WO2025051220A9 - Anticorps anti-ror1/anti-4-1bb et leurs utilisations - Google Patents

Anticorps anti-ror1/anti-4-1bb et leurs utilisations

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Publication number
WO2025051220A9
WO2025051220A9 PCT/CN2024/117323 CN2024117323W WO2025051220A9 WO 2025051220 A9 WO2025051220 A9 WO 2025051220A9 CN 2024117323 W CN2024117323 W CN 2024117323W WO 2025051220 A9 WO2025051220 A9 WO 2025051220A9
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Prior art keywords
seq
amino acid
acid sequence
peptide
sequence identity
Prior art date
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Application number
PCT/CN2024/117323
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WO2025051220A1 (fr
Inventor
Chanjuan Liu
Xi Chen
Zhengyi WANG
Xiaojun Xing
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Tj Biopharma Shanghai Co Ltd
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Tj Biopharma Shanghai Co Ltd
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Priority to CN202480056848.8A priority Critical patent/CN121843967A/zh
Publication of WO2025051220A1 publication Critical patent/WO2025051220A1/fr
Publication of WO2025051220A9 publication Critical patent/WO2025051220A9/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Receptor Tyrosine Kinase-Like Orphan Receptor 1 is a 106-kDa member of the receptor tyrosine kinase family. Structurally, the extracellular domain of the ROR1 receptor is composed of three distinct domains: a membrane-distal Immunoglobulin-Like Domain; a membrane-proximal Kringle Domain; and an intervening Frizzled Domain.
  • ROR1 is expressed in the process of embryo and fetal development, and controls cell polarity, cell migration and neurite growth, etc. The expression is gradually reduced according to progress of development. It is hardly expressed in adults, and it is temporarily expressed in the process of development of B cell, and only little expression has been reported in adipocytes. While ROR1 expression is tightly regulated in normal adult tissues, high levels have been noted in both hematological and solid tumors. ROR1 is normally expressed during early development, but becomes activated by tumor specific mechanisms and may contribute to disease progression in adults.
  • the ligands of ROR1 are believed to be wnt5a and NKX1-2.
  • Wnt5a has been shown to bind to the Frizzled Domain in the extracellular part of ROR1 and, in transfected cells, has been shown to modulate NF- ⁇ activation and proliferation of normal and lung tumor cell lines.
  • Binding of NKX1-2 to ROR1 has been shown to play a role in the survival of lung cancer cell lines through both kinase-dependent and kinase-independent mechanisms.
  • ROR1 has been shown to interact with EGFR through the Kringle domain, and this interaction modulates signaling pathways that control apoptosis in lung cancer cell lines.
  • ROR1 expression does correlate with a worse prognosis in ovarian cancer, no link between ROR1 expression and clinical stage or reduced survival has been shown for lung cancer. Furthermore, although ROR1 siRNA knockdown of lung tumor cell lines leads to reduced viability in vitro, there is no evidence that targeting of ROR1 on primary lung cancer cells results in increased cell death.
  • 4-1BB (CD137 and TNFRSF9) , which was first identified as an inducible costimulatory receptor expressed on activated T cells, is a membrane spanning glycoprotein of the Tumor Necrosis Factor (TNF) receptor superfamily.
  • TNF Tumor Necrosis Factor
  • Current understanding of 4-1BB indicates that expression is generally activation dependent and encompasses a broad subset of immune cells including activated NK and NKT cells, regulatory T cells, dendritic cells (DC) including follicular DC; stimulated mast cells, differentiating myeloid cells, monocytes, neutrophils, eosinophils, and activated B cells.
  • 4-1BB expression has also been demonstrated on tumor vasculature and atherosclerotic endothelium.
  • the ligand that stimulates 4-1BB (4-1BBL) is expressed on activated antigen-presenting cells (APCs) , myeloid progenitor cells and hematopoeitic stem cells.
  • APCs
  • TRAF 1 and TRAF 2 which are pro-survival members of the TNFR-associated factor (TRAF) family, are recruited to the 4-1BB cytoplasmic tail, resulting in downstream activation of NFkB and the Mitogen Activated Protein (MAP) Kinase cascade including Erk, ink, and p38 MAP kinases.
  • MAP Mitogen Activated Protein
  • the cancer cell-specific expression of ROR1 and immune cell expression of 4-1BB indicates that these two targets can be a potential cancer target for antibody therapy.
  • the present disclosure provides multi-specific antibodies having binding specificity to the human Receptor tyrosine kinase-like orphan receptor 1 (ROR1) protein and 4-1BB protein. These antibodies and fragments are useful in the treatment of diseases and conditions such as cancers.
  • ROR1 human Receptor tyrosine kinase-like orphan receptor 1
  • a multi-specific antibody comprising (1) a first antibody moiety that specifically binds to a tumor associated antigen (TAA) , and (2) a second antibody moiety that specifically binds to a human 4-1BB protein.
  • TAA tumor associated antigen
  • the multi-specific antibody further comprises a Fc domain with maintained or improved effector function.
  • the second antibody moiety is a sdAb.
  • the second antibody moiety comprises a HCDR1 of SNCMG (SEQ ID NO: 47) , a HCDR2 of VICTGGGSPSYADSVKG (SEQ ID NO: 48) , and a HCDR3 of DLLRAGTPLSSYEFNY (SEQ ID NO: 49) .
  • the second antibody moiety comprises an amino acid sequence of SEQ ID NO: 9 or a peptide having at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 9.
  • the Fc domain is derived from any one selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
  • the Fc domain is derived from IgG1.
  • the Fc domain comprises an amino acid sequence having at least 80%identity with any one of SEQ ID NOs: 52 and 140-143.
  • the first antibody moiety is a Fab’ fused to the N-terminus of the IgG Fc domain and the second antibody moiety is a sdAb fused to the C-terminus of the IgG Fc domain.
  • the second antibody moiety is fused to the IgG Fc domain via a linker.
  • the multi-specific antibody further comprises a third antibody moiety that specifically binds to a second tumor associated antigen.
  • the first antibody moiety is a Fab’ fused to the N-terminus of the IgG Fc domain and the second antibody moiety is a sdAb fused to the C-terminus of the IgG Fc domain, and the third antibody moiety is a scFv fused to the N-terminus of the first antibody moiety.
  • the second antibody moiety is fused to the IgG Fc domain via a linker
  • the third antibody moiety is fused to the first antibody moiety via a linker
  • the first antibody moiety is a Fab’ fused to the N-terminus of a IgG Fc domain and the second antibody moiety is a sdAb fused to the C-terminus of the IgG Fc domain
  • the third antibody moiety is a scFv fused to the N-terminus of a IgG heavy chain constant region (IgG’ (CH) )
  • the second antibody moiety is a sdAb fused to C-terminus of the IgG’ (CH)
  • the IgG’ (CH) comprises a pairing IgG Fc domain and forms a heterodimer with the IgG Fc domain.
  • the second antibody moiety is fused to the IgG1 Fc domain via a linker, and the second antibody moiety is fused to the IgG’ (CH) via a linker.
  • the first and the third antibody moiety specifically binds to a human receptor tyrosine kinase-like orphan receptor 1 (ROR1) protein but at different epitopes.
  • ROR1 human receptor tyrosine kinase-like orphan receptor 1
  • the first antibody moiety or the third antibody moiety comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising heavy chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the group consisting of:
  • HCDR1 SYAMS (SEQ ID NO: 10) , or RYAMS (SEQ ID NO: 11) ,
  • HCDR2 SISSGGTTYYPDTVKGR (SEQ ID NO: 12) or SISSGGNTYYPDTVKGR (SEQ ID NO: 13) or SISSGGTRYYPDTVKGR (SEQ ID NO: 14) ,
  • HCDR3 DSLYYGSSLYYAMDY (SEQ ID NO: 15) , DALYYGGSLYYAMDY (SEQ ID NO: 16) or DALYYGSSLYYAMDY (SEQ ID NO: 74) ,
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17) ,
  • LCDR2 RANRLVDG (SEQ ID NO: 18) , RENRLVDA (SEQ ID NO: 19) or RANRLVDA (SEQ ID NO: 75) , and
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20) ;
  • HCDR1 TYVMH (SEQ ID NO: 21) or NYVMH (SEQ ID NO: 22) ,
  • HCDR2 YINPYNGGIRYNEKFKG (SEQ ID NO: 23) , YINPYNGVIRYNEKFKG (SEQ ID NO: 24) or YINPYSGGIRYNEKFKG (SEQ ID NO: 24) ,
  • HCDR3 RERGVYYGMDE (SEQ ID NO: 26) , RERGVYYGMSE (SEQ ID NO: 27) or RERGVTAGMDE (SEQ ID NO: 28) ,
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29) or KSSQSLLHSNDKTYLN (SEQ ID NO: 30) ,
  • LCDR2 LVSKLESG (SEQ ID NO: 31) or LVSKLSSG (SEQ ID NO: 32) , and
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) or YQATYFPYT (SEQ ID NO: 34) ;
  • HCDR2 AIDTSDSSTRNNQKFKG (SEQ ID NO: 36) ,
  • HCDR3 GARTGTGFGY (SEQ ID NO: 37) ,
  • LCDR1 KSSQSLLHINGKTYLN (SEQ ID NO: 38) ,
  • LCDR2 LVSKLESG (SEQ ID NO: 31) .
  • LCDR3 LQATHFPYT (SEQ ID NO: 40) ;
  • HCDR2 VIWAGGHTNYNSDLMSR (SEQ ID NO: 42) ,
  • HCDR3 RSIYGDYALDY (SEQ ID NO: 43) ,
  • LCDR1 VTYMH (SEQ ID NO: 44) ,
  • LCDR2 DISKLASG (SEQ ID NO: 45) .
  • LCDR3 QQWNYPLMT (SEQ ID NO: 46) .
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody moiety or the third antibody moiety are
  • HCDR1 SYAMS (SEQ ID NO: 10)
  • HCDR2 SISSGGTTYYPDTVKGR (SEQ ID NO: 12)
  • HCDR3 DSLYYGSSLYYAMDY (SEQ ID NO: 15)
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RANRLVDG (SEQ ID NO: 18)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20)
  • HCDR1 SYAMS (SEQ ID NO: 10)
  • HCDR2 SISSGGTTYYPDTVKGR (SEQ ID NO: 12)
  • HCDR3 DALYYGSSLYYAMDY
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RANRLVDA (SEQ ID NO: 75)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20)
  • HCDR1 SYAMS (SEQ ID NO: 10)
  • HCDR2 SISSGGTTYYPDTVKGR (SEQ ID NO: 12)
  • HCDR3 DALYYGGSLYYAMDY (SEQ ID NO: 16)
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RANRLVDA (SEQ ID NO: 75)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20) ,
  • HCDR1 RYAMS (SEQ ID NO: 11)
  • HCDR2 SISSGGNTYYPDTVKGR (SEQ ID NO: 13)
  • HCDR3 DALYYGSSLYYAMDY (SEQ ID NO: 74)
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RENRLVDA (SEQ ID NO: 19)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20) , or
  • HCDR1 SYAMS (SEQ ID NO: 10)
  • HCDR2 SISSGGTRYYPDTVKGR (SEQ ID NO: 14)
  • HCDR3 DALYYGSSLYYAMDY (SEQ ID NO: 74)
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RENRLVDA (SEQ ID NO: 19)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20) .
  • the first antibody moiety or the third antibody moiety comprises
  • a VH comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 2;
  • a VH comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 58, and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59;
  • a VH comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59;
  • VH comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 63
  • VL comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 64;
  • a VH comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 65
  • a VL comprising the amino acid sequence of SEQ ID NO: 64 or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 64.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody moiety or the third antibody moiety are
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYNGGIRYNEKFKG (SEQ ID NO: 23)
  • HCDR3 RERGVYYGMDE (SEQ ID NO: 26)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) ;
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYNGVIRYNEKFKG (SEQ ID NO: 24)
  • HCDR3 RERGVYYGMDE (SEQ ID NO: 26)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLSSG (SEQ ID NO: 32)
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) ;
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYNGGIRYNEKFKG (SEQ ID NO: 23)
  • HCDR3 RERGVYYGMSE (SEQ ID NO: 27)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 YQATYFPYT (SEQ ID NO: 34) ;
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYSGGIRYNEKFKG (SEQ ID NO: 25)
  • RERGVYYGMSE SEQ ID NO: 27
  • LCDR1 KSSQSLLHSNDKTYLN (SEQ ID NO: 30)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 YQATYFPYT (SEQ ID NO: 34) ;
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYSGGIRYNEKFKG (SEQ ID NO: 25)
  • HCDR3 RERGVTAGMDE (SEQ ID NO: 28)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) ;
  • HCDR1 NYVMH (SEQ ID NO: 22)
  • HCDR2 YINPYSGGIRYNEKFKG (SEQ ID NO: 25)
  • HCDR3 RERGVYYGMDE (SEQ ID NO: 26)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) .
  • the first antibody moiety or the third antibody moiety comprises
  • a VH comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 3, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 4;
  • a VH comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 60
  • a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61;
  • a VH comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 66, and a VL comprising the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 67;
  • a VH comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 68, and a VL comprising the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 69;
  • a VH comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 70
  • a VL comprising the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 71;
  • a VH comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 72
  • a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61;
  • a VH comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 73, and a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61; or
  • a VH comprising the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 76
  • a VL comprising the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 77.
  • the first antibody moiety comprises a heavy chain variable region comprising an amino acid of SEQ ID NO: 5, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 6, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 6.
  • the first antibody moiety comprises a heavy chain variable region comprising an amino acid of SEQ ID NO: 7, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 8, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 8.
  • the first antibody moiety comprises
  • a VH comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 2;
  • a VH comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 58, and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59;
  • a VH comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59;
  • a VH comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 63
  • a VL comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 64; or
  • a VH comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 65
  • a VL comprising the amino acid sequence of SEQ ID NO: 64 or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 64;
  • a VH comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 3, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 4;
  • a VH comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 60
  • a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61;
  • a VH comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 66, and a VL comprising the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 67;
  • a VH comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 68, and a VL comprising the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 69;
  • a VH comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 70
  • a VL comprising the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 71;
  • a VH comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 72
  • a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61;
  • a VH comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 73, and a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61; or
  • a VH comprising the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 76
  • a VL comprising the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 77.
  • the third antibody moiety comprises
  • a VH comprising the amino acid sequence of SEQ ID NO: 1, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 2;
  • a VH comprising the amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 58, and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59;
  • a VH comprising the amino acid sequence of SEQ ID NO: 62, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 59;
  • a VH comprising the amino acid sequence of SEQ ID NO: 63, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 63
  • a VL comprising the amino acid sequence of SEQ ID NO: 64, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 64; or
  • a VH comprising the amino acid sequence of SEQ ID NO: 65, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 65
  • a VL comprising the amino acid sequence of SEQ ID NO: 64 or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 64;
  • a VH comprising the amino acid sequence of SEQ ID NO: 3, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 3, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 4;
  • a VH comprising the amino acid sequence of SEQ ID NO: 60, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 60
  • a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61;
  • a VH comprising the amino acid sequence of SEQ ID NO: 66, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 66, and a VL comprising the amino acid sequence of SEQ ID NO: 67, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 67;
  • a VH comprising the amino acid sequence of SEQ ID NO: 68, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 68, and a VL comprising the amino acid sequence of SEQ ID NO: 69, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 69;
  • a VH comprising the amino acid sequence of SEQ ID NO: 70, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 70
  • a VL comprising the amino acid sequence of SEQ ID NO: 71, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 71;
  • a VH comprising the amino acid sequence of SEQ ID NO: 72, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 72
  • a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61;
  • a VH comprising the amino acid sequence of SEQ ID NO: 73, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 73, and a VL comprising the amino acid sequence of SEQ ID NO: 61, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 61; or
  • a VH comprising the amino acid sequence of SEQ ID NO: 76, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 76
  • a VL comprising the amino acid sequence of SEQ ID NO: 77, or a peptide having at least 90% (or at least 95, at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 77.
  • the linker comprises an amino acid sequence of SEQ ID NO: 56.
  • the first antibody moiety and the third antibody moiety forms a heterodimer via Fc region pairing.
  • the first antibody moiety comprises an IgG heavy chain constant region (IgG (CH) ) comprising an amino acid sequence of SEQ ID NO: 51 and the third antibody moiety comprises an IgG’ (CH) comprising an amino acid sequence of SEQ ID NO: 53; or
  • the first antibody moiety comprises an IgG (CH) comprising an amino acid sequence of SEQ ID NO: 55 and the third antibody moiety comprises an IgG’ (CH) comprising an amino acid sequence of SEQ ID NO: 54.
  • the present disclosure provides a composition comprising the multi-specific antibody provided herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides an isolated cell comprising one or more polynucleotide encoding the multi-specific antibody provided herein.
  • the present disclosure provides a polynucleotide encoding one or more chains of the multi-specific antibody provided herein.
  • the present disclosure provides a method of treating a cancer in a patient in need thereof, comprising administering to the patient the multi-specific antibody provided herein.
  • the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
  • the method comprising administering to the patient a therapy for treating said cancer.
  • the method further comprises administering to the patient a therapy for treating said cancer.
  • the said therapy is selected from the group consisting of immunotherapy, chemotherapy and radiotherapy.
  • the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
  • FIG. 1A-1C show the formats of the bi-specific (A) and bi-paratopic (B-C) anti-ROR1/anti-4-1BB antibodies.
  • FIG. 2A-2B show the cellular binding activity of the bi-specific anti-ROR1/anti-4-1BB antibodies as measured by FACS.
  • FIG. 3A-3C show the ROR1 dependent 4-1BB activation of the bi-specific anti-ROR1/anti-4-1BB antibodies in a reporter gene assay.
  • FIG. 4A-4C show the epitope binning of the anti-ROR1 antibodies.
  • FIG. 5A-5C show the ROR1 dependent 4-1BB activation of the bi-paratopic anti-ROR1/anti-4-1BB antibodies in a reporter gene assay.
  • FIG. 6A-6D show (A-B) the cellular binding activity to ROR1 of the anti-ROR1 mAb as measured by FACS, before and after humanization, and (C-D) ROR1 dependent 4-1BB activation in a reporter gene assay of the bi-specific anti-ROR1/anti-4-1BB antibodies, before and after humanization.
  • FIG. 7A-7F show (A-C) cellular binding activity of the bi-specific and bi-paratopic anti-ROR1/anti-4-1BB antibodies as measured by FACS and (D-F) ROR1 dependent 4-1BB activation of the bi-specific and bi-paratopic anti-ROR1/anti-4-1BB antibodies in a reporter gene assay using ROR1 positive and negative tumor cell lines.
  • FIG. 8A-8C show SPR results of the humanized bi-specific and bi-paratopic anti-ROR1/anti-4-1BB antibodies.
  • FIG. 9A-9B show the in vivo efficacy study of the humanized bi-specific and bi-paratopic anti-ROR1/anti-4-1BB antibodies.
  • A study design;
  • B in vivo anti-tumor efficacy in 4-1BB knock-in mice.
  • FIG. 10A-10E show the ROR1 dependent 4-1BB activation of the affinity matured bi-specific anti-ROR1/anti-4-1BB antibodies in a reporter gene assay.
  • FIG. 11A-11C show the ROR1 dependent 4-1BB activation of the affinity matured bi-paratopic anti-ROR1/anti-4-1BB antibodies in a reporter gene assay.
  • FIG. 12A-12B show the ROR1 dependent 4-1BB induced cytokine release of the affinity matured bi-specific and bi-paratopic anti-ROR1/anti-4-1BB antibodies in a reporter gene assay.
  • FIG. 13A-13C show the in vivo efficacy study of the affinity matured bi-paratopic anti-ROR1/anti-4-1BB antibodies.
  • A study design;
  • B in vivo anti-tumor efficacy in 4-1BB knock-in C57 mice inoculated with hROR1 expressing MC38 cells;
  • C summarized tumor growth inhibition (TGI) for each treatment group.
  • FIG. 14A-14B illustrate tumor inhibition in hu4-1BB mice after treatment with CLDN6 x 4-1BB BsAbs as disclosed herein.
  • a or “an” entity refers to one or more of that entity; for example, “an antibody, ” is understood to represent one or more antibodies.
  • the terms “a” (or “an” ) , “one or more, ” and “at least one” can be used interchangeably herein.
  • an “antibody” or “antigen-binding moiety” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
  • An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof.
  • antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen.
  • CDR complementarity determining region
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991) .
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • Several of the major antibody classes are divided into subclasses such as lgG1 ( ⁇ 1 heavy chain) , lgG2 ( ⁇ 2 heavy chain) , lgG3 ( ⁇ 3 heavy chain) , lgG4 ( ⁇ 4 heavy chain) , lgA1 ( ⁇ 1 heavy chain) , or lgA2 ( ⁇ 2 heavy chain) .
  • half antibody refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain.
  • a half-antibody may encompass a fragment thereof and may also have an antigen binding domain consisting of a single variable domain, e.g., originating from a camelidae.
  • single chain half-antibody refers to a single chain polypeptide comprising a VL domain, optionally a CL domain, a tether, a VH domain, optionally a CH1 domain, a hinge domain, a CH2 domain and a CH3 domain, wherein said domains are positioned relative to each other in an N-terminal to C-terminal direction as follows: VL-tether-VH-hinge-CH2-CH3, VL-tether-VH-partial hinge-CH2-CH3, VL-tether-VH-hinge variant -CH2-CH3, or VL-CL-tether-VH-CH1-hinge-CH2-CH3.
  • single domain antibodies or “single variable domain (SVD) antibodies” generally refers to antibodies in which a single variable domain (VH or VL) can confer antigen binding. In other words, the single variable domain does not need to interact with another variable domain in order to recognize the target antigen.
  • single domain antibodies include those derived from camelids (lamas and camels) and cartilaginous fish (e.g., nurse sharks) and those derived from recombinant methods from humans and mouse antibodies (Nature (1989) 341: 544-546; Dev Comp Immunol (2006) 30: 43-56; Trend Biochem Sci (2001) 26: 230-235; Trends Biotechnol (2003) : 21: 484-490; WO 2005/035572; WO 03/035694; Febs Lett (1994) 339: 285-290; WO00/29004; WO 02/051870) .
  • the sdAb contains only a heavy chain, it can be exchangable used with “VHH” or “single heavy chain variable domain antibody” or “nanobody” .
  • antibody fragment or “antigen-binding fragment” , as used herein, is a portion of an antibody such as F (ab’) 2 , F (ab) 2 , Fab’, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.
  • antibody fragment includes aptamers, spiegelmers, and diabodies.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • a “Fab” with regard to an antibody refers to a monovalent antigen-binding fragment of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • Fab can be obtained by papain digestion of an antibody at the residues proximal to the N-terminus of the disulfide bond between the heavy chains of the hinge region.
  • a “Fab’ ” refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of an antibody at the residues proximal to the C-terminus of the disulfide bond between the heavy chains of the hinge region and thus is different from Fab in a small number of residues (including one or more cysteines) in the hinge region.
  • a “F (ab) 2 ” refers to a dimer of Fab’ that comprises two light chains and part of the two heavy chains.
  • a “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of immunoglobulins.
  • the regions are connected with a short linker peptide of ten to about 25 amino acids.
  • the linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V H with the C-terminus of the V L , or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
  • antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (g, m, a, d, e) with some subclasses among them (e.g., g l-g4) . It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • the immunoglobulin subclasses isotypes) e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5 , etc.
  • immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
  • the four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • Antibodies, antigen-binding moieties, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab’ and F (ab’) 2 , Fd, Fvs, single-chain Fvs (scFv) , single-chain antibodies, disulfide-linked Fvs (sdFv) , fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein) .
  • anti-Id antigen-binding moieties, variants, or derivatives thereof of the disclosure
  • Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Light chains are classified as either kappa or lambda (K, l) .
  • Each heavy chain class may be bound with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CK) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CK domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
  • variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and VH domain, or subset of the complementarity determining regions (CDRs) , of an antibody combine to form the variable region that defines a three dimensional antigen-binding site.
  • This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) .
  • a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363: 446-448 (1993) .
  • each antigen-binding domain is short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen-binding domains referred to as “framework” regions, show less inter-molecular variability.
  • the framework regions largely adopt a b-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the b -sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • the amino acids comprising the CDRs and the framework regions, respectively can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest, ” Kabat, E., et al., U.S. Department of Health and Human Services, (1983) ; and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987) ) .
  • CDR complementarity determining region
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself.
  • “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983) .
  • CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue) , includes approximately 5-7 amino acids, and ends at the next tryptophan residue.
  • CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue.
  • CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid.
  • CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue) ; includes approximately 10-17 residues; and ends at the next tryptophan residue.
  • CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues.
  • CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2 (i.e., following a cysteine residue) ; includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.
  • Antibodies disclosed herein may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • the variable region may be condricthoid in origin (e.g., from sharks) .
  • heavy chain constant region includes amino acid sequences derived from an immunoglobulin heavy chain.
  • a polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain.
  • a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain.
  • an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain) .
  • a CH2 domain e.g., all or part of a CH2 domain
  • the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
  • the heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules.
  • a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG l molecule and a hinge region derived from an IgG 3 molecule.
  • a heavy chain constant region can comprise a hinge region derived, in part, from an IgG l molecule and, in part, from an IgG 3 molecule.
  • a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG l molecule and, in part, from an IgG 4 molecule.
  • the term “light chain constant region” includes amino acid sequences derived from antibody light chain.
  • the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.
  • a “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
  • CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
  • CH1 domain (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system) .
  • Hinge region includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain, a region in IgG corresponding to Glu216 to Pro230 of human IgG1, EU numbering system (Burton, Molec. Immunol. 22: 161-206 (1985) ) .
  • Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161: 4083 (1998) ) .
  • CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) .
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
  • CH3 domain (also referred to as “C3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e., from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG, EU numbering system) .
  • Fc region , “Fc domain” or “fragment crystallizable region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B) , IgG3 and IgG4.
  • disulfide bond includes the covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system) .
  • chimeric antibody will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.
  • Humanized antibody is used herein to describe an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g. a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like, " i.e., more similar to human germline variable sequences.
  • a “humanized antibody” is an antibody or a variant, derivative, analog, or fragment thereof, which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody.
  • FR framework
  • CDR complementary determining region
  • the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of a non-human antibody CDR.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F (ab') 2 , Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • a humanized antibody contains the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanized antibody only contains a humanized light chain.
  • a humanized antibody only contains a humanized heavy chain.
  • a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
  • an antibody By “specifically binds” or “has specificity to, ” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B, ” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D. ”
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable.
  • “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • phrases such as “to a patient in need of treatment” or “asubject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
  • the present disclosure provides multi-specific antibodies containing one or more antigen-binding moieties that have binding specificity to the human ROR1 protein.
  • the murine clones, 3C5, 8F2, 10C9 and 9C9 were selected for construction of the multi-specific antibodies.
  • antigen-binding moieties that include the heavy chain and light chain variable domains with the CDR regions.
  • the CDRs are summarized in Table 1A below (Kabat numbering) .
  • variable regions are summarized in Table 1B below.
  • the VH CDR1, CDR2, and CDR3 are selected from any set of VH CDR1, CDR2, and CDR3 shown in Table 1
  • the VL CDR1, CDR2, and CDR3 are selected from any set of VL CDR1, CDR2, and CDR3 shown in Table 1.
  • the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 are selected from those derived from the same antigen-binding moieties.
  • At least one, or two, or three, or four, or five, or six of the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 of the above are modified by one, two or three amino acid additions, deletions, substitutions, or the combinations thereof.
  • the antibody are a humanized.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F (ab’) 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin (Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-329 (1988) ; and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992) ) .
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ) , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) , wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • the CDR residues underwent post-translational modification (PTM) during the humanization process.
  • PTM post-translational modification
  • the CDR3 of the heavy chain variable region of 3C5 can be mutated from DSLYYGSSLYYAMDY (SEQ ID NO: 15) to D A LYYGSSLYYAMDY (SEQ ID NO: 74) .
  • the CDR2 of the light chain variable region of 3C5 can be mutated from RANRLVDG (SEQ ID NO: 18) to RANRLVD A (SEQ ID NO: 75) .
  • the antibodies and the fragment thereof are humanized and comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 58, or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence of SEQ ID NO: 58.
  • the antibodies and the fragment thereof are humanized and comprise a light chain variable region comprising an amino acid sequence of SEQ ID NO: 59 or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence of SEQ ID NO: 59.
  • the antibodies and the fragment thereof are humanized and comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 58, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 59.
  • the antibodies and the fragment thereof are humanized and comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60 or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence of SEQ ID NO: 60.
  • the antibodies and the fragment thereof are humanized and comprise a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61 or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence of SEQ ID NO: 61.
  • the antibodies and the fragment thereof are humanized and comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 61.
  • Antibodies may be produced by a process of affinity maturation in which a modified antibody is generated that has an improvement in the affinity of the antibody for antigen, compared to an unmodified parent antibody.
  • Affinity-matured antibodies may be produced by procedures known in the art, e.g., Marks et al., Rio/Technology 10: 779-783 (1992) ; Barbas et al. Proc Nat. Acad. Sci. USA 91 : 3809-3813 (1994) ; Schier et al. Gene 169: 147-155 (1995) ; Yelton et al. J. Immunol. 155: 1994-2004 (1995) ; Jackson et al., J. Immunol. 154 (7) : 331 0-15 9 (1995) ; and Hawkins et al, J. Mol. Biol. 226: 889-896 (1992) .
  • affinity matured anti-ROR1 antibody or fragment thereof includes the following CDRs:
  • HCDR1 SYAMS (SEQ ID NO: 10)
  • HCDR2 SISSGGTTYYPDTVKGR (SEQ ID NO: 12)
  • HCDR3 DALYYGGSLYYAMDY (SEQ ID NO: 16)
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RANRLVDA (SEQ ID NO: 75)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20) ;
  • HCDR1 RYAMS (SEQ ID NO: 11)
  • HCDR2 SISSGGNTYYPDTVKGR (SEQ ID NO: 13)
  • HCDR3 DALYYGSSLYYAMDY (SEQ ID NO: 74)
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RENRLVDA (SEQ ID NO: 19)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20) , or
  • HCDR1 SYAMS (SEQ ID NO: 10)
  • HCDR2 SISSGGTRYYPDTVKGR (SEQ ID NO: 14)
  • HCDR3 DALYYGSSLYYAMDY (SEQ ID NO: 74)
  • LCDR1 RASQDIYSYLS (SEQ ID NO: 17)
  • LCDR2 RENRLVDA (SEQ ID NO: 19)
  • LCDR3 LQYDEFPYT (SEQ ID NO: 20) .
  • affinity matured anti-ROR1 antibody or fragment thereof includes the following CDRs:
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYNGVIRYNEKFKG (SEQ ID NO: 24)
  • HCDR3 RERGVYYGMDE (SEQ ID NO: 26)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLSSG (SEQ ID NO: 32)
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) ;
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYNGGIRYNEKFKG (SEQ ID NO: 23)
  • HCDR3 RERGVYYGMSE (SEQ ID NO: 27)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 YQATYFPYT (SEQ ID NO: 34) ;
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYSGGIRYNEKFKG (SEQ ID NO: 25)
  • HCDR3 RERGVTAGMDE (SEQ ID NO: 28)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) ;
  • HCDR1 NYVMH (SEQ ID NO: 22)
  • HCDR2 YINPYSGGIRYNEKFKG (SEQ ID NO: 25)
  • HCDR3 RERGVYYGMDE (SEQ ID NO: 26)
  • LCDR1 KSSQSLLHSNGKTYLN (SEQ ID NO: 29)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 LQATYFPYT (SEQ ID NO: 33) ; or
  • HCDR1 TYVMH (SEQ ID NO: 21)
  • HCDR2 YINPYSGGIRYNEKFKG (SEQ ID NO: 25)
  • RERGVYYGMSE SEQ ID NO: 27
  • LCDR1 KSSQSLLHSNDKTYLN (SEQ ID NO: 30)
  • LCDR2 LVSKLESG (SEQ ID NO: 31)
  • LCDR3 YQATYFPYT (SEQ ID NO: 34) .
  • variable regions of 3C5 and 8F2 undergone affinity maturation and removal of the potential deamidation amino acids are listed in Table 1E below.
  • the antibodies and the fragment thereof are humanized and affinity matured and comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-63 and 65, or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 62-63 and 65.
  • the antibodies and the fragment thereof are humanized and affinity matured and comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 59 and 64 or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 59 and 64.
  • the antibodies and the fragment thereof are humanized and affinity matured and comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 68, 70 and 72-73, or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 66, 68, 70 and 72-73.
  • the antibodies and the fragment thereof are humanized and affinity matured and comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 67, 69 and 71, or a peptide having at least 90%, at least 95%, or at least 98%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 67, 69 and 71.
  • One or more disulfide bonds are introduced into the variable regions.
  • a cystine pair is introduced to the heavy chain variable region and light chain variable region, respectively.
  • One amino acid substitution at G44C in SEQ ID NO: 70 (SEQ ID NO: 76) and one amino acid substitution at Q105C in SEQ ID NO: 71 (SEQ ID NO: 77) are introduced respectively to form a disulfide bond.
  • the present disclosure provides an antibody or antigen-binding fragment thereof having specificity to a human ROR1 protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3.
  • the HCDR1 includes the amino acid sequence of SEQ ID NO: 35
  • the HCDR2 includes the amino acid sequence of SEQ ID NO: 36
  • the HCDR3 includes the amino acid sequence of SEQ ID NO: 37
  • the LCDR1 includes the amino acid sequence of SEQ ID NO: 38
  • the LCDR2 includes the amino acid sequence of SEQ ID NO: 31
  • the LCDR3 includes the amino acid sequence of SEQ ID NO: 40.
  • the VH comprises the amino acid sequence of SEQ ID NO: 5 and the VL includes the amino acid sequence of SEQ ID NO: 6.
  • the HCDR1 includes the amino acid sequence of SEQ ID NO: 41
  • the HCDR2 includes the amino acid sequence of SEQ ID NO: 42
  • the HCDR3 includes the amino acid sequence of SEQ ID NO: 43
  • the LCDR1 includes the amino acid sequence of SEQ ID NO: 44
  • the LCDR2 includes the amino acid sequence of SEQ ID NO: 45
  • the LCDR3 includes the amino acid sequence of SEQ ID NO: 46.
  • the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL includes the amino acid sequence of SEQ ID NO: 8.
  • the present disclosure provides multi-specific antibodies containing one or more antigen-binding moieties that have binding specificity to the human 4-1BB protein.
  • antigen-binding moieties that include a single domain antibody (sdAb) with the CDR regions.
  • sdAb single domain antibody
  • Table 2A Exemplary single heavy chain variable domains are shown in Table 2B.
  • At least one, or two, or three, or four, or five, or six of the VH CDR1, CDR2, and CDR3 of the above are modified by one, two or three amino acid additions, deletions, substitutions, or the combinations thereof.
  • the HCDR1 includes the amino acid sequence of SEQ ID NO: 47
  • the HCDR2 includes the amino acid sequence of SEQ ID NO: 48
  • the HCDR3 includes the amino acid sequence of SEQ ID NO: 49.
  • the VH comprises the amino acid sequence of SEQ ID NO: 9 or a peptide having at least 90% (or at least 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 9.
  • the CDRs, heavy chain variable regions, light chain variable regions or single heavy chain variable domains of the present disclosure can be further modified.
  • the modified heavy chain variable region, light chain variable region or single heavy chain variable domain retains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%or 99%sequence identity and is still capable of binding to the target site.
  • the modification is substitution at no more than one hot spot position from each of the CDRs. In some embodiments, the modification is substitution at one, two or three such hot spot positions. In one embodiment, the modification is substitution at one of the hot spot positions. Such substitutions, in some embodiments, are conservative substitutions.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids.
  • antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived.
  • a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical to the starting sequence.
  • an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody.
  • the term "multispecific antibody” as used herein refers to an antibody comprising an antigen-binding domain that has polyepitopic specificity (i.e., is capable of binding to two, or more, different epitopes on one molecule or is capable of binding to epitopes on two, or more, different molecules) .
  • multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigen binding sites (such as a bispecific antibody) .
  • the first antigen-binding domain and the second antigen-binding domain of the multispecific antibody may bind the two epitopes within one and the same molecule (intramolecular binding) .
  • the first antigen-binding domain and the second antigen-binding domain of the multispecific antibody may bind to two different epitopes on the same ROR1 molecule.
  • the two different epitopes that a multispecific antibody binds are epitopes that are not normally bound at the same time by one monospecific antibody, such as e.g., a conventional antibody or one immunoglobulin single variable domain.
  • the first antigen-binding domain and the second antigen-binding domain of the multispecific antibody may bind epitopes located within two distinct molecules (intermolecular binding) .
  • the first antigen-binding domain of the multispecific antibody may bind to one epitope on one ROR1 molecule
  • the second antigen-binding domain of the multispecific antibody may bind to another epitope on a different ROR1 molecule, thereby cross-linking the two molecules.
  • a multispecific antibody provided herein may be a bispecific antibody.
  • the term "bispecific antibody” as used herein refers to a multispecific antibody comprising an antigen-binding domain that is capable of binding to two different epitopes on one molecule or is capable of binding to epitopes on two different molecules.
  • a bispecific antibody may also be referred to herein as having “dual specificity” or as being “dual specific. " Exemplary bispecific antibodies may bind both ROR1 and any other antigen.
  • one of the binding specificities is for ROR1 and the other is for 4-1BB.
  • bispecific antibodies may bind to two different epitopes of the same ROR1 molecule.
  • bispecific antibodies may bind to two different epitopes on two different ROR1 molecules.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express ROR1.
  • Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983) , WO 93/08829, and Traunecker et al, EMBO J. 10: 3655 (1991) ) , and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168, WO2009/089004, US2009/0182127, US2011/0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26 (6) : 649-658, and Kontermann (2005) Acta Pharmacol. Sin., 26: 1-9) .
  • the present disclosure provides the anti-ROR1 antigen-binding moieties and the anti-4-1BB antigen-binding moieties as bispecific or multi-specific antibodies.
  • the 4-1BB pathway activation can be enhanced by targeting the ROR1 antigen simultaneously, through increasing the 4-1BB clustering.
  • the design of bi-paratopic antigen targeting to the ROR1 induced even stronger 4-1BB activation.
  • the anti-ROR1 antigen-binding moiety provided herein has an full-length antibody/IgG format and the anti-4-1BB antigen-binding moiety provided herein has a single domain antibody (sdAb) /VHH/nanobody format.
  • sdAb single domain antibody
  • the VHH is fused to N-terminus or C-terminus of the IgG. In certain embodiments, the VHH is fused to N-terminus of the IgG heavy chain variable region. In certain embodiments, the VHH is fused to C-terminus of the IgG heavy chain constant region (IgG (CH) ) .
  • the multi-specific antibody provided herein include a first polypeptide comprising from N-to C-terminus: VH (ROR-1) -IgG (CH) -VHH (4-1BB) , and a second polypeptide comprising from N-to C-terminus: VL (ROR-1) -IgG light chain constant region (IgG (CL) ) .
  • the first and second polypeptides are paired via the VH (ROR-1) -VL (ROR-1) pairing.
  • the multi-specific antibody provided herein include two first polypeptides comprising from N-to C-terminus: VH (ROR-1) -IgG (CH) -VHH (4-1BB) , and two second polypeptides comprising from N-to C-terminus: VL (ROR-1) -IgG light chain constant region (IgG (CL) ) (see FIG. 1A) .
  • the first and second polypeptides are paired via the VH (ROR-1) -VL (ROR-1) pairing.
  • the two first polypeptides can be paired via the IgG (CH) pairing.
  • the present disclosure also provides two or more anti-ROR1 antigen-binding moieties as bi-paratopic.
  • a bi-paratopic antibody is a subset of bispecific antibody that recognizes two distinct epitopes on the same antigen.
  • the anti-ROR1 antigen-binding moieties provided herein include a first anti-ROR1 antigen-binding moiety and a second anti-ROR1 antigen-binding moiety.
  • the first anti-ROR1 antigen-binding moiety has a different epitope on the ROR1 antigen from that of the second anti-ROR1 antigen-binding moiety.
  • the first and second anti-ROR1 antigen-binding moieties provided herein have an full-length antibody/IgG format, respectively, and the anti-4-1BB antigen-binding moiety provided herein has a single domain antibody (sdAb) /VHH/nanobody format.
  • the first anti-ROR1 antigen-binding moiety comprises a pair of VH and VL
  • the second anti-ROR1 antigen-binding moiety comprises a pair of VH and VL.
  • the paired VH and VL are fused to IgG constant regions (IgG (CH+CL) ) , respectively.
  • the anti-ROR1 antigen-binding moieties provided herein have an IgG format and a scFv format, respectively, and the anti-4-1BB antigen-binding moiety provided herein has a single domain antibody (sdAb) /VHH/nanobody format.
  • the first anti-ROR1 antigen-binding moiety comprises a pair of VH and VL, and the second anti-ROR1 antigen-binding moiety comprises a scFv.
  • the pair of VH and VL of the first anti-ROR1 antigen-binding moiety are fused to an IgG constant region, wherein the VH is fused to the IgG heavy chain constant region (IgG (CH) ) , and the VL is fused to the IgG light chain constant region (IgG (CL) ) .
  • the scFv of the second anti-ROR1 antigen-binding moiety is fused to the VH of the first anti-ROR1 antigen-binding moiety, or fused to another IgG heavy chain constant region (IgG’ (CH) ) .
  • the IgG (CH) is different from the IgG’ (CH) . In certain embodiments, the IgG (CH) is the same as the IgG’ (CH) .
  • the scFv of the second anti-ROR1 antigen-binding moiety comprises from N-to C-terminus: VL-tether-VH.
  • the scFv is fused to the VH of the first anti-ROR1 antigen-binding moiety (scFv-IgG (CH) ) or the IgG’ (CH) via its VH (scFv-IgG’ (CH) ) .
  • the scFv-IgG (CH) format includes from N-to C-terminus: (VL-tether-VH) - (VH-CH1-hinge-CH2-CH3) .
  • An IgG Fc domain provided herein may comprise CH2-CH3, or hinge-CH2-CH3. In certain embodiments of the present disclosure, an IgG Fc domain starts at position Cys226 to the carboxyl-terminus thereof (EU numbering system) .
  • the scFv-IgG’ (CH) format includes from N-to C-terminus: (VL-tether-VH) -hinge-CH2-CH3, (VL-tether-VH) -partial hinge-CH2-CH3, or (VL-tether-VH) -hinge variant -CH2-CH3.
  • the IgG’ (CH) may comprise hinge-CH2-CH3, partial hinge-CH2-CH3, or hinge variant -CH2-CH3.
  • the hinge variant may include a two amino acid residue linker AA before the hinge region start with “EPKSC” (SEQ ID NO: 138) , wherein the C residue is mutated to S for there is no light chain to form disulfide bond with it.
  • the scFv of the second anti-ROR1 antigen-binding moiety comprises from N-to C-terminus: VH-tether -VL.
  • the scFv is fused to the VH of the first anti-ROR1 antigen-binding moiety (scFv-IgG (CH) ) or the IgG’ (CH) via its VL (scFv-IgG’ (CH) ) .
  • the scFv-IgG (CH) format includes from N-to C-terminus: (VH-tether -VL) - (VH-CH1-hinge-CH2-CH3) .
  • the scFv-IgG’ (CH) format includes from N-to C-terminus: (VH-tether -VL) -hinge-CH2-CH3, (VH-tether -VL) -partial hinge-CH2-CH3, or (VH-tether -VL) -variant hinge-CH2-CH3.
  • the IgG’ (CH) may comprise hinge-CH2-CH3, partial hinge-CH2-CH3, or hinge variant-CH2-CH3.
  • the single domain antibody (sdAb) /VHH of the anti-4-1BB antigen-binding moiety is fused to the C-terminus of the IgG (CH) or the C-terminus of the IgG’ (CH) .
  • the multi-specific antibody provided herein include a first polypeptide comprising from N-to C-terminus: scFv (2 nd ROR-1) -VH (1 st ROR-1) -IgG (CH) -VHH (4-1BB) , and a second polypeptide comprising from N-to C-terminus: VL (1 st ROR-1) -IgG (CL) (see FIG. 1C) .
  • the first polypeptide is paired with the second polypeptide via the VH (1 st ROR-1) -VL (1 st ROR-1) pairing.
  • Two first polypeptides can be paired via the IgG (CH) pairing or more specifically, Fc region pairing.
  • the multi-specific antibody provided herein include a first polypeptide comprising from N-to C-terminus: VH (1 st ROR-1) -IgG (CH) -VHH (4-1BB) , a second polypeptide comprising from N-to C-terminus: VL (1 st ROR-1) -IgG (CL) , a third polypeptide comprising from N-to C-terminus: scFv (2 nd ROR-1) -IgG’ (CH) -VHH (4-1BB) (see FIG. 1B) .
  • the first polypeptide is paired with the second polypeptide via the VH (1 st ROR-1) -VL (1 st ROR-1) pairing, and the first polypeptide is paired with the third polypeptide via the IgG (CH) -IgG’ (CH) pairing.
  • two identical Fc regions form a homodimer.
  • two different Fc regions can form a heterodimer with mutations to the one or two individual chain via, for example, a knob-into-hole (KIH) , a disulfide bond (-S-S-) , or via hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.
  • the IgG (CH) -IgG’ (CH) pairing can be achieved by forming a heterodimer via knob-into-hole (KIH) , hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.
  • the Fc domain provided herein comprises a knob mutation and the pairing Fc domain comprises hole mutation (s) , or vice versa.
  • knock-into-hole or “KIH” technology as used herein refers to the technology directing the pairing of two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact.
  • KIHs have been introduced in the Fc: Fc binding interfaces, CL: CH1 interfaces or VH/VL interfaces of antibodies (see, e.g., US 201 1/0287009, US2007/0178552, WO 96/02701 1, WO 98/050431, Zhu et al, 1997, Protein Science 6: 781-788, and WO2012/106587) .
  • KIHs drive the pairing of two different heavy chains together during the manufacture of multispecific antibodies.
  • multispecific antibodies having KIH in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains.
  • KIH technology can be also be used to pair two different receptor extracellular domains together or any other polypeptide sequences that comprises different target recognition sequences (e.g., including affibodies, peptibodies and other Fc fusions) .
  • knock mutation refers to a mutation that introduces a protuberance (knob) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide.
  • the other polypeptide has a hole mutation.
  • hole mutation refers to a mutation that introduces a cavity (hole) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide.
  • the other polypeptide has a knob mutation.
  • a knob mutation in an IgG1 constant region comprises T366W (EU numbering) .
  • a hole mutation in an IgG1 constant region comprises one or more mutations selected from T366S, L368A, and Y407V (EU numbering) .
  • a hole mutation in an IgG1 constant region comprises T366S, L368A, and Y407V (EU numbering) .
  • a knob mutation in an IgG1 constant region comprises S354C and T366W (EU numbering) .
  • a hole mutation in an IgG1 constant region comprises one or more mutations selected from Y349C, T366S, L368A, and Y407V.
  • Multispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1) ; cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al, Science, 229: 81 (1985) ) ; using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al, J. Immunol, 148 (5) : 1547-1553 (1992) ) ; using "diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci.
  • the IgG (CH) includes a CH1, a hinge region, a CH2 and a CH3. In certain embodiments, the IgG (CH) comprises an amino acid sequence of SEQ ID NO: 50. Two IgG (CH) scan form a homodimer.
  • the IgG (CH) comprises an amino acid sequence of SEQ ID NO: 51 with substitutions at S237C/T249W (corresponding to knob mutation S354C/T366W by EU numbering) , or SEQ ID NO: 55 with substitutions at S237C/T249W and M311L/N317S (a LS mutation corresponding to M428L/N434S by EU numbering that prolongs the half-life of antibody in vivo) .
  • the IgG (CH) includes a CH1, a hinge region and a Fc domain.
  • the Fc domain of the present disclosure comprises an amino acid sequence of SEQ ID NO: 141 with substitutions at S237C/T249W (corresponding to the knob mutation S354C/T366W by EU numbering) , or SEQ ID NO: 142 with substitutions at S237C/T249W and M311L/N317S (a LS mutation corresponding to M428L/N434S by EU numbering that prolongs the half-life of antibody in vivo) .
  • the IgG’ (CH) includes a CH1, a hinge region, a CH2 and a CH3. In certain embodiments, the IgG’ (CH) lacks partial CH1, lacks CH1, or lacks CH1+partial hinge region. In certain embodiments, the IgG’ (CH) includes a partial CH1, a hinge region, a CH2 and a CH3. In certain embodiments, the IgG’ (CH) includes a hinge region, a CH2 and a CH3.
  • the IgG’ includes a hinge region variant, a CH2 and a CH3.
  • the hinge region variant may include a two amino acid residue-short linker AA before the hinge region starts with “EPKS C ” (SEQ ID NO: 138) , wherein the C residue is mutated to S for there is no light chain to form disulfide bond with it.
  • the IgG’ (CH) comprises an amino acid sequence of SEQ ID NO: 53 that has substitutions at Y136C/T153S/L155A/Y194V (a hole mutations with Y349C/T366S/L368A/Y407V by EU numbering) .
  • the IgG’ (CH) comprises an amino acid sequence of SEQ ID NO: 54 that has substitutions at Y136C/T153S/L155A/Y194V and M215L/N221S (a LS mutation corresponding to M428L/N434S by EU numbering that prolongs the half-life of antibody in vivo) .
  • the IgG’ (CH) includes a hinge region variant and a pairing Fc domain.
  • the pairing Fc domain of the present disclosure comprises an amino acid sequence of SEQ ID NO: 143 that has substitutions at Y136C/T153S/L155A/Y194V (a hole mutations with Y349C/T366S/L368A/Y407V by EU numbering) .
  • the IgG’ comprises an amino acid sequence of SEQ ID NO: 52 that has substitutions at Y136C/T153S/L155A/Y194V and M215L/N221S (a LS mutation corresponding to M428L/N434S by EU numbering that prolongs the half-life of antibody in vivo) .
  • An IgG (CH) and an IgG’ (CH) can form a heterodimer.
  • a Fc domain and a pairing Fc domain can form a heterodimer.
  • the IgG (CH) and the IgG’ (CH) forms knob-into-hole (KIH) :
  • the IgG (CH) of SEQ ID NO: 51 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity thereof) can be paired with IgG’ (CH) of SEQ ID NO: 53 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity thereof) ; or
  • the IgG (CH) of SEQ ID NO: 55 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity thereof) can be paired
  • the Fc domain and the pairing Fc domain form knob-into-hole (KIH) :
  • the Fc domain of SEQ ID NO: 141 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity thereof) can be paired with pairing Fc domain of SEQ ID NO: 143 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity thereof) ; or
  • the Fc domain of SEQ ID NO: 142 (or a variant thereof having at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity thereof) can be paired with pairing Fc domain of SEQ ID NO: 52
  • the first antibody moiety or the second antibody moiety comprises an Fc region (also referred to herein as “Fc fragment” ) .
  • the Fc region is an Fc domain, namely, an Fc region possessing some or all effector functions, including for example complement-dependent cytotoxicity (CDC) and antibody-dependent cell mediated cytotoxicity (ADCC) functions.
  • the Fc domain is derive from IgG1 or IgG3.
  • one or more amino acid modifications may be introduced into the Fc domain, thereby generating an Fc domain variant.
  • the Fc domain variant may comprise a human Fc domain sequence (e.g., derived from human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the Fc domain variant change one or more functional and/or pharmacokinetic properties of the antibody.
  • the Fc region can also be engineered to enhance or eliminate effector function.
  • IgG antibodies can induce direct anti-tumor effects by way of indirect anti-tumor effects via the Fc-mediated effector functions that engage other immune cells or killer mechanisms.
  • Effective functions or “antibody effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor (Fc ⁇ RIIa or Fc ⁇ RIIIa) .
  • effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and antibody dependent cell mediated phagocytosis (ADCP) , where nonspecific cytotoxic cells that express Fc ⁇ receptors (Fc ⁇ Rs) recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody dependent cell mediated phagocytosis
  • the Fc regions provided herein are maintained or improved with effector function, such as ADCC and/or CDC.
  • IgG1 and IgG3 induce the strongest Fc-effector functions.
  • IgG1 has the longest half-life and is more stable than IgG3, most therapeutic antibodies with Fc-mediated functions are of IgG1 isotype.
  • IgG2 and IgG4 isotypes have significantly lower binding affinity to Fc ⁇ Rs. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects.
  • the Fc domain is derived from human IgG1. In some embodiments, the human IgG1 derived Fc domain does not comprise L234A mutation and/or a L235A mutation. In some embodiments, the human IgG1 derived Fc domain comprises a L234A mutation and/or a L235A mutation. In some embodiments, the Fc domain is derived from human IgG3. In some embodiments, the Fc domain is derived from human IgG2 or IgG4. In some embodiments, the Fc domain is derived from human IgG4. In some embodiments, the human IgG4 derived Fc domain comprises a S228P, F234A, and/or a L235A mutation. In some embodiments, the human IgG4 derived Fc domain does not comprise a S228P, F234A, and/or a L235A mutation.
  • alterations are made in the Fc domain that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC) , e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .
  • CDC Complement Dependent Cytotoxicity
  • the multispecific construct comprises a variant Fc domain comprising one or more amino acid substitutions which alters half-life and/or changes binding to the neonatal Fc receptor (FcRn) .
  • Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) ) , are described in US2005/0014934A1 (Hinton et al. ) .
  • Those antibodies comprise an Fc region with one or more substitutions therein which alters binding of the Fc region to FcRn.
  • Fc variants include those with substitutions at one or more of Fc region residues, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826) .
  • Fc receptor (FcR) binding assays can be conducted to determine whether the antibody possesses FcgR binding (hence likely ADCC activity) , and/or retains FcRn binding ability.
  • FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) .
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83: 7059-7063 (1986) ) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82: 1499-1502 (1985) ; 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987) ) .
  • non-radioactive assays methods may be employed (see, for example, ACTI TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI) .
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95: 652-656 (1998) .
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) ; Cragg, M.S. et al., Blood 101: 1045-1052 (2003) ; and Cragg, M.S. and M.J. Glennie, Blood 103: 2738-2743 (2004) ) .
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18 (12) : 1759-1769 (2006) ) .
  • Different antibody domains can be fused via one or more linkers.
  • the fusion of the scFv to the VH, or the fusion of the VHH to the IgG (CH) is via a linker.
  • specific formats of the multi-specific antibody provided herein include
  • the tether in the scFv can be the same as the linker.
  • Linkers within the scope of the present disclosure are characterized in terms of amino acid content, length, rigidity and secondary structure. Linkers within the scope of the present disclosure separate a function polypeptide and another functional polypeptide and allow proper folding and functioning of each domain. In this manner, a linker can be tailored to the particular functional polypeptide and the other functional polypeptide. According to one aspect, functional independence of the structural and fused (heterologous) domains is maximized by a suitable linker to limit steric interference between domains during the export and assembly processes of the bacterial cell.
  • longer and more flexible linkers of the type (GGGGS) n wherein n is an integer from 1 to 20 (SEQ ID NO: 39) are exemplary.
  • cell stress is minimized by limiting the overall length of the fusion protein. Longer linker sequences and higher induction levels stress the biosynthetic machinery of the cells, inhibiting cell growth and leading to cell lysis in extreme cases.
  • Linkers within the scope of the present disclosure facilitate functioning of the antibody domains.
  • Linkers within the scope of the present disclosure allow efficient protein processing and export through the bacterial curli secretion machinery as well as provide the proper spatial and physicochemical separation of the respective antibody domains to retain their respective functions.
  • Linkers within the scope of the present disclosure include amino acid residues.
  • the amino acid residues may be any of the naturally occurring amino acid residues.
  • Amino acid residues may also be synthetic amino acids known to those of skill in the art.
  • Representative amino acids which may be used in linkers include Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Cysteine, Selenocysteine, Threonine, Methionine, Proline, Phenylalanine, Tyrosine, Tryptophan, Histidine, Lysine, Arginine, Aspartate, Glutamate, Asparagine, and Glutamine.
  • the linker length can be any length which may be expressed from a cell, such as a bacterial cell when linking the antibody domains.
  • a linker sequence is a polypeptide sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48 or more amino acids.
  • the linker sequence comprises from about 3 amino acids to about 50 amino acids.
  • the linker sequence comprises from about 3 amino acids to about 40 amino acids.
  • the linker sequence comprises from about 5 amino acids to about 30 amino acids.
  • the linker sequence comprises from about 5 amino acids to about 25 amino acids.
  • the linker sequence comprises from about 5 amino acids to about 24 amino acids.
  • the linker sequence comprises from about 5 amino acids to about 23 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 22 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 21 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 20 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 19 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 18 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 17 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 16 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 15 amino acids. In certain embodiments, the linker sequence of the present disclosure has 15 amino acids.
  • the linker sequence comprises a flexible polypeptide, e.g. a polypeptide not having a rigid secondary and/or tertiary structure.
  • the linker sequence comprises glycine and serine residues.
  • at least 50%of the amino acids comprised by the linker sequence are glycine or serine residues, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more are glycine or serine residues.
  • the linker sequence consists of glycine and serine residues.
  • the linker sequence of the present disclosure has an amino acid sequence of SEQ ID NO: 56.
  • the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below.
  • an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label) .
  • Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope.
  • the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.
  • the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • the antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
  • a therapeutic agent which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.
  • the present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the disclosure.
  • the polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.
  • the humanized antibody may be designed to minimize unwanted immunological response toward rodent anti-human antibodies, which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
  • the humanized antibody may have one or more amino acid residues introduced into it from a source that is non-human. These non-human residues are often referred to as "import" residues, which are typically taken from a variable domain. Humanization may be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. For example, see U.S. Patent No. 4,816,567, the contents of which are herein incorporated by reference.
  • the humanized antibody may be a human antibody in which some hypervariable region residues, and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.
  • the antibodies, variants or derivatives of the present disclosure may be used in certain treatment methods.
  • the present disclosure is further directed to antibody-based therapies which involve administering the antibodies of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein.
  • Therapeutic compounds of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein) .
  • the method in one embodiment, entails administering to the patient an effective amount of an antibody of the present disclosure.
  • antibodies of the present disclosure for use in the treatment of a cancer in a patient in need thereof.
  • Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
  • Additional diseases or conditions associated with increased cell survival include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) ) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ) , polycythemia vera, lymphomas (e.g., Hodgkin’s disease and non-Hodgkin’s disease) , multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sar
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient’s age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art.
  • the amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
  • Methods of administration of the antibodies, variants or include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc. ) and may be administered together with other biologically active agents.
  • compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch) , bucally, or as an oral or nasal spray.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.
  • Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the antigen-binding polypeptides or compositions of the disclosure may be administered locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care must be taken to use materials to which the protein does not absorb.
  • compositions comprise an effective amount of an antibody, and an acceptable carrier.
  • the composition further includes a second anticancer agent (e.g., an immune checkpoint inhibitor) .
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the compounds of the disclosure can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • ROR1 ⁇ 4-1BB bispecific antibodies are constructed as shown in FIG. 1A and Table 3 below.
  • FACS binding activity shows that ROR1 ⁇ 4-1BB BsAbs binding with ROR1 endogenous expressed tumor cell A549 and 4-1BB overexpressed HEK293 cell in a dose-dependent manner (FIG. 2A-B) .
  • 3C5 and 8F2 showed non-competitive or non-overlapping binding epitope with ROR1 antigen, as detected by Octet (see FIG. 4A-C) .
  • bi-paratopic ROR1 ⁇ 4-1BB antibodies were designed on the hypothesis that bi-paratopic antigen targeting antibody could make more 4-1BB clustering and induce stronger 4-1BB pathway activation.
  • Humanized anti-ROR1 antibodies were used to design mono-topic (bispecific) and bi-paratopic ROR1 ⁇ 4-1BB TsAb (see Table 4) .
  • the cellular binding of humanized anti-ROR1 mAbs as measured by FACS, and ROR1 dependent 4-1BB activation of humanized ROR1 ⁇ 4-1BB BsAbs were tested similarly as above.
  • the humanized mAbs showed better or comparable cellular binding (FIG. 6A-B)
  • the humanized BsAbs showed better or comparable ROR1 dependent 4-1BB activation as compared to the chimeric antibodies (FIG. 6C-D)
  • the 4-1BB VHH control Uelumab does not activate 4-1BB in the ROR1 positive tumor cell lines in the corresponding concentration range.
  • the comparison between the humanized antibodies showed that the bi-paratopic antibody 4-BiR1 has higher cellular binding of ROR1 in the ROR1-positive tumor cell lines (FIG. 7A-C) and higher ROR1 dependent 4-1BB activation (FIG. 7D-F) , as compared with 4B-3C5 and 4B-8F5, while 4B-3C5 and 4B-8F5 showed similar cellular binding affinity and ROR1 dependent 4-1BB activation. There is no ROR1 independent 4-1BB activation for all the tested antibodies in the ROR1 negative tumor cell line.
  • a surface plasmon resonance assay showed the binding affinity to ROR1 of the humanized antibodies 4B-h3C5 with a K D value of 7.93E-08 M, 4B-h8F5 with a K D value of 2.37E-07M, and a 4B-hBiR1 with a K D value of 1.21E-08 M (FIG. 8A-C) .
  • a syngeneic model (4-1BB humanized mice (Biocytogen) ) was used to evaluate the in vivo efficacy of mono-topic and bi-paratopic ROR1 ⁇ 4-1BBs inoculated with the ROR1-MC38 cell line. All the tested humanized antibodies showed significant inhibition to the tumor growth (FIG. 9A-B) .
  • 4B-2773, 4B-27 and 4B-73 are selected as a representative of engineered mono-topic and bi-paratopic ROR1 ⁇ 4-1BB antibodies to compare with 4B-hBiR1 and the benchmark (Clone ID: BA6 (NA) ⁇ 1A10M12) in a cytokine release assay.
  • 4B-hBiR1 and the benchmark Clone ID: BA6 (NA) ⁇ 1A10M12
  • 1 ug/ml anti-CD3 (OKT3) were coated on plate, MDA-MB-231 cell line was used as target cell, and PBMC and gradient-diluted antibodies were co-cultured for 72 hours. IFN- ⁇ and IL-2 induced by 4-1BB were detected.
  • the results showed that all the test engineered ROR1 ⁇ 4-1BB antibodies released higher cytokines than the bench marker and the urelumab (FIG. 12A-B) .
  • a syngeneic model (4-1BB humanized mice (Biocytogen) ) was used to evaluate the in vivo efficacy of the engineered mono-topic and bi-paratopic ROR1 ⁇ 4-1BBs inoculated with the ROR1-MC38 cell line. All the tested humanized antibodies showed significant inhibition to the tumor growth (FIG. 13A-C) .
  • bispecific antibodies were prepared that targeted claudin 6 (CLDN6) in addition to 4-1BB.
  • CLDN6 claudin 6
  • the single-domain 4-1BB antibody was fused to the C-terminus of the Fc domain (IgG1 Fc wild-type (WT) or IgG1 Fc with N297A (NA) , EU numbering) , while the anti-CLDN6 Fab’s were disposed at the N-terminus.
  • CT26 cells which endogenously express CLDN6 were subcutaneously implanted into BALB/c humanized 4-1BB mice.
  • the mice were intraperitoneally treated with (a) human IgG1, (b) CLDN6-1 x 4-1BB NA (2 mg/kg) , (c) CLDN6-1 x 4-1BB WT (2 mg/kg) , or (d) a combination of the parental CLDN6-1 antibody and the 4-1BB sdAb-Fc (1.8 mg/kg and 0.7 mg/kg) .
  • the treatments were administered twice weekly, for a total of 6 doses. Tumor growth was monitored by volumetric measurement. As shown in FIG.
  • CLDN6-1 x 4-1BB WT and CLDN6-1 x 4-1BB NA both exhibited anti-tumor activity, in which CLDN6-1 x 4-1BB WT achieved even stronger activity with tumor growth inhibition (TGI) of 75%.

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Abstract

L'invention concerne des anticorps bispécifiques et multispécifiques qui ciblent un antigène tumoral et 4-1BB avec une région Fc conservée ou améliorée avec une fonction effectrice, des anticorps multispécifiques particuliers ayant une spécificité de liaison à la protéine ROR1 humaine et à la protéine 4-1BB humaine. L'invention concerne également des procédés d'utilisation des anticorps ou des fragments de ceux-ci pour le traitement du cancer.
PCT/CN2024/117323 2023-09-06 2024-09-06 Anticorps anti-ror1/anti-4-1bb et leurs utilisations Pending WO2025051220A1 (fr)

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