, 2B7-VHH, 2C4-VHH, and 2D10-VHH) with the E:T ratio of 4:1.
  • Data was presented as % cytolysis at different concentrations at either 96 hours ( Figures 7A -C) or 72 hours ( Figure 7D).
  • Figure 7E shows the EC50 values of anti-EpCAM VHH BiTE antibodies in killing of different cells at either 96 hours (96h) or 72 hours (72h) calculated by PRISM, with top and bottom constraints set at 100% and 0% respectively. All anti-EpCAM VHH BiTE antibodies used the same anti-CD3 clone as used in the benchmark antibody (“MT 110”).
  • Figure 8A shows the binding kinetics of llama derived anti-EpCAM bi-specific T- cell engagers using 2C4-VHH and its humanized clone hu2C4-VHH paired with anti-CD3 clone Okt3.
  • Anti-EpCAM 2C4-VHH/Okt3, hu2C4-VHH/Okt3 BiTE antibodies were tested for their binding to biotinylated human EpCAM protein antigen using binding ELISA and the antigen binding EC50 values were tabulated.
  • Figure 8B shows the binding of the anti-EpCAM 2C4-VHH/Okt3, hu2C4- VHH/Okt3 BiTE antibodies tested by flow cytometry analysis at different concentrations to EpCAM positive HT-29 cells
  • Figure 8C shows the binding of the anti-EpCAM 2C4- VHH/Okt3, hu2C4-VHH/Okt3 BiTE antibodies to EpCAM negative, CDS negative HeyA8 cells
  • Figure 8D shows the binding of the anti-EpCAM 2C4-VHH/Okt3, hu2C4- VHH/Okt3 BiTE antibodies to CD3 positive human T cells. The percentage of cells positively stained by anti-EpCAM BiTE antibodies were plotted.
  • Figure 8E shows the binding EC50 values of anti-EpCAM 2C4-VHH/Okt3 and hu2C4-VHH/Okt3 BiTE antibodies to HT-29 cells as calculated by PRISM, with top and bottom constraints set at 100% and 0% respectively.
  • Figure 9A shows the T cell mediated cytotoxicity of EpCAM positive cells by anti- EpCAM bi-specific T-cell engagers using 2C4-VHH and its humanized clone hu2C4-VHH paired with anti-CD3 clone Okt3
  • Figure 9B shows the T cell mediated cytotoxicity of EpCAM negative cells by anti-EpCAM bi-specific T-cell engagers using 2C4-VHH and its humanized clone hu2C4-VHH paired with anti-CD3 clone Okt3.
  • Figure 9C shows the EC50 values of the benchmark BiTE, 2C4-VHH/Okt3 and hu2C4-VHH/Okt3 BiTE antibodies in killing MCF-7 cells at 48 hours as calculated by PRISM, with top and bottom constraints set at 100% and 0% respectively.
  • Figure 9D shows the ELISA measurement of interferon-y secretion from human T cells activated by different anti-EpCAM BiTE antibodies after 48 hours of co-culture with either EpCAM positive MCF-7 cells or EpCAM negative HeyA8 cells
  • Figure 9E shows the ELISA measurement of IL-2 secretion from human T cells activated by different anti-EpCAM BiTE antibodies after 48 hours of co-culture with either EpCAM positive MCF-7 cells or EpCAM negative HeyA8 cells.
  • Figure 10A shows structural formats of anti-GPC3 CAR T (5C4) or GE CAR- BiTE T cells (anti-GPC3 CAR T secreting anti-EpCAM BiTE, “MT110”, “NbO1 -O13A” or “Nb01 -013B” was used as BiTE) or 19E CAR-BiTE T cells (anti-CD19 CAR T secreting anti-EpCAM BiTE, “Nb01-013A” or “Nb01 -013B” was used as BiTE).
  • Figure 10B shows the results of an in vitro assay of Hep3B (GPC3 High , EpCAM High ) cells by anti-GPC3 CAR T, GE CAR-BiTE T cells or 19E CAR-BiTE T cells.
  • Figure 10C shows the results of an in vitro assay of HT-29 (GPC3 Low , EpCAM High ) cells by anti-GPC3 CAR T, GE CAR-BiTE T cells or 19E CAR-BiTE T cells.
  • Figure 10D shows the shows the results of an in vitro assay of HeyA8 (GPC3 ve , EpCAM ue ) cells by anti- GPC3 CAR T, GE CAR-BiTE T cells or 19E CAR-BiTE T cells.
  • Figure 11 A shows the structure map of HE CAR-BITE T (clone 4D5 or F5) using anti-EpCAM VHH BITE (Nb01-013A).
  • FIG 11 B shows the FACS analysis results of tumor markers (HER2 and EpCAM) expressed on AGS and MDA-MB468 cells.
  • Figure 11C shows the percentage of CAR expression on anti-HER2 CAR T (4D5 or F5) and HE CAR- BITE T (4D5 or F5) cells detected by flow cytometry analysis.
  • Figure 11 F shows the ELISA measurement of Interferon-r and IL-2 measured using culture supernatant collected at 36 hours post co-culture effector CAR T cells with AGS cells.
  • Figure 11G shows the ELISA measurement of Interferon-r and IL-2 measured using culture supernatant collected at 36 hours post co-culture effector CAR T cells with MDA-MB468 cells.
  • Figure 12 shows a graph demonstrating that the anti-EpCAM BITE secreted from GE CAR-BITE T cells can mediate strong T cell killing of cancer cells.
  • Figure 13 shows a graph demonstrating the detection of anti-EpCAM BITE molecule Nb01 -013A secreted from GE CAR-BITE T cells by ELISA.
  • Anti-GPC3 CAR T (5C4) cells or anti-GPC3 CAR T cells secreting anti-EpCAM BITE (Nb01 -013A) (named as “GE CAR-BITE T (Nb01 -013A)”) were cultured in T cell growth medium containing IL- 7 (20 ng/ml) and IL-15 (5 ng/ml). The starting cell density was 0.5 million per ml and the percentages of CAR expression in both cultures were around 60%.
  • Cell culture supernatants were collected daily (24h, 48h, 72h, 96h) for an ELISA to detect the amount of secreted anti-EpCAM BITE molecule NbO1-O13A.
  • human EpCAM-Fc tag protein was used to coat the ELISA plate overnight, after blocking with Casein for 2 hours, the culture supernatants containing the anti-EpCAM BiTE molecule NbO1 -O13A were added to the plate. After 1 -hour incubation, the plate was washed and the bound BiTE molecules were detected by an HRP conjugated secondary antibody against the His-tag.
  • Figure 14 shows a graph demonstrating the detection of anti-EpCAM BiTE molecule Nb01 -013A secreted from HE CAR-BITE T cells by ELISA.
  • Anti-HER2 CAR T (4D5 or F5) cells or anti-HER2 CAR T cells secreting anti-EpCAM BiTE (Nb01 -013A) (named as “HE CAR-BiTE T (4D5 or F5)”) were cultured in T cell growth medium containing IL-7 (20 ng/ml) and IL-15 (5 ng/ml). The starting cell density was 1.5 million per ml and the percentages of CAR expression ranged from 36.8% to 57.2%.
  • Cell culture supernatants were collected at 24h and 48h for an ELISA to detect the amount of secreted anti-EpCAM BiTE molecule NbO1 -O13A.
  • human EpCAM-Fc tag protein was used to coat the ELISA plate overnight, after blocking with Casein for 2 hours, the culture supernatants containing the anti-EpCAM BiTE molecule NbO1 -O13A were added to the plate. After 1 -hour incubation, the plate was washed and the bound BiTE molecules were detected by an HRP conjugated secondary antibody against the His-tag.
  • Figure 15 shows a graph demonstrating that human GE CAR-BiTE T cells using anti-EpCAM VHH constructed BiTEs showed superior tumor killing in in vivo Hep3B xenografts.
  • Two million Hep3B (GPC3 High , EpCAM High ) cells were subcutaneously injected into the right flank of 50 male NSG mice (Day -21 ).
  • mice were regrouped according to the measurable tumor size and eight million CAR T (5C4) cells, GE CAR-BiTE T cells (Anti-EpCAM BiTE secreting anti-GPC3 CAR T cells, “MT110”, “Nb01 -013A” or “NbO1 -O13B” was used as BiTE), 19E CAR-BiTE T cells (Anti-EpCAM BiTE secreting anti-CD19 CAR T cells, “NbO1-O13A” or “NbO1-O13B” was used as BiTE), or Mock T cells were intravenously injected into these mice via tail vein. Tumor sizes of each mouse were measured and recorded every 3 to 7 days. The curve shows tumor sizes up to Day 92.
  • Figure 16 shows a graph demonstrating that human HE CAR-BiTE T cells using anti-EpCAM VHH constructed BiTEs showed superior tumor killing in in vivo AGS xenografts.
  • 1.3 million AGS (Her2 H ' ah , EpCAM High ) cells were subcutaneously injected into the right flank of female NSG mice (Day -9).
  • mice were re-grouped according to the measurable tumor size and five million CAR T (4D5) cells, CAR T (F5) cells, HE CAR-BiTE T (4D5) cells (“NbO1 -O13A” was used as BiTE), HE CAR-BiTE T (F5) cells (“Nb01 -013A” was used as BiTE), or Mock T cells were intravenously injected into these mice via tail vein. Tumor sizes of each mouse were measured and recorded every 3 to 7 days.
  • Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications may be made without deviating from the scope of the invention.
  • Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.
  • Example 1 Discovery of fully human anti-EpCAM antibodies from naive human Fab phage display library
  • EpCAM had high level of expression on all cells tested with epithelial morphology such as a lung adenocarcinoma cell line HT-29, a gastric adenocarcinoma cell line AGS, a hepatoblastoma cell line HepG2, a hepatocellular carcinoma cell line Hep3B, and a breast cancer cell line MCF-7.
  • EpCAM was not expressed on non-epithelial cells, such as human T cells, an ovarian cell line HeyA8, and two glioblastoma cell lines A172 and U-87.
  • biotinylated recombinant human EpCAM protein with a poly-His tag (hEpCAM-His) was used to isolate low-affinity EpCAM binders from a naive library of Fab sequences constructed in SlgN, using phage display technology.
  • hEpCAM-His biotinylated recombinant human EpCAM protein with a poly-His tag
  • Clone 1 B6, 1 C1 , 1 C11 , 1 D4 and 1 H6 share the same sequence in the heavy chain variable regions, however the light chain sequences of the variable region are different, except for clone 1 B6 and 1 C1 which also share the same sequences in their light chain variable region but bearing three amino acid differences in the kappa light chain constant region (see Annex).
  • the results of binding ELISA and flow cytometry analysis of cell binding using Fab supernatants for the 6 unique clones were shown in Figure 2A and Figure 2B, respectively.
  • these Fab antibodies were cloned into lgG1 format and their binding affinity to the antigen protein were determined by affinity binding ELISA (Figure 2C).
  • binding EC50 values ranged between 3 to 6 nM, which are higher than the binding EC50 of the benchmark IgG (1 .161 nM, the sequence of the anti-EpCAM arm of “MT110” was used), suggesting that these anti-EpCAM antibodies derived from the human naive Fab library had a relatively low binding affinity to the EpCAM antigen.
  • the anti-EpCAM antibodies were used to construct anti-EpCAM bi-specific T-cell engagers (BiTE).
  • the Fab region of one arm from each of the 6 anti- EpCAM human antibody clones (1 B6, 1C1 , 1011 , 1 D4, 1 E4 and 1 H6) was replaced by an anti-CD3 scFv fragment, and knob-in-hole mutations were introduced to facilitate correct heavy-chain pairing and LALA mutations to remove Fey receptor binding (Figure 3A).
  • These anti-EpCAM BiTE antibodies were first examined for their binding to the antigen protein by affinity binding ELISA.
  • BiTE anti-EpCAM arm of “MT110” with scFv fragment of anti-CD3 clone Okt3, constructed in the same structure as shown in Figure 3A
  • BiTE antibodies using the 6 newly identified anti-EpCAM clones showed much lower absorbance reading at 450nm and higher EC50 values, suggesting lower binding affinities to the antigen (Figure 3B).
  • results from the above assays, depicted in Figures 1 - 4, showed that the disclosed highly specific human anti-EpCAM antibodies in the format of bispecific T cell engagers could elicit a wide range of low to high potency in inducing cell death of EpCAM expressing cells, which have the potential to be used in treating solid tumors with enhanced safety profiles (based on in vitro binding and killing assay results).
  • the present inventors immunized a male alpaca with recombinant human EpCAM protein with a poly-His tag (hEpCAM- His) for a total of six immunizations with time intervals of 1 -2 weeks.
  • the process of llama immunization was done by VIB Nanobody Core at VUB (Vrije Universiteit Brussel) as a paid service.
  • Subsequent humanization of the llama antibodies was performed at SlgN. Around 150 pg of proteins was used for the two initial immunizations and 100 pg of proteins were used for the last four booster immunizations.
  • peripheral blood lymphocytes were collected from the alpaca for RNA extraction, and cDNA was used as the PCR template to amplify VHH sequences for a VHH library to be constructed.
  • Biotinylated recombinant human EpCAM protein with a poly-His tag (hEpCAM-His) was used to isolate EpCAM binders from the constructed llama VHH library, using phage display technology.
  • VHH, 2B7-VHH, 2C4-VHH and 2D10-VHH were able to kill 90-100% of EpCAM positive HT-29 (Figure 7A), HepG2 (Figure 7B), and Hep3B (Figure 7C) cells within 96 hours at concentrations as low as 10 pM, but spared EpCAM negative cell HeyA8 ( Figure 7D).
  • Figure 7E Among the 5 clones, clone 2B7-VHH, 2C4-VHH and 2D10-VHH all showed very potent cell killing efficacy (Figure 7E), while 2C4-VHH and 2D10-VHH exhibited better killing specificity than 2B7-VHH ( Figure 7D).
  • 2C4-VHH and 2D10-VHH shared same CDR sequences but the framework of 2C4-VHH was closer to human VH sequences, 2C4-VHH was chosen as the lead VHH clone for further development.
  • a humanized clone hu2C4-VHH was constructed and its antigen binding ability was compared with the parental 2C4-VHH clone using BITE antibodies paired with a well-characterized anti-CD3 agonist Okt3. Humanization resulted in 10 times reduction in the binding affinity to the antigen protein (Figure 8A) and about 2.5 times weaker cell binding (Figure 8B and Figure 8E).
  • Example 4 Experiments determine binding affinity of anti-EpCAM human lgG1 antibodies and anti-EpCAM llama VHH antibodies
  • Binding affinities of 6 anti-EpCAM IgGi antibodies was measured via Bio-Layer Interferometry (BLI) analysis using Octet RED96 system and shown in Table 1. All 6 antibody clones exhibited binding affinities to the recombinant EpCAM protein at the nanomolar scales ranging from the lowest affinity at 32.39 nM (clone 1 H6) to the highest affinity at 1.432 nM (clone 1 D11 ).
  • Binding affinities of 5 anti-EpCAM VHH antibodies was measured via Bio-Layer Interferometry (BLI) analysis using Octet RED96 system and shown in Table 2. All 5 VHH antibody clones were expressed as bivalent Fc fusion protein for the assay and exhibited binding affinities to the recombinant EpCAM protein at the nanomolar scales ranging from the lowest affinity at 9.715 nM (clone 2C4-VHH) to the highest affinity at 1.975 nM (clone 1 B8-VHH).
  • Example 5 Experiments demonstrating that human CAR T cells secreting anti- EpCAM BITE molecules showed in v/fro cytotoxicity in killing EpCAM expressing cells.
  • the anti-EpCAM bi-specific T-cell engagers using 2C4-VHH paired with either the anti-CD3 clone Okt3 (named as “Nb01 -013A”) or the anti-CD3 clone used in “MT110” (named as “Nb01 -013B”) were used to construct secreted forms of anti-EpCAM BITE with a His-tag.
  • These anti-EpCAM BiTEs were inserted into different CAR constructs against different tumor antigens, for example, GPC3, CD19 or HER2.
  • anti-EpCAM BITE molecules can be secreted by human T cells and contribute to the cytotoxicity of EpCAM expressing target cell killing.
  • the present inventors constructed anti-GPC3 CAR T cells secreting different anti-EpCAM BiTEs (“MT110”, “Nb01-013A”, and “Nb01 -013B”) and named them as GE CAR-BiTE T cells.
  • the present inventors also constructed anti-CD19 CAR T cells secreting different anti- EpCAM BiTEs (“Nb01 -013A” or “Nb01 -013B”), which were named as “19E CAR-BiTE T” cells ( Figure 10A).
  • the present inventors also constructed HE CAR-BITE T cells by fusing the anti- EpCAM BiTE (sequence of “Nb01 -013A” was used) to the anti-HER2 CAR (clone 4D5 from “Trastuzumab” or our in-house anti-HER2 clone F5) lentiviral construct via a P2A cleavable linker ( Figure 11A).
  • both AGS (HER2 High , EpCAM High ) and MDA-MB468 (HER2 ve , EpCAM High ) cells were used to test the in vitro killing efficacy of anti-HER2 CAR T (4D5 or F5) and HE CAR-BiTE T (4D5 or F5) cells.
  • the HE CAR-BiTE T cells were shown to be able to kill AGS cells at a much faster rate than the anti-HER2 CAR alone ( Figure 11 D), suggesting the participation of the BiTE in target cell killing.
  • clone 4D5 constructed anti-HER2 CAR T cells could not initiate efficient killing of AGS cells ( Figure 11 D) with low level of Interferon-y secretion ( Figure 11 F), suggesting that the cytotoxic function of the HE CAR-BiTE T cells (clone 4D5) was mainly contributed by the secreted anti-EpCAM BiTE. More interestingly, anti-HER2 CAR T cells (clone 4D5) showed comparable level of killing of HER2 negative MDA-MB468 cells, while the anti- HER2 CAR T cells constructed using our patented clone F5 showed high specificity by sparing the HER2 negative cells ( Figure 11 E and 11 G).
  • Example 6 Experiments showing that anti-EpCAM BiTE secreted by CAR T cells can elicit strong T cell mediated killing of cancer cells
  • the time-course measurement of BiTE elicited T cell-mediated cytotoxicity was performed by xCelligence impedance assay.
  • Example 7 Experiments demonstrating that secretion of anti-EpCAM llama VHH BITE from different CAR-BITE T cells can be detected from the in vitro cultures
  • Both anti-GPC3 CAR T (5C4) and GE CAR-BiTE T (Nb01 -013A) cells were cultured in T cell growth medium containing IL-7 and IL-15 at the starting cell density of 0.5 million per ml. Cell culture supernatants were collected daily for a consecutive four days, followed by an ELISA to detect the amount of secreted anti-EpCAM BITE molecule Nb01 -013A. The results clearly showed that the amount of secreted NbO1-O13A molecules increased over time, suggesting a continuous secretion and accumulation of anti-EpCAM VHH BiTE from the GE CAR-BiTE T (Nb01 -013A) cell cultures ( Figure 13).
  • the Nb01 -013A BiTE molecules can also be detected from HE CAR- BiTE T (4D5) and HE CAR-BiTE T (F5) cells from the in vitro cultures.
  • Anti-HER2 CAR T (4D5 or F5) cells or HE CAR-BiTE T (4D5 or F5) cells were cultured in T cell growth medium containing IL-7 and IL-15 at the starting cell density of 1.5 million per ml. Cell culture supernatants were collected at 24h and 48h followed by an ELISA to detect the amount of secreted anti-EpCAM BiTE molecule NbO1 -O13A.
  • Example 8 Experiments demonstrating that human CAR T cells secreting anti- EpCAM BiTE molecules showed superior in vivo cytotoxicity in killing EpCAM expressing tumors.
  • Hep3B GPC3 Highh , EpCAM Highh xenograft models in NSG mice to test anti-GPC3 CAR T cells secreting different anti-EpCAM BiTE molecules (“MT110”, “Nb01 -013A”, and “NbO1-O13B”) as well as anti-CD19 CAR T cells secreting different anti-EpCAM BiTE molecules (“Nb01 -013A” or “Nb01 -013B”) ( Figure 15).
  • GE CAR-BiTE T (“Nb01-013A” or “Nb01 - 013B” used as BiTE) cells generated using the same T cell donor showed much potent tumor control efficacies, suggesting that replacing the anti-EpCAM arm with the presently disclosed anti-EpCAM 2C4-VHH significantly improved the in vivo expansion as well as the in vivo killing potency of the GE CAR-BiTE T cells.
  • this example indicates that human CAR T cells secreting anti- EpCAM BiTE molecules derived from in-house anti-EpCAM 2C4-VHH showed superior in vivo cytotoxicity in killing EpCAM expressing tumors.
  • antigen binding proteins variants and fragments thereof may be useful for one or more of the following:
  • Anti-EpCAM BiTEs incorporating the disclosed human Fab clones can be used in treating EpCAM high solid tumors due to their low affinity to the EpCAM antigen protein;
  • Anti-EpCAM BiTEs incorporating the llama VHH clones or humanized VHH clone can be used as locally secreted molecules in treating EpCAM positive solid tumors

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Abstract

La présente invention concerne des protéines de liaison à l'antigène, des variants et des fragments de celles-ci spécifiques à la molécule d'adhérence cellulaire épithéliale (EpCAM) et des protéines de liaison à l'antigène multispécifiques, telles que des activateurs de lymphocytes T bispécifiques (BiTE), qui se lient à EpCAM et à d'autres cibles également. L'invention concerne également des compositions comprenant les protéines de liaison à l'antigène, des variants et des fragments de celles-ci, une cellule exprimant/sécrétant celle-ci, une méthode de traitement utilisant les protéines de liaison à l'antigène, des variants et des fragments de celles-ci, ainsi que d'autres utilisations de celles-ci. Dans un mode de réalisation, la cellule est une cellule immunitaire, par exemple choisie dans le groupe comprenant un lymphocyte T, un macrophage, un monocyte et une cellule NK. Dans un mode de réalisation plus spécifique, la cellule est une cellule CAR T.
PCT/SG2024/050383 2023-06-07 2024-06-07 Protéine de liaison à un antigène multispécifique contre epcam Ceased WO2024253595A1 (fr)

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KR1020267000266A KR20260032536A (ko) 2023-06-07 2024-06-07 EpCAM에 대한 다중특이 항원 결합 단백질
AU2024284754A AU2024284754A1 (en) 2023-06-07 2024-06-07 MULTISPECIFIC ANTIGEN BINDING PROTEIN AGAINST EpCAM
CN202480051122.5A CN121666399A (zh) 2023-06-07 2024-06-07 针对EpCAM的多特异性抗原结合蛋白

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